U.S. patent application number 12/500854 was filed with the patent office on 2010-01-14 for compositions and methods for detection and isolation of phosphorylated molecules.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Brian Agnew, Joseph Beechem, Kyle Gee, Richard Haugland, Wayne Patton, Thomas Steinberg.
Application Number | 20100009381 12/500854 |
Document ID | / |
Family ID | 34590729 |
Filed Date | 2010-01-14 |
United States Patent
Application |
20100009381 |
Kind Code |
A1 |
Agnew; Brian ; et
al. |
January 14, 2010 |
Compositions and methods for detection and isolation of
phosphorylated molecules
Abstract
The present invention relates to phosphate-binding compounds
that find use in binding, detecting and isolating phosphorylated
target molecules including the subsequent identification of target
molecules that interact with phosphorylated target molecules or
molecules capable of being phosphorylated. A binding solution is
provide that comprises a phosphate-binding compound, an acid and a
metal ion wherein the metal ion simultaneously interacts with an
exposed phosphate group on a target molecule and the metal
chelating moiety of the phosphate-binding compound forming a bridge
between the phosphate-binding compound and a phosphorylated target
molecule resulting in a ternary complex. The binding solution of
the present invention finds use in binding and detecting
immobilized and solubilized phosphorylated target molecules,
isolation of phosphorylated target molecules from a complex mixture
and aiding in proteomic analysis wherein kinase and phosphatase
substrates and enzymes can be identified.
Inventors: |
Agnew; Brian; (Eugene,
OR) ; Beechem; Joseph; (Eugene, OR) ; Gee;
Kyle; (Springfield, OR) ; Haugland; Richard;
(Eugene, OR) ; Steinberg; Thomas; (Eugene, OR)
; Patton; Wayne; (Newton, MA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
34590729 |
Appl. No.: |
12/500854 |
Filed: |
July 10, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11697103 |
Apr 5, 2007 |
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12500854 |
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10703816 |
Nov 7, 2003 |
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11697103 |
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10428192 |
May 2, 2003 |
7102005 |
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10703816 |
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60377733 |
May 3, 2002 |
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60393059 |
Jun 28, 2002 |
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60407255 |
Aug 30, 2002 |
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60440252 |
Jan 14, 2003 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 33/5008 20130101;
C12Q 1/42 20130101; G01N 33/502 20130101; Y10T 436/147777 20150115;
Y10T 436/201666 20150115; C12Q 1/485 20130101; G01N 2333/4709
20130101; Y10T 436/145555 20150115; G01N 33/5091 20130101; Y10T
436/142222 20150115 |
Class at
Publication: |
435/7.1 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made in part with government support
under grant number 1 R33 CA093292-01, awarded by the National
Cancer Institute. The United States Government may have certain
rights in this invention.
Claims
1. A binding solution comprising: a) a phosphate-binding compound
having formula (A)m(L)n(B) wherein A is a chemical moiety, L is a
linker, B is a metal-chelating moiety, m is an integer from 1 to 4
and n is an integer from 0 to 4; and b) a salt comprising metal
ions.
2. The binding solution of claim 1, further comprising an acid.
3. The binding solution of claim 1, wherein the metal ions are a
cationic transition metal.
4. The binding solution of claim 1, wherein said linker comprises:
--C(O)NH--.
5. The binding solution of claim 1, wherein the chemical moiety is
a label.
6. The binding solution of claim 5, wherein the label is a dye
selected from the group consisting of a benzofuran, a
quinazolinone, a xanthene, an indole, a benzazole and a
borapolyazaindacene.
7. The binding solution of claim 5, wherein the label is selected
from the group consisting of a dye, an enzyme and a hapten.
8. The binding solution of claim 1, wherein m is 1 and n is 1.
9. The binding solution of claim 2, wherein the acid is acetic
acid.
10. The binding solution of claim 2, wherein the acid is present at
a concentration of 1%-20%.
11. The binding solution of claim 1, wherein the binding solution
has a pH about 3 to about pH 6.
12. The binding solution of claim 1, further comprising a buffering
agent.
13. The binding solution of claim 12, wherein the buffering agent
is selected from the group consisting of salts of formate, acetate,
2-(N-morpholino)ethanesulfonic acid, imidazole,
N-(2-hydroxyethyl)piperazinylethanesulfonic acid,
tris-(hydroxymethyl) aminomethane acetate, or
tris(hydroxymethyl)aminomethane, and hydrochloride.
14. The binding solution of claim 1, further comprising a
water-miscible organic solvent.
15. The binding solution of claim 14, wherein the water-miscible
organic solvent is an alcohol.
16. The binding solution of claim 15, wherein the alcohol is
methanol.
17. The binding solution of claim 15, wherein the alcohol is
present in a concentration of less than about 20%.
18. The binding solution of claim 1, wherein the phosphate-binding
compound has the formula: (A)-[C(X)NH(CH.sub.2).sub.e]--(B);
wherein X is O and e is 0-6.
19. The binding solution of claim 1, wherein the metal chelating
moiety is immobilized on a solid or semi-solid matrix.
20. The binding solution of claim 1, wherein the metal chelating
moiety is capable of binding a trivalent metal ion.
21. A method for detecting a phosphorylated target molecule
immobilized on a gel comprising the following steps: immobilizing a
sample on a gel; contacting the gel of with a binding solution
comprising: a) a phosphate-binding compound having formula
(A)m(L)n(B) wherein A is a chemical moiety, L is a linker, B is a
metal-chelating moiety, m is an integer from 1 to 4 and n is an
integer from 0 to 4; b) a salt comprising metal ions; and, c) an
acid; incubating the gel and the binding solution for sufficient
time to allow the phosphate-binding compound to associate with the
phosphorylated target molecule; and, visualizing the
phosphate-binding compound whereby the phosphorylated target
molecule is detected.
22. The method of claim 21, further comprising contacting the gel
with a fixing solution directly after the immobilizing step.
23. The method of claim 21, further comprising adding a second (or
third) stain to the gel to detect either total protein or proteins
of another class, such as glycoproteins, or both.
24. A kit for binding a phosphorylated target molecule in a sample,
said kit comprising: i) a binding solution comprising: a) a
phosphate-binding compound having formula (A)m(L)n(B) wherein A is
a chemical moiety, L is a linker, B is a metal-chelating moiety, m
is an integer from 1 to 4 and n is an integer from 0 to 4; and b) a
salt comprising metal ions; ii) at least one of instructions,
molecular weight markers that comprise phosphorylated and
non-phosphorylated polypeptides, a fixing solution, a detection
reagent, standards, a wash solution, a matrix, a gel, or a
phosphatase or kinase substrate.
25. The kit of claim 24, wherein the binding solution further
comprises an acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/703,816, filed Nov. 7, 2003 which is a continuation-in-part of
U.S. Ser. No. 10/428,192, filed May 2, 2003 which claims priority
to U.S. Ser. No. 60/377,733, filed May 3, 2002; U.S. Ser. No.
60/393,059, filed Jun. 28, 2002; U.S. Ser. No. 60/407,255, filed
Aug. 30, 2002; and U.S. Ser. No. 60/440,252, filed Jan. 14, 2003,
which disclosures are herein incorporated by reference. Any
disclaimer that may have occurred during the prosecution of the
above-referenced application(s) is hereby expressly rescinded.
FIELD OF THE INVENTION
[0003] The present invention relates to metal-chelating
compositions and methods for use in the detection and isolation of
phosphorylated target molecules. The invention has applications in
the fields of proteomics, molecular biology, high-throughput
screening and diagnostics.
BACKGROUND OF THE INVENTION
[0004] Phosphorylation and dephosphorylation are processes by which
phosphate groups are added or removed from a target molecule,
typically a protein. The process of reversible phosphorylation is a
key feature of cellular regulation, including signal transduction,
gene expression, cell cycle regulation, cytoskeletal regulation and
apoptosis. See, e.g., PROTEIN PHOSPHORYLATION (Marks F. ed., 1996);
Hunter, "Signaling--2000 and beyond," Cell 100:113-127 (2000).
Principally, two classes of enzymes (kinases and phosphatases)
modulate reversible protein phosphorylation, adding phosphate
groups and removing phosphate groups, respectively, from molecules.
Phosphorylation reactions are key features of protein function, and
thus phosphorylated proteins must be able to be identified if the
proteome is to be fully understood; however, to date no practical
methods exist for the systematic parallel analysis of the
phosphorylation status of large sets of proteins involved in the
regulatory circuitry of cells and tissues. See, Wilkins et al.,
Genetic Eng. Rev. 13:19 (1995).
[0005] Signal transduction is an example of a process involving
protein phosphorylation that is critical for cellular regulation.
After an extracellular stimulatory factor binds to its recognized
cell surface receptor, signal transduction is initiated, often by a
specific set of cellular protein kinases. These kinases
subsequently phosphorylate the target molecule, resulting in an
altered activity and a continued cellular response to the signal.
See, e.g., Nishizuka, "Studies and perspectives of protein kinase
C," Science 233:305-312 (1986). It is not enough for researchers to
simply identify whether a protein is a phosphorylated protein or
not. It has become additionally essential for researchers to
identify the sites of phosphorylation on proteins and to determine
the stoichiometry of phosphorylation. Serine, threonine and
tyrosine amino acid residues are the most common sites of
phosphorylation in eukaryotic cells. See, e.g., Guy et al.
"Analysis of Cellular Phosphoproteins by Two-Dimensional Gel
Electrophoresis: Applications for Cell Signaling in Normal and
Cancer Cells," Electrophoresis 15:417-440 (1994). Thus, the focus
for researchers in understanding protein phosphorylation events
occurs at two levels. The first level of analysis requires a
determination of whether a protein is a phosphoprotein, including
identifying molecules responsible for phosphorylation, and the
second level of analysis requires the identification of which amino
acid is phosphorylated and how many amino acids are phosphorylated.
The present invention provides materials and methods for both
levels of analysis. The present invention also provides materials
and methods for analysis of certain other phosphate and
thiophosphate-containing materials including esters of
carbohydrates, nucleotides and lipids.
[0006] Currently, phosphoproteins are most often detected by
autoradiography after incorporation of .sup.32P or .sup.33P into
cultured cells or after incorporation into subcellular fractions by
protein kinases. See, e.g., Yan et al., "Protein Phosphorylation:
Technologies for the Identification of Phosphoamino Acids," J.
Chromatogr. A. 808:23-41 (1998); Guy, G., Phillip, R. and Tan, Y.
Electrophoresis 15:417-440 (1994). Such approaches are restricted
to a limited range of biological materials, such as tissue culture
samples and analysis of clinical samples would require in vivo
labeling of patients, which is not feasible. Several alternatives
to radiolabeling have also been developed over the years.
[0007] Phosphoproteins can also be detected by immunoblotting and
immunoprecipitation. See, e.g., Soskic et al., "Functional
Proteomics Analysis of Signal Transduction Pathways of the
Platelet-Derived Growth Factor Beta Receptor," Biochemistry
38:1757-1764 (1999); Watty et al., "The In Vitro and In Vivo
Phosphotyrosine Map of Activated MuSK," Proc. Natl. Acad. Sci. USA.
97:4585-4590 (2000). Immunoblotting is best performed after
blocking unoccupied sites on the solid-phase support with protein
solutions, which interferes with microchemical analysis. Removal of
the antibody and stain require relatively harsh treatments (i.e.,
heating to 65.degree. C., incubation with 0.1% SDS and 1 mM DTT).
This also poses problems with subsequent use of the protein for
sequencing and mass spectrometry. For immunoprecipitation, only the
anti-phosphotyrosine antibodies display binding that is tight
enough to allow effective isolation. Though high-quality antibodies
to phosphotyrosine are commercially available, antibodies that
specifically recognize phosphoserine and phosphothreonine residues
have been more problematic, often being sensitive to amino acid
sequence context. The reliability of these antibodies has been
questioned because of potential steric hindrances between the
interaction of these antibodies and the phosphoproteins. Moreover,
when phosphoproteins are not enriched prior to detection with the
antibody, the presence of unrelated proteins co-migrating with the
protein of interest may lead to false positive signals. Therefore,
identification of phosphorylated proteins using immunoblotting and
immunoprecipitation techniques is effectively limited to proteins
containing phosphorylated tyrosine residues. See McLachlin &
Chait, supra.
[0008] Alternatively, phosphorylated proteins can be identified
using chromogenic dyes, but with limited success. The cationic
carbocyanine dye "Stains-All"
(1-ethyl-2-[3-(3-ethylnaphtho[1,2d]thiazolin-2-ylidene)-2-methylpropenyl]-
-naphtho[1,2d]thiazolium bromide) stains RNA, DNA, phosphoproteins
and calcium-binding proteins blue while unphosphorylated proteins
are stained red. See, e.g., Green et al., "Differential Staining of
Phosphoproteins on Polyacrylamide Gels with a Cationic Carbocyanine
Dye," Anal. Biochem. 56:43-51 (1973); Hegenauer et al., "Staining
Acidic Phosphoproteins (Phosvitin) in Electrophoretic Gels," Anal.
Biochem. 78:308-311 (1977); Debruyne, "Staining of Alkali-Labile
Phosphoproteins and Alkaline Phosphatases on Polyacrylamide Gels,"
Anal. Biochem. 133:110-115 (1983); "Staining of phosphoproteins in
polyacrylamide gels with acridine orange", Seikagaku 45:327-35
(1973). Stains-All is not routinely used to detect phosphoproteins
due to poor specificity and low sensitivity. Stains-All is at least
10 times less sensitive than Coomassie Brilliant Blue as a general
protein stain and several orders of magnitude less sensitive than
.sup.32P-autoradiography or the techniques described in this
patent. Another chromogenic method, the GelCode.TM. Phosphoprotein
detection kit (Pierce Chemical Company, Rockford, Ill.), is
designed to detect phosphoproteins in gels; however, this method
has many limitations. According to this method, phosphoproteins are
detected in gels through alkaline hydrolysis of phosphate esters of
serine or threonine, precipitation of the released inorganic
phosphate with calcium ions, formation of an insoluble
phosphomolybdate complex and then visualization of the complex with
a dye such as methyl green, malachite green or rhodamine B [as
described in Cutting and Roth (1973)]. The detection sensitivity of
the staining method is considerably poorer than Coomassie Blue
staining, with 80-160 ng of phosvitin, a protein containing roughly
100 phosphoserine residues, being detectable by the commercialized
kit. The staining procedure is quite complex (involving seven
different reagents) and alkaline hydrolysis requires heating gels
to 65.degree. C., which causes the gel matrix to hydrolyze and
swell considerably. Since phosphotyrosine residues are not
hydrolyzed by the alkaline treatment, proteins phosphorylated at
this amino acid residue escape detection by the method. Dyes for
the phosphate-selective fluorescence labeling in which a BODIPY dye
is covalently attached to a reactive imidazole group has been
developed for the detection of pepsin phosphorylation. See, U.S.
Pat. No. 5,512,486; Wang & Giese, "Phosphate-Specific
Fluorescence Labeling of Pepsin by BO-IMI," Anal. Biochem.
230:329-332 (1995).
[0009] In addition to detecting phosphoproteins, two methods for
the chemical derivatization and enrichment of phosphopeptides
resulting in isolation of phosphopeptides from complex mixtures
exist. See, e.g. Goshe et al., "Phosphoprotein Isotope-Coded
Affinity Tag Approach For Isolating and Quantitating
Phosphopeptides in Proteome-Wide Analyses," Anal. Chem.
73:2578-2586 (2001). The first method involves oxidation of
cysteine residues with performic acid, alkaline hydrolysis to
induce .beta.-elimination of phosphate groups from phosphoserine
and phosphothreonine residues, addition of ethanedithiol, coupling
of the resulting free sulfhydryl residues with biotin, purification
of phosphoproteins by avidin affinity chromatography, proteolytic
digestion of the eluted phosphoproteins, a second round of avidin
purification and then analysis by mass spectrometry (Oda, Y.,
Nagasu, T., and Chait, B. Nature Biotechnol. 19:379 (2001)). The
first method uses .beta.-elimination to remove phosphate groups
that are replaced with a tag, as exemplified with biotinylated
thiol groups wherein the peptides could then be isolated by
chromatography on avidin resins. An alternative method requires
proteolytic digestion of the sample, reduction and alkylation of
cysteine residues, N-terminal and C-terminal protection of the
peptides, formation of phosphoramidate adducts at phosphorylated
residues by carbodiimide condensation with cystamine, capture of
the phosphopeptides on glass beads coupled to iodoacetate, elution
with trifluoroacetic acid and evaluation by mass spectrometry (Zhou
et al., "A Systematic Approach to the Analysis of Protein
Phosphorylation," Nat. Biotechnol. 19:375-378 (2001). These methods
are time consuming, require purified phosphopeptides, and are
limiting in what can be isolated. Both procedures identified the
monophosphorylated trypsin peptide fragment from the test protein
.beta.-casein, but both failed to detect the tetraphosphorylated
peptide fragment.
[0010] Alternatively, a method for combining chemical modification
and affinity purification has been shown for the characterization
of serine and threonine phosphopeptides in proteins based on the
conversion of phosphoserine and phosphothreonine residues to
S-(2-mercaptoethyl)cysteinyl or
.beta.-methyl-S-(2-mercaptoethyl)cysteinyl residues by
.beta.-elimination/1,2-ethanedithiol addition, followed by
reversible biotinylation of the modified proteins. After trypsin
digestion, the biotinylated peptides are affinity-isolated and
enriched, followed by their subsequent structural characterization
by liquid chromatography/tandem mass spectrometry (LC/MS/MS). See
Adamczyk et al., "Selective Analysis of Phosphopeptides Within a
Protein Mixture by Chemical Modification, Reversible Biotinylation
and Mass Spectrometry," Rapid. Commun. Mass Spectrom. 15:1481-1488
(2001).
[0011] Fluorescence detection methods appear to offer the best
solution to global protein quantitation in proteomics. However,
currently, there is no satisfactory method for the specific and
reversible fluorescent detection of gel-separated phosphoproteins
from complex samples. Derivatization and fluorophore labeling of
phosphoserine residues by blocking free sulfhydryl groups with
iodoacetate or performate, alkaline .beta.-elimination of the
phosphate residue, addition of ethanedithiol, and reaction of the
resulting free sulfhydryl group with 6-iodoacetamidofluorescein has
been demonstrated in capillary electrophoresis using laser-induced
fluorescence detection and similar reactions have been performed on
protein microsequencing membranes. However, neither method has been
shown to be suitable for detection of phosphoproteins directly in
gels. One problem with the approach is that a delicate balance must
be struck between the base and the ethanedithiol in order to
achieve elimination of the phosphate group from the serine residue
and addition of the ethanedithiol to the resulting dehydroalanine
residue without hydrolysis of the peptide backbone.
[0012] Several instrument-based methods are also available for the
determination of protein phosphorylation such as .sup.31P-NMR, mass
spectrometry [See, e.g., Resing & Ahn, "Protein Phosphorylation
Analysis by Electrospray Ionization-Mass Spectrometery," Methods
Enzymol. 283:29-44 (1997); Aebersold and Goodlett, "Mass
Spectrometry in Proteomics," Chem. Rev. 101:269-295 (2001).
Affolter, M., Watts, J., Krebs, D., and Aebersold, R. Anal.
Biochem. 223:74 (1994); Liao, P., Leykam, J., Andrews, P., Gage,
D., and Allison, J. Anal. Biochem. 219:9 (1994); Oda, Y., Huang,
K., Cross, F., Cowburn, D., and Chait, B. Proc. Natl. Acad. Sci.
USA 96:6591 (1999)) and protein sequencing. Mass spectrometry has
been used to provide the molecular mass of an intact phosphorylated
protein by comparing the mass of the unphosphorylated protein to
that of the phosphorylated protein. See, e.g., McLachlin &
Chait, "Analysis of Phosphorylated Proteins and Peptides by Mass
Spectrometry," Current Opin. Chem. Biol. 5:591-602 (2001). This is
limiting in that researchers must have purified amounts of both
proteins. While these procedures accurately characterize the
phosphorylation status of proteins and peptides, they are
unsuitable for high-throughput screening of phosphorylated
substrates. The techniques are generally used after a
phosphoprotein has been identified by autoradiography or
immunoblotting with an anti-phosphotyrosine antibody. Though
methods have recently been introduced to directly quantify the
relative abundance of phosphoproteins in two different samples by
mass spectrometry through culturing different cell populations in
.sup.15N-enriched and .sup.14N-enriched medium, the linear dynamic
range of such methods has explicitly been demonstrated over only a
10-fold range. Ion suppression phenomena associated with mass
spectrometry prevents stoichiometric comparison of different
phosphoproteins by such techniques.
[0013] For analysis of the site(s) of phosphorylation on molecules,
a more detailed analysis of the sites of phosphate attachment and
stoichiometry often requires the examination of peptide fragments
of the phosphoprotein of interest. Such fragments are usually
generated by digestion of the phosphoprotein with proteases such as
trypsin. However, mass spectroscopic analysis of proteolytic
digests of proteins rarely provides full coverage of the protein
sequence and regions of interests often go undetected. In addition,
protein phosphorylation is often sub-stoichiometric, such that the
phosphoproteins are present in lower abundance than other peptides
from the protein of interest. Therefore, the identification and
characterization of phosphoproteins would be improved greatly by
highly selective methods of enriching phosphopeptides prior to
analysis with mass spectrometry. It would be particularly useful to
detect phosphoproteins by reagents that do not chemically alter the
structure or mass of the phosphoproteins.
[0014] Currently, selective enrichment of phosphopeptides from
complex mixtures is performed using immobilized metal affinity
chromatography, known as IMAC. Using this technique, metal ions
such as Fe.sup.3+ or Ga.sup.+3 are bound to a chelating support
prior to the addition of a complex mixture of peptides or proteins.
See, e.g., Posewitz & Tempst, "Immobilized Gallium(III)
Affinity Chromatography of Phosphopeptides," Anal. Biochem.
71:2883-2892 (1999). Phosphopeptides that bind to the column can be
released using high pH or phosphate buffer, though the latter step
usually requires a further desalting step before analysis with mass
spectrometry. Resins with iminodiacetic acid and nitrilotriacetic
acid chelators are known and are available commercially. See, e.g.,
Neville et al., "Evidence for Phosphorylation of Serine 753 in CFTR
Using a Novel Metal-Ion Affinity Resin and Matrix-Assisted Laser
Desorption Mass Spectrometry," Protein Sci. 6:2436-2445 (1997).
However, there are several complications using current techniques,
including loss of phosphopeptides that do not bind to the column
(low affinity), difficulty in the subsequent elution of
phosphorylated peptides, and background from non-phosphorylated
peptides that have affinity for immobilized metal ions (low
specificity).
[0015] Mass spectrometry-based detection of separated peptides and
direct matrix-assisted laser desorption/ionization (MALDI) analysis
of phosphopeptides bound to an IMAC support has been demonstrated.
See Zhou et al., "Detection and Sequencing of Phosphopeptides
Affinity Bound to Immobilized Metal Ion Beads by Matrix-Assisted
Laser Desorption/lonization Mass Spectrometry," J. Am. Soc. Mass.
Spectrom. 11:273-283 (2000). IMAC has also been coupled directly to
mass spectrometry instruments on-line, or with superseding
separation techniques, such as HPLC and capillary electrophoresis
(CE), for the detection and analysis of phosphopeptides.
[0016] The present invention overcomes the limitations of the
current methods by utilizing a cationic transition metal and a
compound that comprises a metal-chelating moiety and a chemical
moiety, typically a reactive group or label such as a fluorophore,
or a combination thereof, to detect phosphoproteins and
phosphopeptides. There are a variety of chelating moieties that use
poly-carboxylate binding sites to selectively bind monovalent and
divalent metal cations, and these are often used in fluorescent
calcium ion indicators. Examples of these indicators are, for
example, quin-2, fura-2, indo-1 (U.S. Pat. No. 4,603,209); fluo-3
and rhod-2 (U.S. Pat. No. 5,049,673), and FURA RED.TM. (U.S. Pat.
No. 4,849,362). A family of BAPTA-based indicators that are
selective for calcium ions are described in HAUGLAND, HANDBOOK OF
FLUORESCENT PROBES AND RESEARCH CHEMICALS (9.sup.th edition,
CD-ROM, September 2002). Examples of BAPTA-based metal-chelators
are also described in U.S. Pat. Nos. 5,773,227; 5,453,517;
5,516,911; 5,501,980; 6,162,931 and 5,459,276.
[0017] Indicators of free calcium concentrations are based upon
selective calcium binding to fluorescent dyes. Though it is well
known that BAPTA compounds bind certain divalent cations, such as
calcium, as analogs of the common EGTA chelator, the BAPTA
compounds are also known to bind almost all inorganic polyvalent
cations with an affinity that may be higher or lower than that of
the compound for calcium ions. Their selectivity and utility for
measuring calcium in biological cells results from the general
absence or low abundance of these other polycations in living
systems. The affinity and selectivity of the BAPTA-based indicators
for polycations, including gallium and similar metals of utility to
this invention, can be modified by shifts in pH, solvent
composition, ionic strength and other experimental variables. This
shift in cation selectivity and affinity is critical to all aspects
of the disclosed invention, including both detection and isolation
of phosphorylated targets.
[0018] The present invention overcomes the limitations and
disadvantages of currently disclosed methods for identifying,
isolating, analyzing and quantitating phosphorylated proteins and
thus provide methods, compounds and compositions to alleviate
long-felt needs for rapid and effective high-throughput methods for
detecting and isolating phosphoproteins for further analysis. The
present invention can accurately identify phosphopeptides and
phosphoproteins comprising as few as one phosphate group and in a
simple method that does not require multiple steps or pre-treatment
of the sample. Importantly, the present invention is the first
known method to provide a means for accurately identifying the
phosphorylated proteome and allows for the quantitative
identification of increased phosphorylation of proteins. The
present invention is an important tool for identifying novel
phosphorylated proteins in the proteome. The technology has
unsurpassed quantitative characteristics, particularly when used in
combination with reagents for the detection of total proteins. In
addition, as will be described below, the materials and methods of
the present invention are not limited to the detection and/or
separation of phosphorylated proteins.
SUMMARY OF THE INVENTION
[0019] The present invention provides phosphate-binding compounds
and methods for specifically detecting, isolating and/or
quantitating phosphorylated target molecules. These compounds, when
in a moderately acidic environment, and in the presence of an
appropriate metal ion will selectively bind phosphate groups on
phosphorylated target molecules that are either immobilized, such
as in a gel, or adsorbed on a solid or semisolid matrix, or are
dissolved or suspended in a mostly aqueous solution to form a
ternary complex.
[0020] The phosphate-binding compounds comprise a metal chelating
moiety that is optionally bonded to a label or a chemically
reactive group. Thus, in one aspect the phosphate-binding compounds
have the formula (A)m(L)n(B), wherein A is a chemical moiety, L is
a linker, B is a metal-chelating moiety, m is an integer from 1 to
4 and n is an integer from 0 to 4. Without being bound by theory,
it appears that the metal-chelating moiety of these particular
phosphate-binding compounds simultaneously binds a trivalent metal
ion and a phosphate group on a target molecule in a reaction that
forms a ternary complex. In this way, the metal ion provides a
bridge between the phosphate group and the metal-chelating moiety
wherein the chemical moiety A is effectively bound to the phosphate
target molecule by ionic interactions. Thus, it is a requirement of
the present invention that the metal-chelating moiety bind a metal
ion that has simultaneous affinity for the phosphorylated target
molecule, under appropriate conditions.
[0021] The utility of the compositions and methods of this
invention is principally the result of the chemical moieties A that
are covalently attached to the metal-chelating moieties by a linker
to form the phosphate binding compounds of the present invention.
Typically, chemical moieties A are a reactive group or a label. The
reactive groups function to covalently attach another natural or
synthetic substance to the metal-chelating moieties or
alternatively covalently bind the phosphorylated target molecule
after the metal-chelating moiety and metal ion has brought the
reactive group in close proximity to the phosphorylated target
molecule. Particularly useful substances that the reactive group A
covalently binds to the metal chelating moiety of formula
(A)m(L)n(B) include without limitation particles, polymers,
peptides and proteins. In this way, a particle could have many
phosphate-binding compounds attached.
[0022] For detection purposes, A is typically a detectable label
that is a dye including pigments, chromophores and fluorophores,
haptens, enzymes, or radioactive isotopes, although an extensive
assortment of other detectable labels that fall within the scope of
this invention is known. For isolation purposes of phosphate
containing targets, A is typically a label or a reactive group that
is bound to a polymer such as agarose, a surface, a magnetic
particle or a microsphere. The polymer, in combination with the
metal chelating moiety and a metal ion is selected to form a
soluble or insoluble ternary complex with the phosphorylated target
molecule. Such ternary complexes are particularly useful for the
selective isolation of phosphorylated targets from complex mixtures
or as components of various detection schemes. In a further aspect
of the invention, A is a chemical moiety that alters the solubility
of the ternary complex or alternatively comprises an amine-reactive
group used to form a covalent bond with an amine-containing
molecule, including polymers and phosphate target molecules.
[0023] The "binding solution" of the invention (which we define to
include true solutions, suspensions, emulsions, dispersions and
immobilized variants thereof) of the present invention comprises a
phosphate-binding compound, typically having the formula
(A)m(L)n(B), a salt comprising selected metal ions, and an acid.
The preferred salt and metal ion composition and concentration of
the binding solution or suspension will depend to some extent on
the metal-chelating moiety of the compound. A particularly useful
binding solution is the combination of a BAPTA-based chelating
moiety of the phosphate-binding compound and a gallium salt.
Unexpectedly, we have determined that trivalent gallium ions
simultaneously bind BAPTA moieties and phosphorylated target
molecules to form a ternary complex with a useful affinity only in
the presence of a moderately acidic environment. However we have
also shown, other metal-chelating moieties such as DTPA, IDA and
phenanthroline to simultaneously bind gallium trivalent ions and
phosphate groups. Thus, one requirement of the binding solution is
the presence of an acid, wherein the binding solution preferably
has a pH of about 3 to about 6; typically the pH is about 3 to
about 4. The nature of the acid used to obtain this pH appears to
be irrelevant; however, a phosphoric acid, phosphonic acid or
polyphosphoric acid should not be used to obtain this pH, as they
could reduce the stability of the ternary complex. Typically, the
phosphate-binding compound is free in the binding solution or
suspension; however, the phosphate-binding compound can be
immobilized on a solid or semi-solid matrix such that when the
metal ion and acid are added a binding solution is formed and a
ternary complex of the invention is subsequently formed if a
phosphorylated target is present in a sample.
[0024] The methods of the invention comprise contacting a sample
with a binding solution comprising the phosphate-binding compound,
the metal ion and the acid, incubating the sample and the binding
solution for sufficient time to allow the compound of the binding
solution to associate with said phosphorylated target molecule
whereby said phosphorylated target molecule forms a ternary
complex.
[0025] Typically, for detection purposes, the resulting ternary
complex that comprises the compound is illuminated to measure a
detectable optical property of the chemical moiety A, whereby the
presence of the phosphorylated target molecules is detected. The
phosphorylated target molecules can be detected in solution or when
immobilized on a solid or semi-solid matrix. The compositions and
methods of this invention can be used to detect phosphorylated
target molecules present in a complex sample of phosphorylated and
nonphosphorylated target molecules or to detect a change in the
number of phosphate groups on a target molecule. Differences in the
degree of phosphorylation can be due to intrinsic differences in
the degree of phosphorylation of the biopolymer, which can cause
differences in folding of proteins, or to an in vivo process such
as a disease state or in conjunction with an in vitro assay to
identify specific kinases and phosphatases.
[0026] Alternatively, when the method is utilized to selectively
isolate phosphorylated target molecules from solution, visualizing
the complex may not be necessary. To isolate the phosphorylated
target molecules, the ternary complex can be precipitated,
immobilized, separated by a chromatographic or electrophoretic
technique or by a magnetic field or remain in solution. In some
cases, organic extraction can be used to separate the
metal-chelating moiety from the phosphorylated target molecule.
When the ternary complex is precipitated or otherwise immobilized,
the phosphorylated target molecules can be separated from the
nonphosphorylated target molecules and other components of the
sample by affinity chromatography, such as by simple washing with
an aqueous, organic or mixed aqueous/organic wash solution. In some
cases, it is advantageous to further analyze the extracted
phosphorylated target molecules while they are still immobilized on
a matrix. Isolation of phosphorylated target molecules is useful
for further analysis of the target molecules, such as by liquid
chromatography/mass spectrography, an electrophoretic separation
technique, by detection of the bound target molecules by an
antibody to any part of the target molecule or by a variety of
other techniques. In particular, isolation of the phosphorylated
target molecules simplifies the subsequent analysis of the sample
by removing interfering components of the original sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1: Shows the selective detection of a phosphorylated
target molecule (ovalbumin) (1A) in a polyacrylamide gel using a
binding solution comprising gallium chloride and Compound 2
compared to the same gel post-stained with a total protein stain
(1B) (SYPRO.RTM. Ruby protein gel stain) (See, Example 2). The
protein mixture was loaded at ca. 500 .mu.g and contained nine
total proteins, one of which was a phosphoprotein (ovalbumin) that
contains two phosphate groups. The figure demonstrates selective
detection of ovalbumin against a background of very low or no
staining of eight proteins known to be non-phosphorylated.
[0028] FIG. 2: Shows the selective detection of a phosphorylated
target molecule (ovalbumin) (2A) on a PVDF membrane using a binding
solution comprising gallium chloride and Compound 1 compared to the
same membrane post-stained with a total protein stain (2B)
(SYPRO.RTM. Ruby protein blot stain) (See, Example 8). The figure
demonstrates selective detection of ovalbumin against a background
of very low or no staining of five non-phosphorylated proteins.
[0029] FIG. 3: Shows the sensitivity and linear dynamic range of
detecting phosphorylated proteins in a gel using a binding solution
comprising gallium chloride and Compound 2 (See, Example 2); (3A)
is a comparison of five proteins with different ratios of phosphate
groups and (3B) compares pepsin to bovine serum albumin (BSA).
Proteins were loaded in two-fold dilution series on SDS
polyacrylamide mini-gels from 2 ng-1000 ng; each protein sample was
done in series in four replicate gels. The phosphoproteins were
.alpha.-casein (7 or 8 phosphates); dephosphorylated .alpha.-casein
(1 or 2 phosphates); .beta.-casein (5 phosphates); ovalbumin (2
phosphates) and pepsin (1 phosphate). BSA contains no phosphates
and was used as a negative control. The results demonstrate that
the methods and binding solution of the present invention can
detect as little as 1-2 ng of a pentaphosphorylated protein
(.beta.-casein), and 8 ng of a monophosphorylated protein
(pepsin).
[0030] FIG. 4: Shows the detection of protein phosphatase activity
wherein .alpha.-casein and pepsin were used as a phosphatase
substrate. The gels were incubated with a binding solution
comprising gallium chloride and Compound 2 to demonstrate a
reduction in phosphate groups, compared to a control, after the
substrates were incubated with a protein phosphatase, See example
6.
[0031] FIG. 5: Shows the isolation of phosphopeptides (pT/pY and
RII; MWs 1670 and 2193) (Panel B and C) from a solution containing
non-phosphorylated peptides (angiotensin I and II, MWs 1297 and
1046) (Panel A) when the solution of peptides was incubated with a
binding solution comprising gallium chloride and Compound 5. The
mixture was incubated for 1 hour and centrifuged for 5 minutes. The
resulting supernatants (bottom spectra in all panels) and pellet
precipitates (top spectra in all panels) were analyzed by MALDI-TOF
mass spectrometry. Panel A shows the non-phosphorylated peptides
exclusively in the supernatants, while figures B and C show the two
phosphopeptides of greater than 95% purity in the pellets.
[0032] FIG. 6: Shows the analysis of phosphorylated peptides
(.alpha.-casein) eluted from an affinity chromatography matrix
column containing Compound 13 or Compound 14 that had been charged
with gallium ions. Panel A shows differential MALDI-TOF MS analysis
of purified .alpha.-casein phosphoserine peptides after
dephosphorylation (left peaks in pairs) and subsequent
derivatization with methylamine (right peaks in pairs). Results
show that all three peptides are phosphoserine derivatives by
methylamine addition. A and B of Panel A were monophosphorylated
(+31 amu for methylamine) and C was triphosphorylated (+93 amu for
3 methylamines). Panel B shows a MALDI-TOF MS profile of eluted
phosphopeptides from a BAPTA-agarose (Compound 13 or Compound 14)
column versus commercially available metal affinity columns (Pierce
Chemical Co.). Under the conditions used, the BAPTA-agarose column
shows all expected phosphopeptides (arrows) purified from a complex
peptide mix. Panel C shows the Control peptide (MW=1870) with one
phosphothreonine and one phosphotyrosine residue after treatment
with strong base (-98 amu) and methylamine. Results show
elimination of a single phosphate only (-98 amu from threonine)
with no subsequent addition of methylamine (+32 amu), confirming a
single phosphothreonine residue. Phosphotyrosine is determined by a
lack of modification under these elimination conditions.
[0033] FIG. 7: Shows the detection of a phosphoprotein
(.beta.-casein) on a HydroGel microarray (Perkin Elmer, Foster
City, Calif.) when the microarray was incubated with a binding
solution comprising Compound 2 and gallium chloride, see Example
18. The protein was loaded in a two-fold dilution series from 166
pg-0.324 pg on the microarray. The results show the detection of
0.65 pg of a pentaphosphorylated protein on a HydroGel
microarray.
[0034] FIG. 8: Shows the detection of a phosphopeptide (pDISP) on a
HydroGel microarray (Perkin Elmer) when the microarray was
incubated with a binding solution comprising Compound 2 and gallium
chloride, see Example 19. The peptide was loaded in a two-fold
dilution series from 12 pg-0.18 pg on the microarray. The results
demonstrate that as little as 300 fg of a monophosphorylated
peptide can be detected on a HydroGel microarray.
[0035] FIG. 9: Shows the detection of protein kinase activity (9A;
CaMPKII) and (9B; Abl tyrosine kinase) by the detection of peptides
that were phosphorylated on a HydroGel microarray (Perkin Elmer)
that was incubated with a binding solution comprising Compound 2
and gallium chloride, see Examples 20 and 21. The peptide glycogen
synthase 1-10 was detected to demonstrate the kinase activity of
CaMPKII and the peptide Abl was detected to demonstrate the kinase
activity of Abl tyrosine kinase.
[0036] FIG. 10: Shows the detection of a (10A) phosphoprotein and
(10B) phosphopeptide in solution by comparison of the polarization
values with binding solution alone and binding solution with
phosphorylated and non-phosphorylated protein or peptide, ovalbumin
and delta sleep-inducing peptide, see Example 14. The binding
solution alone and binding solution in the presence of
non-phosphorylated protein or peptide demonstrates very similar
fluorescence polarization and anisotropies. However, in the
presence of the phosphoprotein or phosphopeptide, there is a
significant increase in the fluorescence polarization values. This
result demonstrates selective binding of the phosphoprotein and
phosphopeptide to the Compound 2-Ga.sup.3+ complex in solution but
not to the non-phosphorylated protein or peptide.
[0037] FIG. 11: Shows the ratiometric analysis of proteins labeled
with a binding solution of the present invention and the SYPRO.RTM.
Ruby protein gel stain, demonstrating that non-specific staining
and low-abundance phosphoproteins can be distinguished from
non-phosphorylated proteins, see Example 22. A protein mixture
containing phosphorylated and non-phosphorylated proteins was
separated on a polyacrylamide gel and the phosphoproteins were
detected with a binding solution comprising Compound 2 and gallium
chloride. All the proteins were detected when the gel was
post-stained with SYPRO.RTM. Ruby protein gel stain. FIG. 11A)
shows the ratio of fluorescence intensities for ternary complexes
of phosphorylated proteins compared to total proteins. FIG. 11B)
shows the fluorescence intensities of the phosphorylated protein
complexes and total proteins plotted against the protein
concentration, resulting in a constant Y-intercept value. FIG. 11C)
shows the ratio of the Y-intercept values when plotted against the
protein concentration resulting in phosphoproteins with a ratio
value 50-100 times greater than that of the non-phosphorylated
proteins.
[0038] FIG. 12: Shows the ability to detect phosphorylated kinase
substrate in solution when the kinase substrate is conjugated to
either Oregon Green dye label or Alexa Fluor 488 dye label using
Compound 2 as the phosphate-binding compound wherein the dye labels
on the substrate are quenched when a ternary complex is formed with
Compound 2 and GaCl.sub.3. FIG. 12A, is pp 60-Oregon Green 488 dye
(OG); B, p-Abl-Oregon Green dye; C, pp 60-Alexa Fluor 488 dye
(A488); D, pStat3-Oregon Green dye label wherein the circles
represent samples without GaCl.sub.3 addition, and the squares
represent samples with GaCl.sub.3 addition.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0039] The present invention provides phosphate-binding compounds,
a phosphate-binding solution and methods for selectively detecting
and/or isolating phosphorylated target molecules. The
phosphate-binding compounds of the present invention, when present
in the binding solution, selectively bind to phosphorylated target
molecules and permit detection and/or isolation of the target
molecules. The binding solution comprises three critical components
for binding phosphorylated target molecules: 1) phosphate-binding
compounds; 2) a salt comprising a metal ion and 3) an acid. The
phosphate-binding compounds comprise a metal chelating moiety that
is capable of binding the metal ion. Typically the
phosphate-binding compounds are represent, but not limited, by the
formula (A)m(L)n(B) wherein A is a chemical moiety, L is a linker,
B is a metal-chelating moiety, m is an integer from 1 to 4 and n is
an integer from 0 to 4.
[0040] The binding solution typically includes a buffering agent to
maintain the acidic pH, which is ideally about pH 3 to about pH 6,
and an organic solvent, wherein the use and solvent depends on the
application and will be discussed below. The ternary complex that
comprises the phosphate-binding compound, metal ion and
phosphorylated target molecule is stable in an acidic environment
but when the pH approaches neutral (pH 7) or basic (pH>7.0) the
complex becomes increasingly unstable.
[0041] The binding solution is typically used to noncovalently
attach a phosphate-binding compound, of the present invention to
exposed phosphate groups on phosphorylated target molecules,
wherein the phosphate-binding compound typically comprises a label.
Alternatively, the binding solution is used to covalently attached
a phosphate-binding compound to a phosphorylated target molecule
wherein the present phosphate-binding compound comprises a reactive
group that will form a covalent bond when brought within proximity
to the phosphorylated target molecule. Because this is a highly
directed covalent attachment, the reactive group is typically a
photoactivatable group. These bound target molecules can be
subsequently detected using one of the detection methods described
herein or isolated by a number of methods described below. The
metal ions of the binding solution simultaneously have affinity for
both phosphate groups and the metal-chelating moiety of the
phosphate-binding compounds of the invention when in an acidic
environment.
[0042] Thus, a method of the present invention for the binding of
phosphorylated target molecules by a phosphate-binding compound
comprises the following steps: [0043] i) contacting the sample with
a binding solution, and; [0044] ii) incubating the sample and the
binding solution for sufficient time to allow said compound to
associate with said phosphorylated target molecules, whereby said
phosphorylated target molecule is bound.
[0045] The methods of the present invention can be used in
unlimited assay formats, provided that there is sufficient contact
between the sample and the binding solution. Therefore, this method
is intended to cover an unlimited number of assays, in any format,
wherein the binding solution of the present invention has contact
with an exposed phosphate group on a target molecule, regardless of
the intent of the assay. Thus, the methods of the present invention
contemplate, without limit, the identification of phosphorylated
target molecules, identification of dephosphorylated molecules,
identification of enzymes responsible for phosphorylation or
dephosphorylation, directly or indirectly, identification of
molecules that interact with phosphorylated target molecules and
isolation of phosphorylated target molecules. Detection
includes--where practical--quantitation, discrimination and
subsequent analysis and identification of the phosphorylated target
molecules, with the use of standards and controls, as
appropriate.
DEFINITIONS
[0046] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions or process steps, as such may vary. It must be noted
that, as used in this specification and the appended claims, the
singular form "a", "an" and "the" includes plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a phosphorylated protein" includes a plurality of
proteins and reference to "a compound" includes a plurality of
compounds and the like.
[0047] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention is related. The
following terms are defined for purposes of the invention as
described herein.
[0048] The term "affinity" as used herein refers to the strength of
the binding interaction of two molecules, such as a metal-chelating
compound and a metal ion.
[0049] Certain compounds of the present invention can exist in
unsolvated forms as well as solvated forms, including hydrated
forms. In general, the solvated forms are equivalent to unsolvated
forms and are encompassed within the scope of the present
invention. Certain compounds of the present invention may exist in
multiple crystalline or amorphous forms. In general, all physical
forms are equivalent for the uses contemplated by the present
invention and are intended to be within the scope of the present
invention.
[0050] Certain compounds of the present invention possess
asymmetric carbon atoms (optical centers) or double bonds; the
racemates, diastereomers, geometric isomers and individual isomers
are encompassed within the scope of the present invention.
[0051] The compounds of the invention may be prepared as a single
isomer (e.g., enantiomer, cis-trans, positional, diastereomer) or
as a mixture of isomers. In a preferred embodiment, the compounds
are prepared as substantially a single isomer. Methods of preparing
substantially isomerically pure compounds are known in the art. For
example, enantiomerically enriched mixtures and pure enantiomeric
compounds can be prepared by using synthetic intermediates that are
enantiomerically pure in combination with reactions that either
leave the stereochemistry at a chiral center unchanged or result in
its complete inversion. Alternatively, the final product or
intermediates along the synthetic route can be resolved into a
single stereoisomer. Techniques for inverting or leaving unchanged
a particular stereocenter, and those for resolving mixtures of
stereoisomers are well known in the art and it is well within the
ability of one of skill in the art to choose and appropriate method
for a particular situation. See, generally, Furniss et al. (eds.),
VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5.sup.TH ED.,
Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816;
and Heller, Acc. Chem. Res. 23: 128 (1990).
[0052] The compounds of the present invention may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I) or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present invention,
whether radioactive or not, are intended to be encompassed within
the scope of the present invention.
[0053] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents, which would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is intended to also recite --OCH.sub.2--.
[0054] The term "acyl" or "alkanoyl" by itself or in combination
with another term, means, unless otherwise stated, a stable
straight or branched chain, or cyclic hydrocarbon radical, or
combinations thereof, consisting of the stated number of carbon
atoms and an acyl radical on at least one terminus of the alkane
radical. The "acyl radical" is the group derived from a carboxylic
acid by removing the --OH moiety therefrom.
[0055] The term "alkyl," by itself or as part of another
substituent means, unless otherwise stated, a straight or branched
chain, or cyclic hydrocarbon radical, or combination thereof, which
may be fully saturated, mono- or polyunsaturated and can include
divalent ("alkylene") and multivalent radicals, having the number
of carbon atoms designated (i.e. C.sub.1-C.sub.10 means one to ten
carbons). Examples of saturated hydrocarbon radicals include, but
are not limited to, groups such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,
(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for
example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An
unsaturated alkyl group is one having one or more double bonds or
triple bonds. Examples of unsaturated alkyl groups include, but are
not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,
2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1-
and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The
term "alkyl," unless otherwise noted, is also meant to include
those derivatives of alkyl defined in more detail below, such as
"heteroalkyl." Alkyl groups that are limited to hydrocarbon groups
are termed "homoalkyl".
[0056] Exemplary alkyl groups of use in the present invention
contain between about one and about twenty-five carbon atoms (e.g.
methyl, ethyl and the like). Straight, branched or cyclic
hydrocarbon chains having eight or fewer carbon atoms will also be
referred to herein as "lower alkyl". In addition, the term "alkyl"
as used herein further includes one or more substitutions at one or
more carbon atoms of the hydrocarbon chain fragment.
[0057] The terms "alkoxy," "alkylamino" and "alkylthio" (or
thioalkoxy) are used in their conventional sense, and refer to
those alkyl groups attached to the remainder of the molecule via an
oxygen atom, an amino group, or a sulfur atom, respectively.
[0058] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a straight or
branched chain, or cyclic carbon-containing radical, or
combinations thereof, consisting of the stated number of carbon
atoms and at least one heteroatom selected from the group
consisting of O, N, Si, P and S, and wherein the nitrogen,
phosphorous and sulfur atoms are optionally oxidized, and the
nitrogen heteroatom is optionally be quaternized. The heteroatom(s)
O, N, P, S and Si may be placed at any interior position of the
heteroalkyl group or at the position at which the alkyl group is
attached to the remainder of the molecule. Examples include, but
are not limited to, --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CH--O--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3, and
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3. Up to two heteroatoms may be
consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3 and
--CH.sub.2--O--Si(CH.sub.3).sub.3. Similarly, the term
"heteroalkylene" by itself or as part of another substituent means
a divalent radical derived from heteroalkyl, as exemplified, but
not limited by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--.
[0059] The terms "cycloalkyl" and "heterocycloalkyl", by themselves
or in combination with other terms, represent, unless otherwise
stated, cyclic versions of "alkyl" and "heteroalkyl", respectively.
Additionally, for heterocycloalkyl, a heteroatom can occupy the
position at which the heterocycle is attached to the remainder of
the molecule. Examples of cycloalkyl include, but are not limited
to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl,
cycloheptyl, and the like. Examples of heterocycloalkyl include,
but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),
1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,
3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,
tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,
2-piperazinyl, and the like.
[0060] The term "aryl" means, unless otherwise stated, a
polyunsaturated, aromatic moiety that can be a single ring or
multiple rings (preferably from 1 to 3 rings), which are fused
together or linked covalently.
[0061] The term "heteroaryl" as used herein refers to an aryl group
as defined above in which one or more carbon atoms have been
replaced by a non-carbon atom, especially nitrogen, oxygen, or
sulfur. For example, but not as a limitation, such groups include
furyl, tetrahydrofuryl, pyrrolyl, pyrrolidinyl, thienyl,
tetrahydrothienyl, oxazolyl, isoxazolyl, triazolyl, thiazolyl,
isothiazolyl, pyrazolyl, pyrazolidinyl, oxadiazolyl, thiadiazolyl,
imidazolyl, imidazolinyl, pyridyl, pyridazyl, triazinyl,
piperidinyl, morpholinyl, thiomorpholinyl, pyrazinyl, piperainyl,
pyrimidinyl, naphthyridinyl, benzofuranyl, benzothienyl, indolyl,
indolinyl, indolizinyl, indazolyl, quinolizinyl, quinolinyl,
isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl,
quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl, acridinyl,
phenazinyl, phenothiazinyl, phenoxazinyl, purinyl, benzimidazolyl
and benzthiazolyl and their aromatic ring-fused analogs. Many
fluorophores are comprised of heteroaryl groups and include,
without limitations, xanthenes, oxazines, benzazolium derivatives
(including cyanines and carbocyanines), borapolyazaindacenes,
benzofurans, indoles and quinazolones.
[0062] The above heterocyclic groups may further include one or
more substituents at one or more carbon and/or non-carbon atoms of
the heteroaryl group, e.g., alkyl; aryl; heterocycle; halogen;
nitro; cyano; hydroxyl, alkoxyl or aryloxyl; thio or mercapto,
alkyl- or arylthio; amino, alkyl-, aryl-, dialkyl-, diaryl-, or
arylalkylamino; aminocarbonyl, alkylaminocarbonyl,
arylaminocarbonyl, dialkylaminocarbonyl, diarylaminocarbonyl or
arylalkylaminocarbonyl; carboxyl, or alkyl- or aryloxycarbonyl;
aldehyde; aryl- or alkylcarbonyl; iminyl, or aryl- or alkyliminyl;
sulfo; alkyl- or arylsulfonyl; hydroximinyl, or aryl- or
alkoximinyl. In addition, two or more alkyl substituents may be
combined to form fused heterocycle-alkyl ring systems. Substituents
including heterocyclic groups (e.g., heteroaryloxy, and
heteroaralkylthio) are defined by analogy to the above-described
terms.
[0063] The term "heterocycloalkyl" as used herein refers to a
heterocycle group that is joined to a parent structure by one or
more alkyl groups as described above, e.g., 2-piperidylmethyl, and
the like. The term "heterocycloalkyl" refers to a heteroaryl group
that is joined to a parent structure by one or more alkyl groups as
described above, e.g., 2-thienylmethyl, and the like.
[0064] For brevity, the term "aryl" when used in combination with
other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both
aryl and heteroaryl rings as defined above. Thus, the term
"arylalkyl" is meant to include those radicals in which an aryl
group is attached to an alkyl group (e.g., benzyl, phenethyl,
pyridylmethyl and the like) including those alkyl groups in which a
carbon atom (e.g., a methylene group) has been replaced by, for
example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,
3-(1-naphthyloxy)propyl, and the like).
[0065] Each of the above terms (e.g., "alkyl," "heteroalkyl,"
"aryl" and "heteroaryl") includes both substituted and
unsubstituted forms of the indicated radical. Preferred
substituents for each type of radical are provided below.
[0066] Substituents for the alkyl and heteroalkyl radicals
(including those groups often referred to as alkylene, alkenyl,
heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl,
heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) are
generically referred to as "alkyl group substituents," and they can
be one or more of a variety of groups selected from, but not
limited to: --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR',
-halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'--C(O)NR''R''',
--NR''C(O).sub.2R', --NR--C(NR'R''R''').dbd.NR'''',
--NR--C(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --CN and --NO.sub.2 in a number
ranging from zero to (2 m'+1), where m' is the total number of
carbon atoms in such radical. R', R'', R''' and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g.,
aryl substituted with 1-3 halogens, substituted or unsubstituted
alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a
compound of the invention includes more than one R group, for
example, each of the R groups is independently selected as are each
R', R'', R''' and R'''' groups when more than one of these groups
is present. When R' and R'' are attached to the same nitrogen atom,
they can be combined with the nitrogen atom to form a 5-, 6-, or
7-membered ring. For example, --NR'R'' is meant to include, but not
be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above
discussion of substituents, one of skill in the art will understand
that the term "alkyl" is meant to include groups including carbon
atoms bound to groups other than hydrogen groups, such as haloalkyl
(e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and acyl (e.g.,
--C(O)CH.sub.3, --C(O)CF.sub.3, --C(O)CH.sub.2OCH.sub.3, and the
like).
[0067] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are generically
referred to as "aryl group substituents." The substituents are
selected from, for example: halogen, --OR', .dbd.O, .dbd.NR',
.dbd.N--OR', --NR'R'', --SR', -halogen, --SiR'R'' R''', --OC(O)R',
--C(O)R', --CO.sub.2R', --CONR'R'', --OC(O)NR'R'', --NR''C(O)R',
--NR'--C(O)NR''R''', --NR''C(O).sub.2R',
--NR--C(NR'R''R''').dbd.NR'''', --NR--C(NR'R'').dbd.NR''',
--S(O)R', --S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --CN
and --NO.sub.2, --R', --N.sub.3, --CH(Ph).sub.2,
fluoro(C.sub.1-C.sub.4)alkoxy, and fluoro(C.sub.1-C.sub.4)alkyl, in
a number ranging from zero to the total number of open valences on
the aromatic ring system; and where R', R'', R''' and R'''' are
preferably independently selected from hydrogen, substituted or
unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted aryl and substituted or unsubstituted
heteroaryl. When a compound of the invention includes more than one
R group, for example, each of the R groups is independently
selected as are each R', R'', R''' and R'''' groups when more than
one of these groups is present. In the schemes that follow, the
symbol X represents "R" as described above.
[0068] Two of the substituents on adjacent atoms of the aryl or
heteroaryl ring may optionally be replaced with a substituent of
the formula -T-C(O)--(CRR').sub.q--U--, wherein T and U are
independently --NR--, --O--, --CRR'-- or a single bond, and q is an
integer of from 0 to 3. Alternatively, two of the substituents on
adjacent atoms of the aryl or heteroaryl ring may optionally be
replaced with a substituent of the formula
-A-(CH.sub.2).sub.r--B--, wherein A and B are independently
--CRR'--, --O--, --NR--, --S--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2NR'-- or a single bond, and r is an integer of from 1
to 4. One of the single bonds of the new ring so formed may
optionally be replaced with a double bond. Alternatively, two of
the substituents on adjacent atoms of the aryl or heteroaryl ring
may optionally be replaced with a substituent of the formula
--(CRR').sub.s--X--(CR''R''').sub.d--, where s and d are
independently integers of from 0 to 3, and X is --O--, --NR'--,
--S--, --S(O)--, --S(O).sub.2--, or --S(O).sub.2NR'--. The
substituents R, R', R'' and R''' are preferably independently
selected from hydrogen or substituted or unsubstituted
(C.sub.1-C.sub.6)alkyl.
[0069] As used herein, the term "heteroatom" includes oxygen (O),
nitrogen (N), sulfur (S), phosphorus (P) and silicon (Si).
[0070] The term "amino" or "amine group" refers to the group
--NR'R'' (or NRR'R'') where R, R' and R'' are independently
selected from the group consisting of hydrogen, alkyl, substituted
alkyl, aryl, substituted aryl, aryl alkyl, substituted aryl alkyl,
heteroaryl, and substituted heteroaryl. A substituted amine being
an amine group wherein R' or R'' is other than hydrogen. In a
primary amino group, both R' and R'' are hydrogen, whereas in a
secondary amino group, either, but not both, R' or R'' is hydrogen.
In addition, the terms "amine" and "amino" can include protonated
and quaternized versions of nitrogen, comprising the group
--NRR'R'' and its biologically compatible anionic counterions.
[0071] The term "attachment site" as used herein refers to a site
on a moiety or a molecule, e.g. a quencher, a fluorescent dye, an
avidin, or an antibody, to which is covalently attached, or capable
of being covalently attached, to a linker or another moiety.
[0072] The term "aqueous solution" as used herein refers to a
solution that is predominantly water and retains the solution
characteristics of water. Where the aqueous solution contains
solvents in addition to water, water is typically the predominant
solvent.
[0073] The term "BAPTA" as used herein refers to a metal-chelating
compound that is
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or its
analogs, derivatives, ring-fused variants and conjugates, and all
metallic and nonmetallic salts, partial salts and hydrates thereof,
including any corresponding compounds disclosed in U.S. Pat. Nos.
4,603,209; 4,849,362; 5,049,673; 5,453,517; 5,459,276; 5,516,911;
5,501,980; 6,162,931 and 5,773,227 (supra). When used generically,
"BAPTA" refers to two benzene rings that are joined by a
C.sub.1-C.sub.3 hydrocarbon bridge terminated by oxygen atoms,
including methylenedioxy (--OCH.sub.2O--), ethylenedioxy
(--OCH.sub.2CH.sub.2O--) or propylenedioxy
(--OCH.sub.2CH.sub.2CH.sub.2O--) bridging groups, where each
benzene ring is optionally substituted by one or more substituents
that adjust the metal ion-binding affinity, solubility, chemical
reactivity, spectral properties or other physical properties of the
compound. In a preferred embodiment of the present invention
"BAPTA" is covalently attached to a chemical moiety A that, in
combination with an appropriate trivalent metal ion and an acid,
permits detection or isolation of phosphorylated target molecules
as a ternary complex. BAPTA derivatives additionally include
compounds in which the benzene rings of the BAPTA structure are
substituted by or fused to additional aromatic, or heteroaromatic
rings.
[0074] The term "biotin" as used herein refers to any biotin
derivative, including without limitation, substituted and
unsubstituted biotin, and analogs and derivatives thereof, as well
as substituted and unsubstituted derivatives of caproylamidobiotin,
biocytin, desthiobiotin, desthiobiocytin, iminobiotin, and biotin
sulfone.
[0075] The term "biotin-binding protein" as used herein refers to
any protein that binds selectively to biotin, including without
limitation, antibodies to biotin, substituted or unsubstituted
avidin, and analogs and derivatives thereof, as well as substituted
and unsubstituted derivatives of antibodies, streptavidin, ferritin
avidin, nitroavidin, nitrostreptavidin, Neutravidin.TM. avidin (a
de-glycosylated modified avidin having an isoelectric point near
neutral) and their dye-, enzyme-, or polymer-modified variants and
immobilized forms of the biotin-binding proteins.
[0076] The term "buffer" as used herein refers to a system that
acts to minimize the change in acidity or basicity of the solution
against addition or depletion of chemical substances.
[0077] The term "carbonyl" as used herein refers to the functional
group --(C.dbd.O)--. However, it will be appreciated that this
group may be replaced with other well-known groups that have
similar electronic and/or steric character, such as thiocarbonyl
(--(C.dbd.S)--); sulfinyl (--S(O)--); sulfonyl (--SO.sub.2)--),
phosphonyl (--PO.sub.2--).
[0078] The term "carboxy" or "carboxyl" refers to the group
--R'(COOR.sup.13) where R' is alkyl, substituted alkyl, aryl,
substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, or
substituted heteroaryl. R.sup.13 is hydrogen or a salt.
[0079] The term "chemical moiety A" or "chemical moiety" as used
herein refers to the moiety that is covalently attached to the
metal chelating compound according the Formula (A)m(L)n(B) to form
a phosphate-binding compound of the present invention. The
"chemical moiety A" includes a label, as defined below, that is a
detectable moiety used to facilitate detection and isolation of
phosphorylated target molecules. The term "chemical moiety A" is a
natural or syntheic moiety that is typically a label but can also
be, without limitation, a reactive group, typically a
photoactivatable group or an amine reactive group such as
succinimidyl ester that functions to covalently bind a polymer
including agarose, acrylamide, microparticles, a protein such as an
antibody or an antigen, a phosphate target molecule and a ligand,
including those well known to one skilled in the art, to the
phosphate-binding compounds of the present invention. In addition,
the chemical moiety A can also be a metal-chelating moiety of the
present invention, typically if a metal chelating moiety is
attached to a phosphate-binding compound of the present invention
it will be through a reactive group (conjugation reaction) however
the metal chelating moiety could be covalently attached wherein a
reactive group was not used and is connected by a linker to the
phosphate-binding compound of the present invention.
[0080] The term "complex" as used herein refers to the association
of two or more molecules, usually by non-covalent bonding, e.g.,
with a metal ion-chelator and a metal ion complexed with (i.e.,
noncovalently bound to) a protein or, for instance, of an antibody
and antigen, enzyme and enzyme substrate, ligand and receptor (e.g.
biotin and avidin), nucleic acid and its complementary strand, a
protein with another protein or with a nucleic acid having affinity
for the first protein, and the like.
[0081] The term "detectable response" as used herein refers to an
occurrence of, or a change in, a signal that is directly or
indirectly detectable either by observation or by instrumentation.
Typically, the detectable response is an optical response resulting
in a change in the wavelength distribution patterns or intensity of
absorbance or fluorescence or a change in light scatter,
fluorescence lifetime, fluorescence polarization, or a combination
of these parameters. Alternatively, the detectable response is an
occurrence of a signal wherein the dye is inherently fluorescent
and does not produce a change in signal upon binding to a metal ion
or phosphorylated target molecule. Alternatively, the detectable
response is the result of a signal, such as color, fluorescence,
radioactivity or another physical property of the detectable label
becoming spatially localized in a subset of a sample such as in a
gel, on a blot, or an array, in a well of a micoplate, in a
microfluidic chamber, or on a microparticle as the result of
formation of a ternary complex of the invention that comprises a
phosphorylated target molecule.
[0082] The term "directly detectable" as used herein refers to the
presence of a detectable label or the signal generated from a
detectable label that is immediately detectable by observation,
instrumentation, or film without requiring chemical modifications
or additional substances. For example, a fluorophore produces a
directly detectable response.
[0083] The term "DTPA" as used herein refers to a metal chelating
moiety diethylenetriamine pentaacetic acid or derivatives thereof
and any corresponding moieties disclosed in U.S. Pat. Nos.
4,978,763 and 4,647,447. DTPA is represented by the formula
(CH.sub.2CO.sub.2R.sup.13).sub.ZN[(CH.sub.2).sub.SN(CH.sub.2CO.sub.2R.sup-
.13)].sub.T(CH.sub.2).sub.SN(CH.sub.2CO.sub.2R.sup.13).sub.Z
wherein the linker is attached to a methine carbon or nitrogen atom
and Z is 1 or 2, S is 1 to 5, T is 0 to 4 and R.sup.13 is hydrogen
or a salt.
[0084] The term "dye" as used herein refers to a compound that
emits light to produce an observable detectable signal. "Dye"
includes fluorescent and nonfluorescent compounds that include
without limitations pigments, fluorophores, chemiluminescent
compounds, luminescent compounds and chromophores. The term
"fluorophore" as used herein refers to a composition that is
inherently fluorescent or demonstrates a change in fluorescence
upon binding to a biological compound or metal ion, or metabolism
by an enzyme, i.e., fluorogenic. Fluorophores may be substituted to
alter the solubility, spectral properties or physical properties of
the fluorophore. Fluorophores of the present invention are not
sulfonated. Numerous fluorophores are known to those skilled in the
art and include, but are not limited to benzofurans, quinolines,
quinazolinones, indoles, benzazoles, borapolyazaindacenes and
xanthenes, with the latter including fluoresceins, rhodamines and
rhodols as well as other fluorophores described in RICHARD P.
HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND
RESEARCH CHEMICALS (9.sup.th edition, including the CD-ROM,
September 2002).
[0085] The term "energy transfer" as used herein refers to the
process by which the excited state energy of an excited group, e.g.
fluorescent reporter dye, is conveyed through space or through
bonds to another group, e.g. a quencher moiety or fluorescer, which
may attenuate (quench) or otherwise dissipate or transfer the
energy to another reporter group or emit the energy at a longer
wavelength. Energy transfer typically occurs through fluorescence
resonance energy transfer (FRET).
[0086] The term "energy transfer pair" as used herein refers to any
two moieties that participate in energy transfer. Typically, one of
the moieties acts as a fluorescent reporter, i.e. donor, and the
other acts as an acceptor, which may be a quenching compound or a
compound that absorbs and re-emits energy in the form of a
fluorescent signal ("Fluorescence resonance energy transfer."
Selvin P. (1995) Methods Enzymol 246:300-334; dos Remedios C. G.
(1995) J. Struct. Biol. 115:175-185; "Resonance energy transfer:
methods and applications." Wu P. and Brand L. (1994) Anal Biochem
218:1-13). Fluorescence resonance energy transfer (FRET) is a
distance-dependent interaction between two moieties in which
excitation energy, i.e. light, is transferred from a donor to an
acceptor without emission of a photon. The acceptor may be
fluorescent and emit the transferred energy at a longer wavelength,
or it may be non-fluorescent and serve to diminish the detectable
fluorescence of the reporter molecule (quenching). FRET may be
either an intermolecular or intramolecular event, and is dependent
on the inverse sixth power of the separation of the donor and
acceptor, making it useful over distances comparable with the
dimensions of biological macromolecules. Thus, the spectral
properties of the energy transfer pair as a whole change in some
measurable way if the distance between the moieties is altered by
some critical amount. Self-quenching probes incorporating
fluorescent donor-non-fluorescent acceptor combinations have been
developed primarily for detection of proteolysis (Matayoshi, (1990)
Science 247:954-958) and nucleic acid hybridization ("Detection of
Energy Transfer and Fluorescence Quenching" Morrison, L., in
Nonisotopic DNA Probe Techniques, L. Kricka, Ed., Academic Press,
San Diego, (1992) pp. 311-352; Tyagi S. (1998) Nat. Biotechnol.
16:49-53; Tyagi S. (1996) Nat. Biotechnol 14:303-308). In most
applications, the donor and acceptor dyes are different, in which
case FRET can be detected by the appearance of sensitized
fluorescence of the acceptor or by quenching of donor
fluorescence.
[0087] The term "enzyme" as used herein refers to a protein
molecule produced by living organisms, or through chemical
modification of a natural protein molecule, that catalyzes a
chemical reaction of other substances without itself being
destroyed or altered upon completion of the reactions. Examples of
other substances, include, but are not limited to chemiluminescent,
chromogenic and fluorogenic substances or protein-based
substrates.
[0088] The term "IDA" as used herein refers to imidodiacetic acid
metal chelating moieties having the formula
--N(CH.sub.2CO.sub.2R.sup.13).sub.2 wherein R.sup.13 is hydrogen or
a salt and the linker is attached to the nitrogen atom provided
that the linker is not a single covalent bond attached to an
aromatic ring of a fluorophore.
[0089] The term "isolated" as used herein with reference to the
subject peptides, proteins and protein complexes, refers to a
preparation of a peptide, protein or protein ternary complex that
is essentially free from contaminating nonphosphorylated peptides,
proteins or other associated target molecules that normally would
be present in association with the peptide, protein or complex,
e.g., in a cellular mixture or milieu in which the protein or
complex is found endogenously. In addition, in some embodiments,
"isolated" also refers to the further separation from reagents of
the invention used to isolate the peptide, protein or complex from
cellular mixture. Thus, an isolated protein or protein complex is
separated (isolated) from other components of the sample and
optionally from the phosphate-binding compounds of the invention
(including polymeric matrices) that normally would "contaminate" or
interfere with the study or further processing of the complex in
isolation, such as by mass spectrometry. The term "isolated" can
also refer to phosphorylated target molecules that are spatially or
temporally separated from each other such as by different physical
locations on a gel or array or by having different passage times
through a detector such as in a column or capillary.
[0090] The term "kit" as used herein refers to a packaged set of
related components, typically one or more compounds or
compositions, optionally comprising buffers, separation media,
standards, software and other components.
[0091] The term "label" as used herein refers to a detectable
moiety that is used to facilitate detection and isolation of
phosphorylated target molecules in combination with the
metal-chelating moieties of the present invention. Illustrative
labels include labels that can be directly observed or measured or
indirectly observed or measured such as fluorophores, radioactive
and enzyme reporter labels (Patton, W., et al, J. Chromatography B:
Biomedical Applications (2002) 771:3-31; Patton, W., et al,
Electrophoresis (2000) 21:1123-1144). Such labels include, but are
not limited to, radiolabels that can be measured with
radiation-counting devices; pigments, dyes or other chromogens that
can be visually observed or measured with a spectrophotometer; spin
labels that can be measured with a spin label analyzer; and
fluorescent labels (fluorophores), where the output signal is
generated by the excitation of a suitable molecular adduct and that
can be visualized by excitation with light that is absorbed by the
dye or can be measured with standard fluorometers or imaging
systems, for example or metal particles, e.g. gold or silver
particles or metallic bar codes that can be detected by their
optical or light-scattering properties. The label can be a
chemiluminescent substance, where the output signal is generated by
chemical modification of the signal compound; a metal-containing
substance; or an enzyme, where there occurs an enzyme-dependent
secondary generation of signal, such as the formation of a colored
product from a colorless substrate. The term label can also refer
to a "tag", hapten or other ligand that can bind selectively to a
labeled molecule such that the labeled molecule, when added
subsequently, is used to generate a detectable signal. For example,
one can use biotin as a tag and then use an avidin or streptavidin
conjugate of horseradish peroxidase (HRP) to bind to the tag, and
then use a chromogenic substrate (e.g., tetramethylbenzidine) or a
fluorogenic substrate such as Amplex.RTM. Red reagent (Molecular
Probes, Inc.) to detect the presence of HRP. Numerous labels and
tags and methods for their selective detection are known by those
of skill in the art and include, but are not limited to, particles,
fluorophores, haptens, enzymes and their chromogenic, fluorogenic
and chemiluminescent substrates and other labels that are described
in the MOLECULAR PROBES HANDBOOK, supra. In addition, present
labels can be substituted with substitutents that alter the
ion-binding affinity of the phosphate binding compound, solubility,
chemical reactivity, spectral properties or other physical
properties of the label provided that the label is not
sulfonated.
[0092] The term "Linker" or "L", as used herein, refers to a single
covalent bond or a series of stable covalent bonds incorporating
1-30 nonhydrogen atoms selected from the group consisting of C, N,
O, S and P that covalently attach the phosphate-binding compounds
to another moiety such as a chemically reactive group or a
phosphorylated target molecule. Exemplary linking members include a
moiety that includes --C(O)NH--, --C(O)O--, --NH--, --S--, --O--,
and the like. A "cleavable linker" is a linker that has one or more
cleavable groups that may be broken by the result of a reaction or
condition. The term "cleavable group" refers to a moiety that
allows for release of a portion, e.g., a label or phosphorylated
target molecule, of a conjugate from the remainder of the conjugate
by cleaving a bond linking the released moiety to the remainder of
the conjugate. Such cleavage is either chemical in nature, or
enzymatically mediated. Exemplary enzymatically cleavable groups
include natural amino acids or peptide sequences that end with a
natural amino acid.
[0093] In addition to enzymatically cleavable groups, it is within
the scope of the present invention to include one or more sites
that are cleaved by the action of an agent other than an enzyme.
Exemplary non-enzymatic cleavage agents include, but are not
limited to, acids, bases, light (e.g., nitrobenzyl derivatives,
phenacyl groups, benzoin esters), and heat. Many cleaveable groups
are known in the art. See, for example, Jung et al., Biochem.
Biophys. Acta, 761: 152-162 (1983); Joshi et al., J. Biol. Chem.,
265: 14518-14525 (1990); Zarling et al., J. Immunol., 124: 913-920
(1980); Bouizar et al., Eur. J. Biochem., 155: 141-147 (1986); Park
et al., J. Biol. Chem., 261: 205-210 (1986); Browning et al., J.
Immunol., 143: 1859-1867 (1989). Moreover a broad range of
cleavable, bifunctional (both homo- and hetero-bifunctional) spacer
arms are commercially available.
[0094] An exemplary cleavable group, an ester, is cleavable group
that may be cleaved by a reagent, e.g. sodium hydroxide, resulting
in a carboxylate-containing fragment and a hydroxyl-containing
product.
[0095] The linker can be used to attach the compound to another
component of a conjugate, such as a targeting moiety (e.g.,
antibody, ligand, non-covalent protein-binding group, etc.), an
analyte, a biomolecule, a drug and the like.
[0096] The term "metal chelator" or "metal-chelating moiety" as
used herein refers to a chemical moiety that combines with a metal
ion to form a chelate ring structure. For the purposes of the
present invention the metal chelator has affinity for a metal ion
that has simultaneous affinity for the metal chelator and a
phosphate target molecule in a moderately acidic environment.
Examples of metal-chelating moieties include, but are not limited
to, BAPTA, IDA, DTPA and phenanthroline. The metal chelators are
optionally substituted by substituents that adjust the ion-binding
affinity, solubility, chemical reactivity, spectral properties or
other physical properties of the compound provided that the metal
chelator is not sulfonated.
[0097] The term "metal ion" as used herein refers to any trivalent
metal ion that has simultaneous affinity for a phosphate group of a
target molecule and a metal-chelating compound of the invention at
pH 3 to 6 and that can be used to form a ternary complex of the
phosphate-binding compound and the phosphorylated target molecule.
Such metal ions include, without limitation, Al.sup.3+, Fe.sup.3+
and Ga.sup.3+. For purposes of the present invention, the metal ion
must have simultaneous affinity for both the metal-chelating moiety
and phosphate groups of the target molecule and, as such, confers
affinity to the metal-chelating moiety for the phosphate groups of
the target molecule that would not be present without the metal
ion.
[0098] The term "phosphate-binding compound" or "binding compound"
as used herein refers to a compound that is capable of binding a
metal ion wherein the metal ion has simultaneous affinity for a
phosphorylated target molecule. Such phosphate-binding compounds
are typically represented by, but are not limited to, the formula
(A)m(L)n(B)n wherein A is a chemical moiety, L is a linker, B is
metal-chelating moiety, m is an integer from 1 to 4 and n is an
integer from 0 to 4. These compounds effectively, but
non-covalently, attach a label to a phosphorylated target molecule
when the metal-chelating moiety indirectly binds phosphate groups
on the target molecule.
[0099] The terms "phosphorylated target molecule" or "phosphate
target molecule" as used herein refers to a molecule possessing one
or more phosphate or phosphate analog moieties each attached to
such molecule by a single ester bond or inorganic phosphate.
Phosphate analogs include, without limitation, thiophosphate,
boraphosphate, phosphoramide, H-phosphonate, alkylphosphonate,
phosphorothioate, phosphorodithioate and phosphorofluoridate.
Phosphorylated target molecules include, but are not limited to,
phosphoproteins, phosphopeptides, phospholipids, phosphoglycans,
phosphocarbohydrates, phosphoamino acids, pyrophosphate and
inorganic phosphate and their thiophosphate analogs. Most known
phosphate compounds, and subsequently the phosphorylated target
molecules, can be categorized into one of three groups; 1)
individual phosphate groups (e.g., inorganic phosphate or a
phosphate group (PO.sub.3) on a protein or peptide); 2)
multiple-linked phosphate group (e.g., pyrophosphate or a
nucleotide such as ATP); or 3) bridging phosphate group (i.e.,
nucleic acids). For the purposes of the present invention,
phosphorylated target molecules do not include molecules in the
third group, e.g., DNA or RNA. Typically, phosphoproteins and
phosphopeptides are phosphorylated post-translationally on the
tyrosine, serine or threonine amino acid residues. Other
phosphorylated amino acid residues in peptides and proteins include
1-phospho-histidine, 3-phospho-histidine, phospho-aspartic acid,
phospho-glutamic acid and less commonly N'-phospho-lysine,
N.sup..omega.-phospho-arginine and phospho-cysteine (Kaufmann, et
al (2001) Proteomics 1: 194-199; Yan, J., Paxker, N., Gooley, A.
and Williams, K. (1998) J. Chromatograph. A 808: 23-41). Thus, a
phosphorylated protein or peptide typically comprises at least one
of these amino acid residues. Phosphorylated target molecules also
include phosphorylated proteins that incorporate other non-peptide
regions such as lipids or carbohydrates, e.g., lipoproteins and
lipopolysaccharides. In addition, the lipid or carbohydrate
residues of the proteins can be phosphorylated instead or in
combination with the tyrosine, serine or threonine amino acid
residues of the proteins and peptides such as a
phosphomannose-modified or N-acetylglucosamine-1-phosphate modified
protein. Other modifications include a pyridoxal phosphate Schiff
base to the epsilon-amino group of lysine, and an O-pantetheine
phosphorylation of serine residue. The gamma phosphate of
nucleotide triphosphates is also detectable using the methods of
this invention, making photolabeled proteins and peptides
detectable by this procedure. For the purposes of the present
invention phosphorylated target molecules include phosphorylated
lipids and carbohydrates.
[0100] The term "photoactivatable reactive group" as used herein
refers to a chemical moiety that becomes chemically active by
exposure to an appropriate wavelength, typically a UV wavelength.
Once activated the reactive group is capable of forming a covalent
bond with a proximal moiety on a biological or non-biological
component. In the instant case, the phosphate-binding compounds may
contain a photoactivatable group that can form a covalent bond with
a phosphorylated target molecule when brought within proximity by
the formation of the ternery complex and activated by an
appropriate wavelength. Photoactivatable groups include, but are
not limited to, benzophenones, aryl azides and diazirines.
[0101] The terms "protein" and "polypeptide" are used herein in a
generic sense to include polymers of amino acid residues of any
length. The term "peptide" is used herein to refer to polypeptides
having less than 100 amino acid residues, typically less than 15
amino acid residues. The terms apply to amino acid polymers in
which one or more amino acid residues is an artificial chemical
analogue of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers. The peptide or
protein may be further conjugated to or complexed with other
moieties such as dyes, haptens, radioactive isotopes, natural and
synthetic polymers (including microspheres), glass, metals and
metallic particles, proteins and nucleic acids.
[0102] The term "reactive group" as used herein refers to a group
that is capable of reacting with another chemical group to form a
covalent bond, i.e. is covalently reactive under suitable reaction
conditions, and generally represents a point of attachment for
another substance. The reactive group is a moiety, such as a
photoactivatable group, carboxylic acid or succinimidyl ester, on
the compounds of the present invention that is capable of
chemically reacting with a functional group on a different compound
to form a covalent linkage resulting in a phosphate-binding labeled
component. Reactive groups generally include nucleophiles,
electrophiles and photoactivatable groups.
[0103] Exemplary reactive groups include, but not limited to,
olefins, acetylenes, alcohols, phenols, ethers, oxides, halides,
aldehydes, ketones, carboxylic acids, esters, amides, cyanates,
isocyanates, thiocyanates, isothiocyanates, amines, hydrazines,
hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,
mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic
acids, sulfinic acids, acetals, ketals, anhydrides, sulfates,
sulfenic acids isonitriles, amidines, imides, imidates, nitrones,
hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids,
allenes, ortho esters, sulfites, enamines, ynamines, ureas,
pseudoureas, semicarbazides, carbodiimides, carbamates, imines,
azides, azo compounds, azoxy compounds, and nitroso compounds.
Reactive functional groups also include those used to prepare
bioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and
the like. Methods to prepare each of these functional groups are
well known in the art and their application to or modification for
a particular purpose is within the ability of one of skill in the
art (see, for example, Sandler and Karo, eds. ORGANIC FUNCTIONAL
GROUP PREPARATIONS, Academic Press, San Diego, 1989).
[0104] The term "sample" as used herein refers to any material that
may contain phosphorylated target molecules, natural or synthetic,
as defined above, or contains components that directly interact
with phosphate or phosphorylated target molecules, such as enzymes.
Typically, the sample comprises purified or semi-purified
phosphorylated target molecules and endogenous host cell proteins.
The phosphorylated target molecules can be made synthetically or
obtained in a purified or semi-purified form from cells (eukaryotic
and prokaryotic, without limitation) cell extracts, cell
homogenates, subcellular components as natural or recombinant
molecules. Alternatively, phosphorylated target molecules can be
obtained from tissue homogenate, bodily and other biological
fluids, or synthesized proteins, all of which comprise a sample in
the present invention. The sample may be in an aqueous or mostly
aqueous solution, a viable cell culture or immobilized on a solid
or semi solid surface such as a polymer gel, a membrane, a
microparticle, an optical fiber or on a microarray. In addition
"sample" as use herein also refers to substrates for kinases or
phosphatases or molecules that bind phosphorylated target molecules
that may or may not be phosphorylated. In this way the sample
comprises components that interact with phosphate and
phosphorylated target molecules, particularly including antibodies
to either the phosphorylated target molecules or to other regions
of the target molecule or, for instance, complexes of biotinylated
target molecules with an avidin derivative.
[0105] The term "ternary complex" as used herein refers to a
composition that simultaneously comprises a phosphate-binding
compound, a trivalent metal ion of the present invention and a
phosphate target molecule, wherein the metal ions simultaneously
have affinity for both the metal-chelating moiety of the compound
and the phosphate group on the molecule, and wherein the metal ion
forms a bridge between the two molecules. Unless limited by the
context of their use, the terms "binding" and "complex formation"
in this invention mean the process of formation of this ternary
complex.
The Phosphate-Binding Compounds
[0106] In general, for ease of understanding the present invention,
the phosphate-binding compounds and components of the binding
solution will first be described in detail, followed by the many
and varied methods in which the phosphate-binding compounds and
metal ions find uses, which is followed by exemplified methods of
use and synthesis of certain novel compounds that are particularly
advantageous for use with the methods of the present invention.
[0107] The phosphate-binding compounds of the present invention are
characterized as being capable of binding to a trivalent metal ion
that has simultaneous affinity for a phosphorylated target molecule
to form a ternary complex. Thus, any metal chelating moiety that is
capable of binding a trivalent metal ion wherein the metal ion has
affinity for a phosphorylated target molecule is contemplated and
considered part of the present invention. For detection, isolation
and quantification purposes the metal chelating moiety is typically
covalently attached to a reactive group or a label that typically a
dye, a hapten or an enzyme, wherein the reactive group or label are
collectively defined herein as "chemical moiety A".
[0108] Thus, the present phosphate-binding compounds are typically
represented by, but not limited to, the formula (A)(B) wherein A is
the chemical moiety and B is the metal chelating moiety. More
typically, the present phosphate-binding compounds are represented
by the formula (A)m(L)n(B), wherein A is a chemical moiety and L is
a linker that covalently attaches the chemical moiety to the
metal-chelating moiety (B) and m and n are individually integers
between 0 and 4. The metal-chelating moiety is dictated by metal
ions that have affinity for phosphate and phosphate analog groups;
such ions include, but are not limited to, Ga.sup.3+, Fe.sup.3+ and
Al.sup.3+. It was found that for purposes of the present invention
trivalent gallium ions when in a moderately acidic environment,
e.g. between about pH 3 and about pH 6, have affinity for phosphate
groups on target molecules and certain chelating groups such as
BAPTA, IDA, DTPA and phenanthroline; BAPTA chelating moieties are
the most preferred.
Metal Chelating Moieties
[0109] The metal-chelating moieties are moieties characterized as
being capable of simultaneously binding metal ions that have
affinity for exposed phosphate groups on target molecules, wherein
a ternary complex is formed between the metal-chelating moiety, the
metal ion and the phosphorylated target molecule. Metal ions that
have been found to bind phosphate groups include, without
limitation, trivalent gallium, iron and aluminum. Metal-chelating
moieties that bind these ions, under certain conditions, include,
without limitation, BAPTA, IDA, DTPA and phenanthrolines. Thus, the
metal-chelating moieties must 1) bind metal ions that have affinity
for phosphate groups, 2) not interfere with the binding of the
metal ion for the phosphate groups and 3) maintain a stable ternary
complex. Exemplary metal-chelating moieties that fit these three
criteria include BAPTA, IDA, DTPA and phenanthrolines.
[0110] BAPTA, as used herein, refers to analogs, including
fluorescent and nonfluorescent derivatives, of the metal-chelating
moiety (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)
and salts thereof including any corresponding compounds disclosed
in U.S. Pat. Nos. 4,603,209; 4,849,362; 5,049,673; 5,453,517;
5,459,276; 5,516,911; 5,501,980; and 5,773,227. These BAPTA-based
metal-chelating moieties are well known to those skilled in the
art, primarily as calcium indicators due to their ability to bind
divalent calcium ions under physiological conditions, i.e. a pH of
about 7 and free calcium ion concentrations near the micromolar and
submicromolar range. As calcium indicators these compounds are
typically used in live cells wherein the indicators are derivatized
on a carboxylic group to comprise at least one lipophilic group or
specifically an acetoxymethyl (AM) ester group, wherein AM ester is
represented as --CH.sub.2OCOCH.sub.3, to produce cell permeant
derivatives of the indicators.
[0111] However, we found that calcium is a totally ineffective
metal ion for practice of the methods of the present invention to
detect phosphorylated target molecules described in this invention
with these indicators.
[0112] For the sake of clarity the following structure represents
preferred present BAPTA metal-chelating moieties having Formula
IV:
##STR00001##
[0113] Preferably the two rings are linked by a hydrocarbon bridge
between two oxygen atoms in which p is 0, 1 or 2 and the ring
substituents (R.sup.1-R.sup.8) are selected independently from the
group consisting of hydrogen, halogen, hydroxyl, alkoxy, alicyclic,
heteroalicyclic, alkyl, aryl, amino, aldehyde, carboxyl, nitro,
cyano, thioether, sulfinyl and linker (L). Alternatively, two
adjacent ring substituents in combination constitute a cyclic
substituent that is cycloalkyl, cycloheteroalkyl, aryl, fused aryl,
heteroaryl or fused heteroaryl. Preferably, the BAPTA
metal-chelating moieties have at least two substituents that are
not hydrogen, a most preferred BAPTA metal-chelating moiety is
substituted by a fluorine atom as one of the substituents, most
preferably substituted at the R.sup.6 or R.sup.3 position (e.g.,
Compounds 1, 2, 5, 7, 8 and 12). Typically the linker attaching the
chemical moiety to the BAPTA is at the R.sup.3 or R.sup.6 position.
Equally preferred are BAPTA metal-chelating moieties that comprise
a carbonyl group as a substituent, preferably at the R.sup.7
position, e.g., Compounds 9 and 12. Without being bound by a
particular theory, it appears that an electron withdrawing group
such as fluorine or carbonyl substituted at the R.sup.3, R.sup.4,
R.sup.6 or R.sup.7 position results in BAPTA chelating moieties
that are particularly advantageous for chelating trivalent gallium
ions that then also allows for the simultaneous interaction of the
chelated gallium ion with an exposed phosphate group on the
phosphorylated target molecules, resulting in a stable ternary
complex.
[0114] The bridge substituents R.sup.9, R.sup.10, R.sup.11 and
R.sup.12, are independently selected from the group consisting of
hydrogen, lower alkyl, or adjacent substituents R.sup.9 and
R.sup.10, taken in combination, constitute a 5-membered or
6-membered alicyclic or heterocyclic ring. R.sup.15, R.sup.16,
R.sup.17 and R.sup.18 are independently H or lower alkyl;
preferably R.sup.15, R.sup.16, R.sup.17 and R.sup.18 are all
hydrogen. R.sup.13 and R.sup.14 are independently hydrogen or a
salt.
[0115] It is understood that the chemical moieties of the present
invention are attached to the BAPTA metal-chelating moiety by a
linker at any of R.sup.1-R.sup.12 or alternatively the dye label
comprises one of the aromatic rings of the metal-chelating moieties
wherein no linker is present. Therefore, two adjacent substituents
of R.sup.1-R.sup.12, when taken in combination with each other, and
with the aromatic ring to which they are bound, comprise a
fluorophore or chromophore label. However, a phosphate-binding
compound could have more than one linker, such that a dye label is
attached with no linker and four other linkers are present on the
metal chelating compound to attach other labels or reactive groups.
In one aspect of the invention, two adjacent ring substituents
(R.sup.1-R.sup.4 or R.sup.5-R.sup.8) taken in combination form the
dye label that is a fused benzofuran or heteroaryl- or
carboxyheteroaryl-substituted benzofuran fluorophore. Where the dye
label is fused to the compound of the invention, it is preferably
fused between R.sup.2 and R.sup.3, or between R.sup.6 and
R.sup.7.
[0116] Xanthene derivative dyes are particularly useful dyes of the
present invention wherein, either or both of the benzene rings of
the BAPTA or substituted BAPTA metal-binding compound is bonded to
a xanthene ring through a single chemical bond, as in the common
Ca.sup.2+ indicators fluo-3, fluo-4 and rhod-2 (U.S. Pat. No.
5,049,673, supra) or through the intermediacy of a phenyl or
substituted phenyl spacer as in the Oregon Green.RTM. BAPTA
indicators (U.S. Pat. No. 6,162,931, supra). The xanthene rings are
typically bonded to the BAPTA at positions para to the nitrogen
functions of the BAPTA. Particularly preferred are
xanthene-containing BAPTA derivatives whose fluorophore is a
rhodamine or a halogenated fluorescein. Particularly preferred are
fluorescent BAPTA derivatives in which the 5-position of the BAPTA
chelator is substituted by F, including rhod-5F and fluo-5F.
[0117] DTPA, as used herein, refers to diethylenetriamine
pentaacetic acid chelating moieties and derivatives thereof,
including any corresponding compounds disclosed in U.S. Pat. Nos.
4,978,763 and 4,647,447. DTPA metal-chelating moieties are
represented by Formula V comprising
(CH.sub.2CO.sub.2R.sup.13).sub.ZN[(CH.sub.2).sub.SN(CH.sub.2CO.sub.2R.sup-
.13)].sub.T(CH.sub.2).sub.SN(CH.sub.2CO.sub.2R.sup.13).sub.Z,
wherein a linker is attached to a methine carbon or nitrogen atom,
Z is 1 or 2, S is 1 to 5, T is 0-4 and R.sup.13 is independently a
hydrogen or a salt.
[0118] IDA, as used herein, refers to iminodiacetic acid compounds
and derivatives thereof and is represented by Formula VI comprising
-(L)-N(CH.sub.2CO.sub.2R.sup.13).sub.2 wherein R.sup.13 is
independently a hydrogen or a salt and provided that said linker is
not a single covalent bond. The IDA metal-chelating moieties must
be attached by a linker to a chemical moiety wherein the linker
comprises at least one nonhydrogen atom. Without wishing to be
bound by a theory, it appears that the linker increases the
stability of the ternary complex and possibly tunes the affinity of
the metal-chelating moiety for a metal ion of the present
invention.
[0119] In addition to the above mentioned specific metal chelating
moieties we have also found that phenanthroline based chelators
also form ternary complex with metal ions and phosphate target
molecules in a moderately acidic environment. Phenanthroline, as
used herein, refers to 1,10-phenanthroline compounds and
derivatives thereof and is represented by the structure
##STR00002##
[0120] Any of the aromatic carbon atoms may be substituted with
substituents well known to one skilled in the art, including those
substituents disclosed in U.S. Pat. No. 6,316,267, supra.
Alternatively, a linker can be attached to any of the aromatic
carbon atoms to covalently attach a chemical moiety A to the
phenanthroline moiety to form the phosphate-binding compounds of
the present invention.
Labels
[0121] In certain embodiments, the present phosphate-binding
compounds comprise a label that is covalently bonded to a present
metal chelating moiety. The present labels are characterized as
being any label known to one skilled in the art and when the label
is either covalently linked to a metal-chelating moiety or
comprises part of the metal-chelating moiety wherein no linker is
present, forms a present phosphate-binding compound. Labels
include, without limitation, a chromophore, a fluorophore, a
fluorescent protein, a phosphorescent dye, a tandem dye (energy
transfer pair), a microparticle, a polymer, a hapten, an enzyme and
a radioisotope. Preferred labels include dyes, fluorescent
proteins, haptens, and enzymes. The covalent linkage can be a
single covalent bond or a combination of stable chemical bonds. The
covalent linkage binding the label to the metal-chelating moiety is
typically a single covalent bond, but can also be a substituted
alkyl chain that incorporates 1-30 nonhydrogen atoms, or a
substituted cycloalkyl, selected from the group consisting of C, N,
O, S and P.
[0122] A dye of the present invention is any chemical moiety that
exhibits an absorption maximum beyond 280 nm, that when part of a
phosphate-binding compound retains its unique spectral properties
to provide a detectable signal. The preferred dyes are fluorophores
or chemiluminescence precursors that are directly detectable or
that upon action of an additional reagent or reagents yield
fluorescence or chemiluminescence.
[0123] Dyes of the present invention include, without limitation; a
pyrene, an anthracene, a naphthalene, an acridine, a stilbene, an
indole or benzindole, an oxazole or benzoxazole, a thiazole or
benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), a
cyanine including any corresponding compounds in U.S. Ser. Nos.
09/968,401 and 09/969,853 and U.S. Pat. Nos. 6,403,807 and
6,348,599), a carbocyanine (including any corresponding compounds
in U.S. Ser. No. 09/557,275 and U.S. Pat. Nos. 5,486,616;
5,268,486; 5,569,587; 5,569,766; 5,627,027 and 6,048,982), a
carbostyryl, a porphyrin, a salicylate, an anthranilate, an
azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene
(including any corresponding compounds disclosed in U.S. Pat. Nos.
4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896, supra),
a xanthene (including any corresponding compounds disclosed in U.S.
Pat. Nos. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343
6,221,606; 6,358,684; 6,008,379; 6,111,116; 6,184,379; 6,017,712;
6,080,852; 5,847,162 and U.S. Ser. No. 09/922,333) an oxazine or a
benzoxazine, a carbazine (including any corresponding compounds
disclosed in U.S. Pat. No. 4,810,636), a phenalenone, a coumarin
(including an corresponding compounds disclosed in U.S. Pat. Nos.
5,696,157; 5,459,276; 5,501,980 and 5,830,912), a benzofuran
(including any corresponding compounds disclosed in U.S. Pat. Nos.
4,603,209 and 4,849,362) and benzphenalenone (including any
corresponding compounds disclosed in U.S. Pat. No. 4,812,409) and
derivatives thereof. As used herein, oxazines include resorufins
(including any corresponding compounds disclosed in U.S. Pat. No.
5,242,805), aminooxazinones, diaminooxazines, and their
benzo-substituted analogs.
[0124] Where the dye is a xanthene, the dye is optionally a
fluorescein, a rhodol (including any corresponding compounds
disclosed in U.S. Pat. Nos. 5,227,487 and 5,442,045), a rhodamine
(including any corresponding compounds in U.S. Pat. Nos. 5,798,276
and 5,846,737). As used herein, rhodamine and rhodol dyes include,
among other derivatives, compounds that comprise xanthenes with
saturated or unsaturated "julolidine" rings. As used herein,
fluorescein includes benzo- or dibenzofluoresceins,
seminaphthofluoresceins, or naphthofluoresceins. Similarly, as used
herein rhodol includes seminaphthorhodafluors (including any
corresponding compounds disclosed in U.S. Pat. No. 4,945,171).
[0125] Preferred dyes of the present invention include benzofurans,
quinolines, quinazolinones, xanthenes, indoles, benzazoles and
borapolyazaindacenes. Preferred xanthenes include
julolidine-containing xanthenes, as well as fluoresceins, rhodols,
rhodamines and rosamines. Xanthenes of this invention comprise both
compounds substituted and unsubstituted on the carbon atom of the
central ring of the xanthene by substituents typically found in the
xanthene-based dyes such as phenyl and substituted-phenyl moieties.
It is an important aspect of the current invention that none of the
preferred fluorescent dyes are sulfonated.
[0126] Alternatively, the dye is a xanthene that is bound via an L
that is a single covalent bond at the 9-position of the xanthene.
Preferred xanthenes include derivatives of 3H-xanthen-6-ol-3-one
attached at the 9-position, derivatives of 6-amino-3H-xanthen-3-one
attached at the 9-position, or derivatives of
6-amino-3H-xanthen-3-imine attached at the 9-position.
[0127] Typically the dye contains one or more aromatic or
heteroaromatic rings that are optionally substituted one or more
times by a variety of substituents, including without limitation,
halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl,
alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring
system, benzo, or other substituents typically present on dyes
known in the art.
[0128] In one aspect of the invention, the dye has an absorption
maximum beyond 480 nm. In a particularly useful embodiment, the dye
absorbs at or near 488 nm to 514 nm (particularly suitable for
excitation by the output of the argon-ion laser excitation source)
or near 546 nm (particularly suitable for excitation by a mercury
arc lamp). As is the case for many dyes, they can also function as
both chromophores and fluorophores, resulting in compounds that
simultaneously act both as colorimetric and fluorescent labels for
phosphorylated target molecules. Thus, the described fluorescent
dyes are also the preferred chromophores of the present
invention.
[0129] In addition to dyes, enzymes also find use as labels for the
phosphate-binding compounds having the formula (A)m(L)n(B). Enzymes
are desirable labels because amplification of the detectable signal
can be obtained resulting in increased assay sensitivity. The
enzyme itself does not produce a detectable response but functions
to break down a substrate when it is contacted by an appropriate
substrate such that the converted substrate produces a fluorescent,
calorimetric or luminescent signal. Enzymes amplify the detectable
signal because one enzyme on a labeling compound can result in
multiple substrate molecules being converted to a detectable
signal. This is advantageous where there is a low quantity of
phosphorylated target molecules present in the sample or a
fluorophore does not exist that will give comparable or stronger
signal than the enzyme. Fluorophores are most preferred because
they do not require additional assay steps that can lead to an
unstable ternary complex. The enzyme substrate is selected to yield
the preferred measurable product, e.g. color, fluorescence or
chemiluminescence. Such substrates are extensively used in the art,
many of which are described in the MOLECULAR PROBES HANDBOOK,
supra.
[0130] A preferred calorimetric or fluorogenic substrate and enzyme
combination uses oxidoreductases such as horseradish peroxidase
(HRP) and a substrate such as 3,3'-diaminobenzidine (DAB) or
3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color
(brown and red, respectively). Other calorimetric oxidoreductase
substrates that yield detectable products include, but are not
limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS), o-phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine
(TMB), o-dianisidine, 5-aminosalicylic acid and
4-chloro-1-naphthol. Fluorogenic substrates include, but are not
limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic
acid, reduced phenoxazines and reduced benzothiazines, including
Amplex.RTM. Red reagent and its variants (U.S. Pat. No. 4,384,042)
and reduced dihydroxanthenes, including dihydrofluoresceins (U.S.
Pat. No. 6,162,931) and dihydrorhodamines, including
dihydrorhodamine 123. Peroxidase substrates that are tyramides
(U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158) represent a
unique class of peroxidase substrates in that they can be
intrinsically detectable before action of the enzyme but are "fixed
in place" by the action of a peroxidase in the process described as
tyramide signal amplification (TSA). These substrates are
extensively utilized to label targets in samples that are cells,
tissues or arrays for their subsequent detection by microscopy,
flow cytometry, optical scanning and fluorometry.
[0131] Another preferred colorimetric (and in some cases
fluorogenic) substrate and enzyme combination uses a phosphatase
enzyme such as an acid phosphatase or a recombinant version of such
a phosphatase in combination with a colorimetric substrate such as
5-bromo-4-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl
phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl
phosphate, or o-nitrophenyl phosphate or with a fluorogenic
substrate such as 4-methylumbelliferyl phosphate,
6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S.
Pat. No. 5,830,912), fluorescein diphosphate, 3-O-methylfluorescein
phosphate, resorufin phosphate,
9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate (DDAO
phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos.
5,316,906 and 5,443,986).
[0132] Glycosidases, in particular .beta.-galactosidase,
.beta.-glucuronidase and .beta.-glucosidase, are additional
suitable enzymes. Appropriate colorimetric substrates include, but
are not limited to, 5-bromo-4-chloro-3-indolyl
.beta.-D-galactopyranoside (X-gal) and similar indolyl
galactosides, glucosides, and glucuronides, o-nitrophenyl
.beta.-D-galactopyranoside (ONPG) and p-nitrophenyl
.beta.-D-galactopyranoside. Preferred fluorogenic substrates
include resorufin .beta.-D-galactopyranoside, fluorescein
digalactoside (FDG), fluorescein diglucuronide and their structural
variants (U.S. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424
and 5,773,236), 4-methylumbelliferyl .beta.-D-galactopyranoside,
carboxyumbelliferyl .beta.-D-galactopyranoside and fluorinated
coumarin .beta.-D-galactopyranosides (U.S. Pat. No. 5,830,912).
[0133] Additional enzymes include, but are not limited to,
hydrolases such as cholinesterases and peptidases, oxidases such as
glucose oxidase and cytochrome oxidases and reductases for which
suitable substrates are known.
[0134] Enzymes and their appropriate substrates that produce
chemiluminescence are preferred for some assays. These include, but
are not limited to, natural and recombinant forms of luciferases
and aequorins. Chemiluminescence-producing substrates for
phosphatases, glycosidases and oxidases such as those containing
stable dioxetanes, luminol, isoluminol and acridinium esters are
additionally useful. Several chemiluminescent substrates for
phosphatase enzymes are known, including the BOLD APB
chemiluminescent substrate (Molecular Probes, Inc.).
[0135] In addition to enzymes, haptens such as biotin, digoxigenin
and 2,4-dinitrophenyl are also preferred labels. Biotin is useful
because it can function in an enzyme system to further amplify the
detectable signal, and it can function as a tag to be used in
affinity chromatography for isolation purposes. For detection
purposes, an enzyme conjugate that has affinity for biotin is used,
such as avidin-HRP. Subsequently, a peroxidase substrate is added
to produce a detectable signal. For isolation purposes, a protein
such as avidin that has affinity for biotin is conjugated to
agarose beads. The biotin-labeled metal-chelating moiety, after
contacting a phosphorylated target molecule, is then incubated with
the avidin beads, on a column, bound to a magnetic particle or in
solution, to separate and/or concentrate the phosphorylated target
molecules. A preferred form of biotin is the desthiobiotin analog,
which can be easily adsorbed and released from avidin-based
affinity matrices. A preferred form of avidin for some applications
is CaptAvidin biotin-binding protein (Molecular Probes), which
permits facile release of biotinylated compounds.
[0136] Haptens also include, among other derivatives, hormones,
naturally occurring and synthetic drugs, pollutants, allergens,
affector molecules, growth factors, chemokines, cytokines,
lymphokines, amino acids, peptides, chemical intermediates,
nucleotides and the like.
[0137] Fluorescent or luminescent proteins also find use as labels
for the phosphate-binding compounds of the present invention.
Examples of fluorescent proteins include green-fluorescent protein
(GFP), acquorin and the phycobiliproteins and the derivatives
thereof. The fluorescent proteins, especially phycobiliproteins,
are particularly useful for creating tandem dye-labeled labeling
reagents or for indirect detection of hapten-labeled labeling
compounds or phosphorylated target molecules that are immobilized
on a matrix, such as a microsphere or an array. These tandem dyes
comprise a fluorescent protein and a fluorophore for the purposes
of obtaining a larger Stokes shift, wherein the emission spectra
are farther shifted from the wavelength of the fluorescent
protein's absorption spectra. This property is particularly
advantageous for detecting a low quantity of a target molecule in a
sample wherein the emitted fluorescent light is maximally
optimized; in other words, little to none of the emitted light is
reabsorbed by the fluorescent protein. For this to work, the
fluorescent protein and fluorophore function as an energy transfer
pair wherein the fluorescent protein emits at the wavelength that
the acceptor fluorophore absorbs and the fluorophore then emits at
a wavelength farther from the fluorescent proteins than could have
been obtained with only the fluorescent protein. Alternatively, the
fluorophore functions as the energy donor and the fluorescent
protein is the energy acceptor. Particularly useful fluorescent
proteins are the phycobiliproteins disclosed in U.S. Pat. Nos.
4,520,110; 4,859,582; 5,055,556 and the fluorophore bilin protein
combinations disclosed in U.S. Pat. No. 4,542,104. Alternatively,
two or more fluorophore dyes can function as an energy transfer
pair wherein one fluorphore is a donor dye and the other is the
acceptor dye (including any dye compounds disclosed in U.S. Pat.
Nos. 6,358,684; 5,863,727; 6,372,445 and 5,656,554).
Reactive Groups
[0138] Selected phosphate-binding compounds of the invention
include one or more reactive groups within their structure. The
reactive group provides a locus for attaching a metal chelating
moiety to another species, generally referred to herein as a
"component" of a conjugate. Exemplary components include biological
or non-biological molecules, linkers, solid supports,
phosphorylated target molecules and the like. The reactive group
reacts with a functional group of complementary reactivity at the
"attachment site" of the component of the conjugate. The reaction
leads to the formation of a covalent linkage between the metal
chelating moiety of the phosphate-binding compound and the
component of the conjugate.
[0139] Therefore, these reactive groups function to attach a
biological or non-biological component to the metal chelating
moiety or are used to form a covalent attachment between the
present chelating moieties and the phosphorylated target molecules.
Such components include solid and semi-solid matrices such as
polymeric particles (in particular polystyrene microspheres of a
diameter less that about 16 microns), magnetic particle, polymeric
membranes and glass. These substances are particularly useful when
an assay is utilized wherein the phosphate-binding compound is
immobilized, such as for isolation purposes or when a further assay
is conducted directly on an immobilized phosphorylated target
molecules, as described in this invention.
[0140] In addition, any biological component can be covalently
attached by way of reactive groups to the phosphate-binding
compound; these include but are not limited to, proteins, peptides,
saccharides and polysaccharides, nucleic acids (including
nucleotides and nucleosides), amino acids, organelles, cells and
cellular extract components.
[0141] Therefore, the phosphate-binding compounds of the present
invention can also comprise reactive groups, such as an
amine-reactive group, for the covalent attachment of the
phosphate-binding compound to a matrix, microparticle, a phosphate
target molecule or directly to a biological component. Thus, when
the ternary complex comprising the phosphate-binding compound,
metal ion and phosphorylated target molecules forms, the reactive
groups can form an additional covalent bond with the phosphorylated
target molecule. This effectively increases the complex's stability
and allows for more stringent isolation and analysis of
phosphorylated target molecules, including being able to maintain
the complex's integrity above the moderately acidic pH range.
[0142] Typically, covalent attachment of the phosphate-binding
compound to a molecule is the result of a chemical reaction between
an electrophilic group and a nucleophilic group. However, in a
preferred embodiment, when a reactive group is used that is
photoactivated, the covalent attachment results when the binding
solution is illuminated. This is particularly advantageous to
ensure that only phosphorylated target molecules form a covalent
attachment to the binding compounds of the present invention.
Photoactivatable reactive groups include, without limitation,
benzophenones, aryl azides and diazirines. Exemplary
phosphate-binding compounds comprising a photoactivatable group
include Compounds 34, 36, 39, 42 and 44.
[0143] Typically, the conjugation reaction between the reactive
group and the component to be conjugated results in one or more
atoms of the reactive group to be incorporated into a new linkage
attaching the compound or reagents of the invention to the
biological or non-biological component. Selected components
include, without limitation, these other molecules include without
limitation, labels, biological components (proteins, nucleic
acid,), non-biological components including microparticles, plastic
such as microplate wells, polymers such as PVDF, nitrocellulose,
polysaccharides in particular agarose, dextrans and cellulose
including any compounds disclosed in U.S. Pat. No. 5,453,517.
[0144] Selected examples of nucleophile, electrophiles and
resulting covalent linkages are shown in Table 1.
TABLE-US-00001 TABLE 1 Examples of some routes to useful covalent
linkages with electrophile and nucleophile reactive groups
Electrophilic Group Nucleophilic Group Resulting Covalent Linkage
activated esters* amines/anilines carboxamides acrylamides thiols
thioethers acyl azides** amines/anilines carboxamides acyl halides
amines/anilines carboxamides acyl halides alcohols/phenols esters
acyl nitriles alcohols/phenols esters acyl nitriles amines/anilines
carboxamides aldehydes amines/anilines imines aldehydes or ketones
hydrazines hydrazones aldehydes or ketones hydroxylamines oximes
alkyl halides amines/anilines alkyl amines alkyl halides carboxylic
acids esters alkyl halides thiols thioethers alkyl halides
alcohols/phenols ethers alkyl sulfonates thiols thioethers alkyl
sulfonates carboxylic acids esters alkyl sulfonates
alcohols/phenols ethers anhydrides alcohols/phenols esters
anhydrides amines/anilines carboxamides aryl halides thiols
thiophenols aryl halides amines aryl amines aziridines thiols
thioethers boronates glycols boronate esters carbodiimides
carboxylic acids N-acylureas or anhydrides diazoalkanes carboxylic
acids esters epoxides thiols thioethers haloacetamides thiols
thioethers haloplatinate amino platinum complex haloplatinate
heterocycle platinum complex haloplatinate thiol platinum complex
halotriazines amines/anilines aminotriazines halotriazines
alcohols/phenols triazinyl ethers halotriazines thiols triazinyl
thioethers imido esters amines/anilines amidines isocyanates
amines/anilines ureas isocyanates alcohols/phenols urethanes
isothiocyanates amines/anilines thioureas maleimides thiols
thioethers phosphoramidites alcohols phosphite esters silyl halides
alcohols silyl ethers sulfonate esters amines/anilines alkyl amines
sulfonate esters thiols thioethers sulfonate esters carboxylic
acids esters sulfonate esters alcohols ethers sulfonyl halides
amines/anilines sulfonamides sulfonyl halides phenols/alcohols
sulfonate esters *Activated esters, as understood in the art,
generally have the formula --CO.OMEGA., where .OMEGA. is a good
leaving group (e.g., succinimidyloxy (--OC.sub.4H.sub.4O.sub.2)
sulfosuccinimidyloxy (--OC.sub.4H.sub.3O.sub.2--SO.sub.3H),
-1-oxybenzotriazolyl (--OC.sub.6H.sub.4N.sub.3); or an aryloxy
group or aryloxy substituted one or more times by electron
withdrawing substituents such as nitro, fluoro, chloro, cyano, or
trifluoromethyl, orcombinations thereof, used to form activated
aryl esters; or a carboxylic acid activated by a carbodiimide to
form an anhydride or mixed anhydride --OCOR.sup.a or
--OCNR.sup.aNHR.sup.b, where R.sup.a and R.sup.b, which may be the
same or different, are C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
perfluoroalkyl, or C.sub.1-C.sub.6 alkoxy; or cyclohexyl,
3-dimethylaminopropyl, or N-morpholinoethyl). **Acyl azides can
also rearrange to isocyanates
[0145] Preferred reactive groups incorporated into the compounds of
the invention react with an amine, a thiol or an alcohol. In one
embodiment, the reactive group is a photoactivatable group, an
acrylamide, an activated ester of a carboxylic acid, an acyl azide,
an acyl nitrile, an aldehyde, an alkyl halide, an amine, an
anhydride, an aniline, an aryl halide, an aryl azide, an azide, an
aziridine, a benzophenone, a boronate, a carboxylic acid, a
diazoalkane, a diazirine, a haloacetamide, a halotriazine, a
hydrazine, an imido ester, an isocyanate, an isothiocyanate, a
maleimide, a phosphoramidite, a sulfonyl halide, or a thiol
group.
[0146] Where the reactive group is an activated ester of a
carboxylic acid, the resulting compound is particularly useful for
preparing conjugates of proteins, nucleic acids, e.g., nucleotides
and oligonucleotides, or haptens. Where the reactive group is a
maleimide or haloacetamide the resulting compound is particularly
useful for conjugation to thiol-containing substances. Where the
reactive group is a hydrazide, the resulting compound is
particularly useful for conjugation to periodate-oxidized
carbohydrates and glycoproteins, and in addition is an
aldehyde-fixable polar tracer for cell microinjection. Where the
reactive group is a silyl halide, the resulting compound is
particularly useful for conjugation to silica surfaces,
particularly where the silica surface is incorporated into a fiber
optic probe subsequently used for remote ion detection or
quantitation or forms the substrate of a microarray or biochip.
Where the reactive group is a photoactivatable group (benzophenone,
aryl azide or diazirine) the resulting phosphate-binding compound
is particularly useful for conjugation to phosphorylated target
molecules, See Example 50.
[0147] Preferably, the reactive group is a photoactivatable group,
a succinimidyl ester of a carboxylic acid, a haloacetamide, a
hydrazine, an isothiocyanate, a maleimide group, an aliphatic
amine, a silyl halide, or a psoralen. More preferably, the reactive
group is a photoactivatable group, a succinimidyl ester of a
carboxylic acid, a maleimide, an iodoacetamide, or a silyl
halide.
Linkers
[0148] In an exemplary embodiment, the compounds of the present
invention that include a reactive group or label further comprise a
linker. The linker serves to covalently attach the reactive group
or label to the metal chelating moiety of the phosphate-binding
compound. When present, the linker is a single covalent bond or a
branched- or straight-chain, saturated or unsaturated chain of
atoms. Examples of L include substituted or unsubstituted
polyalkylene, arylene, alkylarylene, arylenealkyl, or arylthio.
[0149] In certain embodiments, no linker is present when the
phosphate-binding compounds comprise a label. In this instance the
label and the metal-chelating moiety share an aromatic ring, e.g.,
benzofuran and BAPTA. Thus, when the label and chelating moiety
share an aromatic ring no linker is present and n of the formula
(A)m(L)n(B) is 0. A preferred embodiment, when the metal chelating
moiety comprise a label, is phosphate-binding compounds wherein no
linker is present; however, linkers as single covalent bonds are
equally preferred.
[0150] Thus, the label or reactive group may be directly attached
(where Linker is a single bond) to the present compounds or
attached through a series of stable bonds. When the linker is a
series of stable covalent bonds the linker typically incorporates
1-30, more preferably 1-20, and most preferred 1-15 non-hydrogen
atoms selected from the group consisting of C, N, O, S and P. In
addition, the covalent linkage can incorporate a platinum atom,
such as described in U.S. Pat. No. 5,714,327.
[0151] The linker may be any combination of chemical bonds,
optionally including, single, double, triple or aromatic
carbon-carbon bonds, as well as carbon-nitrogen bonds,
nitrogen-nitrogen bonds, carbon-oxygen bonds, sulfur-sulfur bonds,
carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen
bonds, and nitrogen-platinum bonds. Exemplary components of the
linker include ether, thioether, thiourea, amine, ester,
carboxamide, sulfonamide, hydrazide bonds and aromatic or
heteroaromatic bonds.
[0152] An exemplary linker is a combination of single carbon-carbon
bonds and carboxamide, sulfonamide or thioether bonds. The bonds of
the linker typically result in the following moieties being in the
linker: ether, thioether, carboxamide, thiourea, sulfonamide, urea,
urethane, hydrazine, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl
and amine moieties.
[0153] Any combination of linkers may be present in the present
phosphate-binding compounds. An exemplary compound of the present
invention, when attached to more than one label, reactive group or
a combination thereof will have one or two linkers attached that
may be the same or different. The linker may also be substituted to
alter a physical property of the present compounds, such as
affinity, hydrophilicity, solubility and spectral properties of the
compound.
[0154] Selected examples of phosphate-binding compounds incorporate
the following three (I, II and III) Linker formulas: Formula (I)
--(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.e(NHC(X)(CH.sub.2).sub.e).sub.d--
and Formula (II)
--((C.sub.6R''.sub.4)O).sub.d(CH.sub.2).sub.e(C(X)NH(CH.sub.2).sub.e).sub-
.g(NH).sub.dC(X)NH(C.sub.6R''.sub.4)(CH.sub.2).sub.e--, Formula
(III)
--(NHC(X)(NH).sub.d(CH.sub.2).sub.e(NH).sub.dC(X)(NH).sub.d(CH.sub.2).sub-
.e(NHC(X)(CH.sub.2).sub.e).sub.d)--, wherein X is O or S, d is 0-1,
e is 0-6, g is 1-4 and R'' is independently H, halogen, alkoxy or
alkyl. It is understood that X, d, e and g are independently
selected within a linker.
[0155] Thus, a selected embodiment of the present invention is the
following phosphate-binding compound formulas (VII, VIII, IX, X and
XI): Formula (VII) (A)(B), no linker; Formula (VIII) (A)-(n)(B),
linker is a single covalent bond; Formula (IX)
(A)-[(CH.sub.2).sub.eC(X)NH(CH.sub.2).sub.e(NHC(X)(CH.sub.2).sub.e).sub.d-
]--(B); Formula (X)
(A)-[(C.sub.6R''.sub.4)O).sub.d(CH.sub.2).sub.e(C(X)NH(CH.sub.2).sub.e).s-
ub.g(NH).sub.dC(X)NH(C.sub.6R''.sub.4)(CH.sub.2).sub.e]--(B) and
Formula (XI)
(A)-[NHC(X)(NH).sub.d(CH.sub.2).sub.e(NH).sub.dC(X)(NH).sub.d(CH.sub-
.2).sub.e(NHC(X)(CH.sub.2).sub.e).sub.d]--(B), wherein A is a
chemical moiety and B is a metal-chelating moiety.
[0156] Any combination of linkers may be used to attach the
chemical moiety and the metal-chelating moiety together. In
addition, a metal-chelating moiety may have more than one linker
that is used to attach either another label, such as an energy
transfer pair, or an additional substance such as agarose, a
microparticle or a reactive group that functions to attach the
linker to the additional substance or to the phosphorylated target
molecule. A preferred embodiment includes a metal-chelating moiety
attached to a label, with or without a linker, and also attached to
a reactive group, a phosphorylated target molecule, a
non-biological component or a biological component. The linker may
also be substituted to alter the physical properties of the
phosphate-binding compound, such as binding affinity of the
metal-chelating moiety and spectral properties of the dye, or
substituted with an amine- or thiol-reactive group.
[0157] Another important feature of the linker is to provide an
adequate space between the chemical moiety A and the chelating
moiety B so as to prevent the chemical moiety from providing a
steric hindrance to the binding of the metal ion for the binding
domain of the metal-chelating moiety and the binding of the metal
ion for the phosphorylated target molecule. It is appreciated that
not all chemical moieties will provide a steric hindrance, as a
preferred embodiment of the present invention is a metal-chelating
moiety that comprises a dye label without a linker. However, some
labels such as biotin and reactive groups are typically attached to
the metal-chelating moiety by a linker. Therefore, the linkers of
the present phosphate-binding compounds are important for (1)
attaching the chemical moiety A to the metal-chelating moiety, (2)
providing an adequate distance between the chemical moiety and the
metal-chelating moiety so as not to sterically hinder the affinity
of the metal-chelating moiety and a phosphate group on a target
molecule and (3) for altering the affinity of the metal-chelating
moiety for the phosphorylated target molecule either by the choice
of the atoms of the linker or indirectly by addition of
substituents to the linker.
[0158] The metal-chelating moieties of the present invention
typically contain 1) no linker, 2) a single covalent bond as a
linker, 3) a linker of Formula I, 4) a linker of Formula II, 5) a
linker of Formula III or a combination thereof. However, it is
appreciated that a wide variety of linkers that do not fall within
the scope of these formulas are also useful as linkers of the
phosphate-binding compounds. These options can be present
individually or in any combination, as embodied by the formula
(A)m(L)n(B), on the metal-chelating moiety to attach chemical
moieties A such as labels or reactive groups to form the
phosphate-binding compounds of the present invention.
Synthesis
[0159] The synthetic strategy of phosphate-binding compounds that
provide optimal signals after formation of a ternary complex
involves selection of appropriate chemical linkages between the
chemical moieties A and the metal-chelating moiety, and also
selection of appropriate substituents on the metal-chelating
moiety. These selections are made such that the resulting
phosphate-binding compound retains optimal simultaneous binding
affinity for both the metal and of the metal for the phosphorylated
target molecules and sufficient solubility to promote a persistent
ternary complex. Improper selections result in phosphate-binding
compounds that do not have sufficient binding affinity and do not
produce a persistent ternary complex. Improper selections also
result in excessive non-selective binding of the phosphate-binding
compound to analytes other than the phosphorylated target
compounds, resulting in a high background and thus a low
signal-to-noise ratio. Compounds that are suitable for practice of
the invention are best screened by the method in Example 1D.
[0160] We have discovered that BAPTA derivatives are particularly
suitable for practice of the various aspects of the invention,
although other metal chelating moieties under appropriate
conditions are equally preferred. The novel phosphate-binding
compounds of the present invention whose synthesis and use is
illustrated in examples include BAPTA chelating moieties with a
quinazolinone fluorescent dye (Compounds 6, 7 and 23), BAPTA
chelating moieties with a borapolyazaindacene fluorescent dye
(Compounds 8, 24 and 27a-f), BAPTA chelating moieties with a
xanthene based dye (Compounds 11, 19, 26 and 25) BAPTA chelating
moieties with a biotin label, wherein the biotin is attached by a
linker (Compounds 9, 12, 15 and 18), BAPTA chelating moieties with
a benzothiazole label (Compound 17), BAPTA chelating moieties with
agarose covalently attached (Compounds 13 and 14), BAPTA compounds
comprising an aniline attached by a linker to the BAPTA compound
(Compound 10) and BAPTA chelating moieties with a label and a
photoactivatable reactive group (Compounds 34, 36, 39, 42 and 44).
Novel compounds also include borapolyazaindacene fluorophore labels
attached by a linker to DTPA chelating moieties (Compounds 20, 21
and 22). These novel phosphate-binding compounds find use in the
detection and isolation of phosphorylated target molecules.
Synthesis of these compounds is exemplified in Examples 30-48 and
52-56.
[0161] The phosphate-binding compounds of the present invention
exhibit sufficient noncovalent binding affinity for the gallium
(III)-phosphorylated target molecule complex to allow for rinsing
away of excess reagents from the persistent ternary complex.
Additionally, it was found that certain phosphate-binding compounds
provided optimal signal after formation of the ternary complex and
are thus more environmentally sensitive. This high signal appears
to be a function of well-tuned hydrophobicity of the
phosphate-binding compound--gallium (III)-phosphorylated target
molecule complex. Therefore, when a detectable response is
desirable, e.g., labeling phosphorylated target molecules in
solution, and where the detectable response is a fluorescence
response, it is typically a change in fluorescence, such as a
change in the intensity, excitation or emission wavelength
distribution of fluorescence, fluorescence lifetime, fluorescence
polarization, or a combination thereof. Typically this change in
fluorescence is a result of energy transfer between a first and
second fluorophore wherein an energy transfer pair is employed, one
attached to the chelating moiety and the other attached to a
phosphorylated molecule, resulting in a shifted wavelength or
quenching of the fluorescent signal. Preferably, when energy
transfer is not employed, the detectable optical response upon
binding the gallium ion and the phosphorylated target molecule to
the chelator is a change in fluorescence intensity that is greater
than approximately 2-fold.
[0162] However, for applications wherein the phosphorylated target
molecule or phosphate-binding compound is immobilized--resulting in
an immobilized ternary structure--an increase in detectable
fluorescence response due to the chelation of the metal-chelating
moiety and subsequent ternary complex formation may not be
necessary. This is due to the stable ternary complex, which allows
for washing and removal of unbound phosphate-binding compounds
wherein the fluorescence response from the phosphate-binding
compound is sufficient to visualize the phosphorylated target
molecule. Therefore, a preferred embodiment in this situation is a
phosphate-binding compound that undergoes little or no change in
fluorescence when bound to a metal ion of the present invention and
a phosphorylated target molecule.
[0163] The combination of metal-chelating moieties, labels and or
reactive groups provides phosphate-binding compounds that are
either environmentally sensitive (i.e., that produce a fluorescence
change upon simultaneously binding a metal ion and the
phosphorylated target molecules to form a ternary complex) or
insensitive (i.e., that produces no change in fluorescence signal).
The most preferred fluorescent dyes of the present invention for
generating a strong detectable signal and facilitating formation of
the ternary complex include benzofuran, quinoline, quinazolone,
xanthene, benzazole, and borapolyazaindacene compounds including
various derivatives thereof. These fluorescent dyes produce a
strong detectable signal when the dye comprises a metal-chelating
moiety. It is an important aspect of the current invention that
none of the preferred fluorescent dyes are sulfonated.
TABLE-US-00002 TABLE 2 Com- pound number Phosphate-binding compound
Properties Com- pound 1 ##STR00003## The fluorophore is a
fluorinated xanthene (fluorescein) derivative and the
metal-chelating moiety is a BAPTA compound (Formula IV) that is
fluorinated at the R.sup.6 position. The linker is a single
covalent bond. The counterion is K.sup.+. Com- pound 2 ##STR00004##
The fluorophore is a xanthene (rhodamine) derivative and the metal-
chelating moiety is a BAPTA compound (Formula IV) that is
fluorinated at the R.sup.6 position. The linker is a single
covalent bond. The counterion is K.sup.+. Com- pound 3 ##STR00005##
The fluorophore is a xanthene (rhodamine) derivative and the metal-
chelating moiety is a BAPTA compound (Formula IV). The linker is a
single covalent bond. The counterion is K.sup.+. Com- pound 4
##STR00006## The fluorophore is a benzofuran that shares an
aromatic ring with the metal-chelating moiety (Formula IV) and
comprises a substituted heteroaryl moiety. The metal-chelating
moiety, BAPTA, is fluorinated at the R.sup.6 position. The
phosphate-binding compound does not comprise a linker. The
counterion is K.sup.+. Com- pound 5 ##STR00007## The fluorophore is
a xanthene (rhodamine) derivative and the metal- chelating moiety
is a BAPTA compound (Formula IV) that is fluorinated at the R.sup.6
position. The linker is a single covalent bond. The counterion is
K.sup.+. Com- pound 6 ##STR00008## The fluorophore is a
quinazolinone with an adjacent hydroxyl group on the
metal-chelating moiety (BAPTA, Formula IV). R.sup.13 and R.sup.14
are independently hydrogen or a salt and the linker is a single
covalent bond. Com- pound 7 ##STR00009## The fluorophore is a
quinazolinone with an adjacent hydroxyl group on the
metal-chelating moiety (BAPTA, Formula IV). The metal-chelating
moiety is fluorinated at the R.sup.6 position and R.sup.13 and
R.sup.14 are independently hydrogen or a salt. The linker is a
single covalent bond. Com- pound 8 ##STR00010## The fluorophore is
a borapolyazaindacene and the metal- chelating moiety is a BAPTA
compound (Formula IV). The metal- chelating moiety is fluorinated
at the R.sup.6 position and R.sup.13 and R.sup.14 are independently
hydrogen or a salt. The linker is represented by Formula I. Com-
pound 9 ##STR00011## The fluorophore is a xanthene (rhodamine)
derivative and the metal- chelating moiety is a BAPTA compound
(Formula IV), wherein R.sup.13 and R.sup.14 are independently
hydrogen or a salt. The linker attaching the fluorophore to the
BAPTA compound is a single covalent bond. A second linker at
R.sup.7 (Formula I) covalently attaches a biotin label to the
phosphate-binding compound. Com- pound 10 ##STR00012## The
fluorophore is a xanthene (rhodamine) derivative that is attached
by a single covalent bond linker to the metal-chelating moiety
(BAPTA, Formula IV), wherein R.sup.13 and R.sup.14 are
independently hydrogen or a salt. A second linker (Formula I) at
R.sup.7 attaches an aniline moiety. Com- pound 11 ##STR00013## The
fluorophore is a xanthene (rhodamine) derivative that is attached
to the metal-chelating moiety (BAPTA, Formula IV) by a single
covalent bond, wherein R.sup.13 and R.sup.14 are independently
hydrogen or a salt. A second linker (Formula I) at R.sup.7 attaches
an amine group. Com- pound 12 ##STR00014## The label is a biotin
that is attached to the metal-chelating moiety (BAPTA, Formula IV)
by a linker (Formula III). The metal-chelating moiety is
fluorinated at the R.sup.3 position and R.sup.13 and R.sup.14 are
independently hydrogen or a salt. Com- pound 13 ##STR00015## The
metal-chelating moiety (BAPTA, Formula IV) is attached to agarose
by a linker, wherein R.sup.13 and R.sup.14 are independently
hydrogen or a salt. Com- pound 14 ##STR00016## The metal-chelating
moiety (BAPTA, Formula IV) is attached to agarose by a linker,
wherein R.sup.13 and R.sup.14 are independently hydrogen or a salt
and the metal-chelating moiety is fluorinated at the R.sup.3
position. Com- pound 15 ##STR00017## The metal-chelating moiety
(BAPTA, Formula IV) is simultaneously attached to biotin and a
xanthene (rhodamine) derivative fluorophore, both by a linker
represented by Formula III. R.sup.13 and R.sup.14 are independently
hydrogen or a salt. Com- pound 16 ##STR00018## The metal-chelating
moiety (BAPTA, formula IV) is attached by a single covalent bond to
a xanthene (rhodamine) derivative. R.sup.13 and R.sup.14 are
independently hydrogen or a salt. Com- pound 20 ##STR00019## The
dye is a borapolyazaindacene that is attached to the
metal-chelating moiety (DTPA, Formula V) by a linker (Formula II).
The dye is substituted by a thienyl group. The counterion is
K.sup.+. Com- pound 21 ##STR00020## The dye is a
borapolyazaindacene that is attached to a metal-chelating moiety
(DTPA, Formula V) by a linker (Formula II). The counterion is
K.sup.+. Com- pound 22 ##STR00021## The dye is a
borapolyazaindacene that is attached to the metal-chelating moiety
(DTPA, Formula V) by a linker represented by Formula II. The
counterion is K.sup.+. Com- pound 29 ##STR00022## The dye is a
xanthene derivative that is attached to the metal-chelating moiety
(Formula IV) by a linker that is a single covalent bond. The
counter ion is K.sup.+.
Binding Solution
[0164] The present binding solution comprising: [0165] a) a metal
chelating moiety; [0166] b) a salt comprising trivalent metal ions,
wherein said metal ion is capable of simultaneously binding said
metal chelating moiety and a phosphorylated target molecule; and,
[0167] c) an acid.
[0168] The metal chelating moiety is optionally attached to a
label, a reactive group or a combination thereof wherein the metal
chelating moiety is typically selected from the group consisting of
BAPTA, IDA, DTPA and phenanthrolines. However, any metal chelating
moiety described above, specifically or generically, is considered
part of the invention to be used in the present binding solution.
The label is typically a member selected from the group consisting
of a dye, an enzyme and a hapten and the reactive group is
preferably a photoactivatable group. In a preferred embodiment the
dye is selected from the group consisting of a benzofuran, a
quinazolinone, a xanthene, an indole, a benzazole and a
borapolyazaindacene provided that said dyes are not sulfonated.
[0169] Thus, in a preferred embodiment, the binding solution of the
present invention comprises the following components: [0170] a) a
phosphate-binding compound having formula (A)m(L)n(B) wherein A is
a chemical moiety, L is a linker, B is a metal-chelating moiety, m
is an integer from 1 to 4 and n is an integer from 0 to 4; [0171]
b) a salt comprising metal ions; and, [0172] c) an acid.
[0173] The binding solution can be prepared in a variety of ways,
which are dependent on the method and the medium in which the
sample is present, as described below. In a preferred embodiment,
the binding solution comprises a phosphate-binding compound having
formula (A)m(L)n(B), a salt comprising a metal ion and acid in an
aqueous solution sufficient to adjust the pH of the binding
solution to 3-6; optionally the binding solution comprises an
organic solvent or a mixture of organic solvents and additional
ionic or nonionic components, e.g. sodium chloride. Any of the
components of the binding solution can be added together or
separately and in no particular order and, as will become evident,
the phosphate-binding compound may be immobilized on a solid or
semi-solid matrix, wherein the metal ion and acid are added to the
matrix to form the binding solution of the present invention.
Therefore, the phosphate-binding compounds do not need to be free
in the binding solution to form the solution but may be immobilized
on a solid or semi-solid matrix surface. We have found that
depending on the method, i.e. detection and isolation, that the
concentration of metal ion and phosphate-binding compound needs to
be adjusted.
[0174] Soluble phosphate-binding compounds are prepared by
dissolution in a solvent, such as water, DMSO, DMF or methanol,
usually at a final concentration of about 0.1 .mu.M to 10 .mu.M;
preferably, the phosphate-binding compound is present in the
binding solution at a concentration of about 0.5 .mu.M to 5 .mu.M
and most preferably at a concentration of about 1.0 .mu.M. However,
in applications in which the binding solution is used to
precipitate phosphorylated target molecules from solution, a higher
concentration of phosphate-binding compounds in the binding
solution is desired--preferably about 0.05 mM to 1 mM. For
precipitation purposes that concentration is increased but the
ratio of phosphate-binding compound to metal ion is comparable to
the ratio of the binding solution used for detection purposes.
[0175] The metal ion-containing salt preferably contains trivalent
gallium ions, such as is prepared from gallium chloride, but can be
any gallium salt known to those skilled in the art. Alternatively
iron and aluminum ions also find use in the binding solution of the
present invention. Gallium salts that can be used with the present
invention include, without limit, acetylacetonate, arsenide,
bromide, chloride, fluoride, iodide, nitrate, nitride, perchlorate,
sulfate and sulfide. The gallium salt is typically present in the
binding solution at a concentration of about 10 nM to about 1 mM;
preferably the concentration of the gallium salt is about 0.5 .mu.M
to 10 .mu.M. However, for precipitation purposes, the gallium salt
is preferably present at a slightly higher concentration of about
0.1 mM to about 0.5 mM.
[0176] Analysis of the stability and specificity of the
phosphate-binding compounds for gallium ions and the gallium ions
for the phosphorylated target molecules was evaluated as a function
of pH (Example 1). Based on these results, it was determined that a
preferred binding solution comprises an acid to provide a
moderately acidic environment for the binding reaction. In fact, an
important and unexpected aspect of the present invention is that
metal-chelating groups bind trivalent cations such as gallium in a
moderately acidic environment, resulting in a titration of
fluorescent signal with an increase in pH level approaching neutral
pH. An acidic environment is defined as a solution having a pH less
than 6.9. Typical suitable acidic components include without
limitation acetic acid, trichloroacetic acid, trifluoroacetic acid,
perchloric acid, or sulfuric acid. The acidic component is
typically present at a concentration of 1%-20% and is buffered to
the appropriate pH by a base. The pH of the binding solution is
preferably about pH 3-6 and most preferred is about pH 4.0. Acetic
acid is a preferred acid for use at or near pH 4. The optimal pH
for each compound used may vary slightly depending on the compound
used; for Compound 2, pH 4.0 is preferred.
[0177] The pH of the binding solution is optionally modified by the
inclusion of a buffering agent in addition to the acidic component.
In particular, we have shown that the presence of a buffering agent
unexpectedly improves binding of phosphorylated target molecules
immobilized in electrophoresis gels, provided that an alcohol is
also included in the formulations. Any buffering agent that
maintains an acidic environment and is compatible with the
phosphorylated target molecules in the sample is suitable for
inclusion in the binding solution.
[0178] Useful buffering agents include salts of formate, acetate,
2-(N-morpholino)ethanesulfonic acid, imidazole,
N-(2-hydroxyethyl)piperazinylethanesulfonic acid,
tris-(hydroxymethyl)aminomethane acetate, or
tris(hydroxymethyl)aminomethane, hydrochloride, wherein the
buffering agent does not chelate gallium ions. An exemplified
buffering agent is sodium acetate. The buffering agent is typically
present in the binding solution at a concentration of about 20 mM
to 500 mM; preferably the concentration is about 50 mM to 200
mM.
[0179] Inclusion of a water-miscible organic solvent, typically an
alcohol, in the binding solution is recommended when the binding
solution contains a pH-buffering agent and a salt. Although the use
of highly polar solvents such as formamide is permitted, typically,
the polar organic solvent is an alcohol having 1-6 carbon atoms, or
a diol or triol having 2-6 carbon atoms. A preferred alcohol is
1,2-propanediol. The polar organic solvent, when present, is
typically included in the binding solution at a concentration of
5-50%. The presence of a polar organic solvent is particularly
advantageous when binding sodium dodecyl sulfate (SDS)-coated
proteins, as is typically the case when binding phosphorylated
proteins or peptides that have been electroblotted from
SDS-polyacrylamide gels. Typically, in the preferred procedure, SDS
is removed from a gel or blot prior to addition of the binding
solution by fixing and washing; however, some SDS may remain and
can interfere with the binding methods of the present invention.
Without wishing to be bound by any theory, it appears that the
presence of an alcohol improves luminescent labeling of
phosphorylated proteins or peptides by removing any SDS that was
not removed by washing or fixing the sample. However,
nitrocellulose membranes may be damaged by high concentrations of
alcohol (for example, greater than about 20%), and so care should
be taken to select solvent concentrations that do not damage the
membranes upon which the phosphorylated proteins or peptides are
immobilized.
Methods of Use
[0180] The phosphate-binding compounds of the present invention can
be used without limitation for the analysis and monitoring of
phosphorylated target molecules. In this way, phosphorylated target
molecules can be detected in unlimited assay formats that provide
information about the number of phosphate groups on the target
molecule, the identification of enzymes involved in phosphorylation
and dephosphorylation, the role that such target molecules have in
the proteome and--with further analysis--the site of attachment of
phosphate groups on the target molecules. Further analysis can be
carried out after the compounds of the present invention are used
to selectively detect and/or isolate phosphorylated target
molecules.
[0181] The methods of the present invention can be carried out on
samples that are immobilized, on samples in which the
phosphate-binding compound is immobilized or where both the sample
and phosphate-binding compounds are in solution. The binding
solution is combined with the sample in such a way as to facilitate
contact between the phosphate-binding compound, trivalent metal ion
and any phosphorylated target molecules present in the sample,
wherein formation of a ternary complex effectively binds a chemical
moiety A to the phosphorylated target molecules that are present.
When the sample is immobilized on a solid or semi-solid support,
the binding solution is typically incubated with the sample under
conditions that maximize contact, such as gentle mixing or
rocking.
[0182] The methods of the present invention for detecting
phosphorylated target molecules that have been immobilized on a gel
comprise the following steps: [0183] i) immobilizing the sample on
a gel; [0184] ii) optionally contacting the gel of step i) with a
fixing solution; [0185] iii) contacting the gel of step ii) with a
binding solution of the present invention [0186] iv) incubating the
gel of step iii) and the binding solution for sufficient time to
allow said compound to associate with said phosphorylated target
molecule; [0187] v) visualizing the phosphate-binding compound
whereby said phosphorylated target molecule is detected; and,
[0188] vi) optionally, a second (or third) stain is added to the
gel to detect either total protein or proteins of another class,
such as glycoproteins, or both.
[0189] Typically, immobilizing the sample on a gel comprises
electrophoretically separating the sample. The gel, without limit,
includes any gel known to one of skill in the art for separating
target molecules from each other, including polymer-based gels such
as agarose and polyacrylamide wherein an electrical current is
passed through the gel and the target molecules migrate based on
charge and size. Thus, gels (reduced and native) also include both
one and two-dimensional gels, and isoelectric focusing gels.
Capillary electrophoresis may be employed using gels, solutions
containing polymers, or solutions alone.
[0190] Optionally, a sample separated on a gel may be transferred
to a polymeric membrane, using techniques well known to one skilled
in the art, wherein the membrane is then contacted with a binding
solution of the present invention to selectively detect
phosphorylated target molecules. A method of the present invention
for detecting phosphorylated target molecules immobilized on a
membrane comprises the following steps: [0191] i)
electrophoretically separating the sample on a gel; [0192] ii)
transferring the separated sample to a membrane; [0193] iii)
optionally contacting the membrane of step ii) with a fixing
solution; [0194] iv) contacting the membrane of step iii) with a
binding solution; [0195] v) incubating the membrane of step iv) and
the binding solution for sufficient time to allow the compound to
associate with the phosphorylated target molecule; and, [0196] v)
visualizing the compound, whereby said phosphorylated target
molecule is detected. [0197] vi) Optionally, a second (and/or
third) stain is added to the membrane to detect either total
protein or proteins of another class, such as glycoproteins.
[0198] Protein gel electrophoresis is typically performed using SDS
as a component of either the sample preparation or in the running
buffer. However, SDS interferes with the binding solution of the
present invention and therefore must be removed from the gel or
membrane prior to addition of the binding solution. Gels and
membranes are fixed and washed, which results in the removal of
most or all of the SDS from the gels or blots. A preferred fixing
solution for gels and membranes comprises methanol and acetic acid;
optionally the fixing solution comprises glutaraldehyde. The
methanol is present at a concentration of about 35-50% and the
acetic acid is present at about 0-15% and the glutaraldehyde is
present at about 0-2%. Typically, washing the gels or membranes
with 100% water follows fixing.
[0199] However, for purposes of the invention, the binding solution
also detects phosphorylated target molecules that have been
separated on a native or non-reduced gel. Therefore, for methods
utilizing these gels that do not contain SDS, the fixing solution
step is not necessary.
[0200] After samples have been separated on a gel or transferred to
a polymeric membrane, optionally fixed, and washed, the gel or blot
is incubated with a binding solution (Examples 2-9). The
phosphorylated proteins or peptides are incubated with the binding
solution for a time sufficient for the phosphate-binding
compound/metal ion complex to bind to the phosphorylated proteins
or peptides that are present. Preferably, this time is not more
than 24 hours, more preferably this time is less than 8 hours and
most preferably this incubation time is less than 2 hours, but not
less than 5 minutes. After incubation with the binding solution the
gels or membranes are typically washed with a mixture that
preferably comprise an acidic buffering agent and acetonitrile;
useful buffering agents to be used with the present invention
include, without limitation, NaOAc, formate and
2-(N-morpholino)ethanesulfonic acid. Typically, the buffering agent
is present in the washing solution at a concentration of about 25
mM to about 100 mM. In addition, it has been found that optional
inclusion of acetonitrile in the washing solution usually reduces
non-specific labeling. Preferably, acetonitrile is present at a
concentration from 1-7%, more preferably 3-4%. An alternative
washing solution is comprised of 10-20% 1,2-propanediol.
[0201] Thus, following binding of the phosphate-binding compound
and washing, the ternary complex can be illuminated directly when
the phosphate-binding complex comprises a fluorophore or
chromophore label, as described above, to visualize the
phosphorylated target molecules. Alternatively the presence and
location of the phosphorylated target molecule on the blot can be
detected using antibodies to the label, such as anti-BAPTA
antibody, an anti-fluorophore antibody, an anti-hapten antibody or
an avidin (when the label is a biotin derivative), which is then
detected by standard means used to detect proteins on Western blots
such as by fluorescence, chemiluminescence or radioactivity,
indicating labeling of the phosphorylated target molecules.
[0202] The phosphate-binding compounds of the binding solution are
chosen depending upon their ability to bind phosphorylated target
molecules in different media. Therefore, preferred
phosphate-binding compounds for binding phosphorylated target
molecules in a gel include compounds 1-4 and 7-11 of the present
invention. Preferred compounds for binding phosphorylated target
molecules on a membrane include compounds 1, 4 and 7 of the present
invention.
[0203] A particular advantage to identifying phosphorylated
proteins or peptides in a 2-D gel is the ability to correctly
identify the phosphoproteome, as well as to quantitate
post-translational modification of proteins for the addition or
subtraction of phosphate groups. Specifically, labeling of
phosphorylated proteins or peptides while doing concurrent, or
subsequent, total protein staining identifies the phosphorylated
proteome, while the intensity of the signal can be correlated to
the level of phosphorylation, when compared to the total protein
stain (see, Examples 6, 7 and 13). Any fluorescent dye specific for
total proteins can be used to stain total proteins in the gel; a
preferred stain is SYPRO.RTM. Ruby dye for gels or any dye
disclosed in U.S. Pat. No. 6,316,276 B1. Other fluorescent dyes
such as MDPF and CBQCA could also be used for detection on
membranes. Because SDS is removed by washing prior to staining with
the staining mixture of the present invention, total protein stains
such as SYPRO.RTM. Ruby dye are preferred because SDS is not
critical for their staining function. However, protocol changes can
be made when using a stain that requires SDS for staining
sensitivity, such as SYPRO.RTM. Orange dye, SYPRO.RTM. Red dye and
SYPRO.RTM. Tangerine dye, by adding SDS back to the gel prior to a
total protein stain step and including SDS in the staining solution
for the total protein stain (Malone et al. Electrophoresis (2001)
22(5):919-32). A preferred mixture for returning SDS back to a gel
is 2% acid/0.0005% SDS, and optionally 40% ethanol, wherein the gel
is incubated for at least one hour. Alternatively, the total
protein stain can be performed prior to the phosphorylated target
molecules staining of the present invention; therefore, in this
case, it is not necessary to add back the SDS to the gel, but
simply to remove the SDS prior to the phosphorylated target
molecule staining step, as contemplated by the present invention.
Therefore, alternative preferable total protein stains for gels
include but are not limited to, SYPRO.RTM. Orange dye, SYPRO.RTM.
Tangerine dye and SYPRO.RTM. Red dye or any dye disclosed in U.S.
Pat. No. 5,616,502 or U.S. Ser. No. 09/632,927. Alternative, but
less preferred, total protein stains for gels include Coomassie
Blue or silver staining, which utilize staining techniques well
known to those skilled in the art. Alternative total proteins
stains useful for staining blots are SYPRO.RTM. Rose Plus dye and
DyeChrome.TM. dye or any dye solution disclosed in U.S. Pat. No.
6,329,205 B1 and U.S. Ser. No. 10/005,050.
[0204] Another very important advantage when labeling
phosphorylated target molecules in a 2-D gel is to include a stain
for glycoproteins, wherein a 3-way analysis of the proteome could
be accomplished (Steinberg et al., "Rapid and Simple Single
Nanogram Detection of Glycoproteins in Polyacrylamide Gels and on
Electroblots," Proteomics 1:841-855 (2001)). A preferred
glycoprotein stain is Pro-Q.TM. Emerald 300 dye or Pro-Q.TM.
Emerald 488 dye, Pro-Q.TM. Fuchsia dye or any other dye disclosed
in U.S. Ser. No. 09/970,215. In addition, if the sample comprises
fusion proteins with oligohistidine affinity peptides, Pro-Q.TM.
Sapphire 365 or 488 dye can be used to simultaneously detect these
proteins or peptides.
[0205] Thus, it is particularly advantageous that the parallel
determination of both protein expression levels and functional
attributes of the proteins such as phosphorylation of proteins can
be achieved with the present invention within a single 2-D gel
electrophoresis experiment. Analysis can be accomplished by using
image analysis software, e.g., Compugen's Z3 program or Phoretix
Progenesis software. Any two images can be re-displayed, allowing
visual inspection of the differences between the images, and
quantitative information can be readily retrieved in tabular form
with differential expression data calculated.
[0206] Alternatively, single-dimension polyacrylamide and
corresponding blots can be simultaneously or subsequently stained
for total proteins or glycoproteins using staining techniques and
dyes described above. A particular advantage for counterstaining a
gel or blot that has been labeled using methods of the present
invention is the ability to distinguish between nonspecific
labeling and labeling of phosphorylated target molecules with a low
number of phosphate groups. This is important for accurately
identifying phosphorylated target molecules that have undergone a
small change in the degree of phosphorylation. Counterstaining a
blot or gel with a total protein stain such as SYPRO.RTM. Ruby
permits a ratiometric analysis of the fluorescent signal generated
from the dyes of the present invention compared to the fluorescent
signal generated from a total protein stain (see, FIG. 11 and
Example 22). This ratiometric analysis also permits the
stoichiometry determination of the phosphorylated target molecule
relating to the overall phosphorylation state of the molecule as
well as the addition or subtraction of phosphate groups.
[0207] Another particular advantage for staining phosphorylated
proteins or peptides separated in polyacrylamide gels is for the
analysis of proteins of interest by combining spot detection with
the compounds of this invention with mass spectrometry techniques
for further analysis. For example, because phosphoproteins may
co-migrate in a gel, further analysis may be essential or desired
to specifically identify and analyze the phosphoprotein of
interest. This further analysis can be achieved by measurement of a
set of peptide masses derived from a protein, i.e., by peptide
mapping with mass spectrometry (MS), or by obtaining amino acid
sequence information from individual peptides, i.e., protein
sequencing by MS/MS or by Edman degradation. Thus, a protein band
or spot, once identified using the compositions and methods of the
present invention, may be excised from the gel, rinsed, optionally
reduced and S-alkylated, and then digested in situ in the gel with
a sequence-specific protease, e.g., trypsin, using standard
protocols. See Shevchenko et al., "Mass Spectrometric Sequencing of
Proteins from Silver Stained Polyacrylamide Gels," Anal. Chem.
68:850-58 (1996). The peptide mixture thus generated may be
extracted from the gel and analyzed by MS, using standard
protocols. Peptide mapping by matrix-assisted laser
desorption/ionization (MALDI) mass spectrometry is often most
sensitive. Methods for the in-gel digestion of proteins are
described in Jensen et al., "Mass Spectrometric Identification and
Microcharacterization of Proteins From Electrophoretic Gels:
Strategies and Applications," PROTEINS: Structure, Function, and
Genetics Suppl. 2:74-89 (1998).
[0208] DNA-binding proteins are key to the regulation and control
of gene expression, replication and recombination. The
electrophoretic mobility shift assay (or gel shift assay) is
considered an essential tool in modern molecular biology for the
study of protein-nucleic acid interactions. Nucleic acids could be
detected with SYBR.RTM. Green II dye, while phosphoproteins are
subsequently detected by methods described in this invention. All
fluorescence staining steps would be performed after the entire
gel-shift experiment is completed, so there is no need to pre-label
either the DNA or the protein and no possibility of the fluorescent
reagents interfering with the protein-nucleic acid interactions. A
third total protein stain might be employed as well, such as
SYPRO.RTM. Ruby dye. In this way the influence of protein
phosphorylation on DNA-binding may be measured. The ability to
independently quantify each molecular species allows more rigorous
data analysis methods to be applied, especially with respect to the
mass of phosphoprotein bound per nucleic acid.
[0209] The present invention is also contemplated to be used in a
wide range of microarray formats, including but not limited to the
methods and arrays disclosed in US Patent Application 2002/0076727;
US Patent Application 2002/0106785; US Patent Application
2002/0055186; WO 99/39210; WO 00/63701; WO 02/25288; WO 01/18545,
WO 00/04380 and U.S. Pat. Nos. 6,403,368; 6,475,809; 6,365,418;
6,409,921; 5,595,915; 6,461,807; 6,399,299. Phosphorylated target
molecules immobilized on an array such as a HydroGel-coated slide
including those disclosed in U.S. Pat. Nos. 6,372,813; 6,391,937;
6,387,631; 6,413,722 and those manufactured by Perkin Elmer; can
also be detected using the methods and compositions of the present
invention (Examples 18 and 19). Alternatively, phosphate-binding
compounds can be immobilized on these arrays.
[0210] The methods of the present invention for detecting
phosphorylated target molecules on an array typically comprise the
steps of: [0211] i) immobilizing said sample on an array; [0212]
ii) contacting said array of step i) with a binding solution,
[0213] iii) incubating said array of step ii) and said binding
solution for sufficient time to allow said compound to associate
indirectly with said phosphorylated target molecule; and, [0214]
iv) illuminating said compound with a suitable light source whereby
said phosphorylated target molecule is detected.
[0215] The sample is immobilized on the array using techniques well
known to one skilled in the art, including but not limited to,
using a piezo array printer, contact printer or other array printer
technology, immobilizing a phosphorylated target molecule, binding
molecule such as an antibody and then added the sample to
non-covalently bind the phosphorylated target molecules to the
array. Typically the array comprises molecules that covalently
attach the sample, or a protein that selectively binds the sample,
such as an amine-reactive group.
[0216] The array is incubated with a binding solution for
sufficient time to form a ternary complex between a
phosphate-binding compound, the metal ion (typically gallium) and
phosphorylated target molecule. Alternatively, the array may
comprise phosphate-binding compounds complexed with the metal ions
immobilized on the surface of the array, wherein a sample is
incubated with the array and detection of phosphorylated target
molecules occurs when the target molecules bind the metal
ion/phosphate-binding complex and are typically illuminated,
unbound sample is washed away. In this way, an assay to detect
phosphatases or kinases is performed with an appropriate peptide or
protein substrate and the resulting phosphorylated or
dephosphorylated peptides or proteins are spotted or synthesized on
the array, wherein phosphate groups on the peptides bind the
phosphate-binding compounds/metal-ion complex on the array.
Alternatively, a kinase and/or phosphatase substrate is spotted or
synthesized on the array and then the enzyme, kinase (and ATP) or
phosphatase, is added to the array. After removing the enzyme, the
array is then contacted with the binding solution. In this way, the
array is used to detect and/or isolate phosphorylated target
molecules and to identify the enzymes responsible for adding and/or
removing phosphate groups from target molecules and their
efficiency in doing so.
[0217] Typically phosphatase and kinase peptide substrates are
immobilized on an array by spotting or synthesis using standard
protocols, the phosphates or kinase enzymes, either comprise an
unknown sample or are isolated enzymes, are added and subsequent
presence of phosphate groups is detected using a binding solution
of the present invention. Thus, the methods and binding solution of
the present invention are useful, for example, with arrays of
protein substrates for various protein kinases (e.g., myosin light
chain, MARCKS, myelin basic protein, casein, src-supressed C kinase
substrate, insulin Receptor Substrate 1, Nuclear factor 90, Rap1,
transcription factor stat5a). A sample comprising phosphatase or
kinase enzymes is incubated with the array comprising enzyme
substrate; following incubation under appropriate conditions and
with appropriate reaction additives for the enzymes the
phosphorylated products can be detected with a binding solution of
the present invention. When the detectable label is a fluorophore,
for example, the coordinates of the fluorescent signals provides a
read-out of the kinases present in the fluid and their activity
against the various enzyme substrates (peptides or proteins) on the
array. Detection of phosphorylated target molecules with an array
offers many possibilities and the above description is not meant to
limit how the present invention can be used in combination with
array technology.
[0218] Current commercial kinase assays are often time-consuming
and require many steps such as electrophoresis, centrifugation,
ELISA or immunoprecipitation. The present invention provides
methods for the rapid, sensitive, and non-radioactive detection of
a variety of selected kinases and phosphatases and provides, in
addition, methods that are well suited for high-throughput
screening. The kinase and phosphatase assays of the present
invention also permit the screening of inhibitors and activators
of, for example, tyrosine kinases and, in addition, also permit the
monitoring and the purification of kinase and phosphatase enzymes.
Moreover, detection of the enzyme substrate on the array makes the
methods of the invention far more sensitive than any known
solution-based assays for kinases and phosphatases and use of
fluorescence or chemiluminescence for detection on the array
permits a higher density of labeling than is possible with
radiochemical detection.
[0219] As described above, a kinase substrate is covalently or
non-covalently attached to a surface, solid or semisolid matrix
including a microwell plate, polymeric beads or an array such as a
HydroGel array slide (or amine microarray substrate, aldehyde
microarray substrate, an epoxy microarray substrate, a
poly-L-lysine microarray substrate or other polyacrylamide
microarray substrates) and the assay is performed in a
non-continuous heterogeneous manner. The kinase substrate comprises
a kinase consensus phosphorylation site, preferably a peptide or a
random polymer (poly(Glu:Tyr), poly(Glu:Ala:Tyr). Optionally the
kinase substrate comprises a fluorophore. A sample suspected of
containing a kinase is combined with the kinase substrate, along
with ATP, wherein an active kinase enzyme will add phosphates to
the kinase substrate. The addition of phosphate groups is measured
after removal of the kinase solution and adequate washing, wherein
a binding solution, as described above, is added to the kinase
substrate. Typically the phosphate-binding compound comprises a
fluorophore and the kinase activity is measured by illuminating the
fluorophore. Alternatively, the phosphate-binding compound
comprises an enzyme such as peroxidase, wherein the kinase activity
would be measured after addition of the appropriate enzyme
substrate and detection with a fluorometers or an instrument to
measure color or chemiluminescence. In addition, using an inhibitor
of the selected kinase or phosphatase in the assay, for example, by
using sodium orthovanadate may enhance the specificity of the
kinase. Furthermore, the assay methods of this invention can be
used to screen for inhibitors or activators of kinases and/or
phosphatases. Alternatively, the assay is easily adaptable to
measure phosphatase activity wherein the phosphatase substrate,
phosphorylated peptides or proteins, would be bound to a solid or
semi-solid matrix such as a microwell plate, polymeric particle or
a hydrogel.
[0220] The materials and methods of the present invention may also
be used to detect and/or quantitate kinases or phosphatases by
employing a FRET-based assay. For example, a peptide labeled with a
fluorophore can be combined with the phosphate-binding
compound/metal-ion (typically gallium) complex derivatized with a
phycoerythrin or other dye label. When the peptide is
phosphorylated, the peptide binds the dye labeled phosphate-binding
compound and the emission maximum shifts in the assay.
Time-resolved fluorescence can be achieved, for example, by
employing a europium-based chelate on the peptide and the
phosphate-binding compound/metal ion complex derivatized with
allophycocyanin. The donor fluorophore can be excited, in this
example, at 335 nm and an emission shift from 620 nm to 665 nm
indicates peptide interaction with the metal-chelator and gallium
complex.
[0221] Thus, in one aspect of the invention, numerous enzymes,
including nitrogenase, phosphoribosyl-pyrophosphate synthetase,
undecaprenyl pyrophosphate synthase, DNA polymerases, RNA
polymerases, farnesyltransferase, nucleoside triphosphate
pyrophosphohydrolases, pyrophosphate-fructose 6-phosphate
1-phosphotransferase (PFPPT), sulfate adenyltransferase,
UTP-glucose 1-phosphate uridinyltransferase (UGPP), asparagine
synthetase, and UDP-glucose pyrophosphorylase involve the
metabolism of inorganic pyrophosphate and thus are potential
targets for quantitation by the disclosed invention.
[0222] In addition, the methods and materials of the present
invention are also useful for studying functional proteomics
involving ligand overlay methodology. For example, arrayed proteins
would be detected after incubation with phosphatidylinositol
4,5-bisphosphate (PIP2) micelles, followed by incubation with the
binding solution. The differences in labeling would highlight an
important class of phosphatidylinositide-binding proteins. Proteins
such as SWI/SNF-like BAF, a chromatin remodeling complex and
cofilin/ADF, a ubiquitous actin-binding protein, are likely to be
identified using the methods of the present invention (Rando et al.
Proc Natl Acad Sci USA 99(5):2824-9 (2002); Ojala et al.
Biochemistry 40(51):15562-9 (2001)). Another example of a ligand
overlay assay would be GTP-binding proteins, wherein the small
GTP-binding proteins can be separated by high-resolution 2-D gel
electrophoresis and subsequently transferred under renaturing
conditions to a nitrocellulose or PVDF membrane and probed with
GTP. The bound GTP would then be subsequently bound with the
binding solution of the present invention, resulting in
identification of GTP-binding proteins. A variety of other membrane
overlay nucleotide-binding assays could be preformed using the
binding solution of the present invention, wherein potentially any
ligand and binding protein, wherein at least one of the pair
contains phosphate group(s), could be used to identify novel
binding proteins (Gromov et al. Electrophoresis (1994)
3-4:478-81).
[0223] In contrast to having either the phosphorylated target
molecules or the phosphate-binding compound immobilized, the
compositions and methods of the present invention are also useful
for binding, detecting and isolation of phosphorylated target
molecules that are free in a solution. A sample suspected of
containing phosphorylated target molecules is incubated with the
binding solution comprising fluorescent dye-labeled
phosphate-binding compounds wherein phosphorylated target molecules
are detected by fluorescence polarization (Example 14) or energy
transfer (Example 51).
[0224] Fluorescence polarization is based upon the finding that the
emission of light by a fluorophore may be depolarized by rotational
diffusion, or the rate at which a molecule tumbles in solution (J.
Phys. Rad. 1:390-401 (1926)). Polarization is the measurement of
the average angular displacement of the fluorophore, which occurs
between the absorption and subsequent emission of a photon. This
angular displacement of the fluorophore is, in turn, dependent upon
the rate and extent of rotational diffusion during the lifetime of
the excited state, which is influenced by the viscosity of the
solution and the size and shape of the diffusing fluorescent
species. If viscosity and temperature are held constant, for
example, then fluorescence polarization is directly related to the
molecular volume or size of the fluorophore. Thus, when detecting
phosphorylated target molecules in solution, the compounds and
methods of the present invention contemplate taking advantage of
fluorescent polarization, as described in U.S. Pat. No. 6,207,397.
The detection of phosphorylated target molecules would be based
upon the observation that changes in polarization occur when a
fluorescent dye-labeled phosphate-binding compound undergoes a
molecular weight change due to the binding of a phosphorylated
target molecule, for example, a phosphoprotein. The solution
containing the sample and binding solution are irradiated with
plane-polarized light of a wavelength that is sufficient to excite
the fluorophore. The light subsequently emitted by the fluorescent
phosphorylated target molecule is polarized to varying degrees,
depending on the molecular size of the fluorescent dye. In the
unbound state in solution, low molecular weight labeled
phosphate-binding compounds will rotate rapidly, and give low
polarization readings. The degree of polarization of the emission
can be measured without the necessity to separate the components in
the solution. See, FIG. 10.
[0225] As discussed above for the kinase assay, FRET or energy
transfer between a first dye label and a second dye label can be
employed for the detection of phosphorylated target molecules in
solution. A first dye label is employed on the phosphate-binding
compound and a second dye label is added to the sample as part of a
phosphorylated or phosphorylatable molecule. When the
phosphate-binding compound binds the dye-labeled molecule then the
first and second dye label are brought within proximity that
facilitates energy transfer. The first dye label has a first
absorption and emission spectra and the second dye label has a
second absorption and emission spectra. Energy transfer occurs
between the two dye labels when overlap between the emission of the
first dye label and the absorption of the second dye label is
present wherein the absorbed energy by the second dye label is
either quenched or re-emitted at a longer wavelength. It is
understood that the first dye label is present either as part of
the phosphate-binding compound or as part of a phosphorylated or
phosphorylatable target molecule; the same is also the case for the
second dye label.
[0226] In this instance, a method for detecting phosphorylated
target molecules in a solution sample comprises: [0227] a)
contacting said sample with a binding solution to form a combined
mixture, wherein said binding solution comprises a
phosphate-binding compound, a salt comprising trivalent metal ions
and an acid, wherein said combined mixture comprises a first dye
label that has a first absorption and emission spectra and a second
dye label that has a second absorption and emission spectra; [0228]
b) incubating said phosphate binding compound and said sample for a
sufficient amount of time for said phosphate binding compound to
bind a phosphorylated target molecule; and, [0229] c) illuminating
said sample with an appropriate wavelength whereby said
phosphorylated target molecule is detected by a change in
fluorescence signal.
[0230] This method is particularly useful when the sample contains
ATP, as is the case for many kinase assays, due to the ability to
discriminate between bound ATP and the kinase substrate. This
method is also useful when a phosphate-binding compound is employed
that does not result in an increase in signal intensity when a
phosphorylated target molecule is bound compared to when no target
molecule is bound. Thus, the use of energy transfer allows for a
homogenous assay system that does not require separation or
additional detection reagents for the detection of phosphorylated
target molecules in solution. For the detection of immobilized
target molecules this is not an issue because unbound
phosphate-binding compounds or unused ATP is washed away, leaving
only the bound phosphate-binding compounds and a resulting
detectable signal. However, we have found that for solution-based
assays both fluorescence polarization and FRET are preferred for
the detection of phosphorylated target molecules.
[0231] Thus in an aspect of the invention, the detection of kinase
enzymes in a solution based assay comprises a dye-labeled kinase
substrate, enzyme and ATP combined with a present binding solution
wherein the phosphate-binding compound comprises a metal chelating
moiety that is covalently bonded to a dye label. The
phosphate-binding compound may still bind the free ATP but there
will be no shift in signal, in this instance, a shift only occurs
when a phosphate-binding compound binds a phosphorylated
dye-labeled kinase substrate. This same methodology can be applied
to phosphatase substrates wherein the removal of phosphate groups
from the substrate results in a signal from the first and second
dye label as would be observed without energy transfer. It is
advantageous that this assay be observed at several set time
intervals to observe the change in fluorescent signal and for a
baseline signal. See, FIG. 12.
[0232] In a further aspect of the invention, the phosphate-binding
compounds that comprise a reactive group can be used to covalently
attach a present phosphate-binding compound to a phosphorylated
target molecule. The phosphate-binding compounds may further
comprise a present label. In this instance, a binding solution is
incubated with a phosphorylated target molecule for a sufficient
amount of time for the phosphate-binding compound to bind the
target molecule. The covalent bond forms when an appropriate
reactive group is brought into proximity with a compatible reactive
group on the target molecule. In a preferred embodiment, the
reactive group is a photoactivatable group such that the group is
only converted to a reactive species after illumination with an
appropriate wavelength. An appropriate wavelength is generally a UV
wavelength that is less than 400 nm. This method provides for
specific attachment to only the target molecules, either in
solution or immobilized on a solid or semi-solid matrix.
[0233] Thus, a preferred method for specifically covalently
labeling phosphorylated target molecules in a sample comprises:
[0234] i) contacting said sample with a binding solution comprising
a phosphate binding compound that is covalently bonded to a label
and a photoactivatable chemically reactive group; a salt comprising
trivalent metal ions and an acid; [0235] ii) incubating said sample
and said binding solution for sufficient time to allow said
phosphate binding compound and said metal salt to associate with
said phosphorylated target molecule; [0236] iii) illuminating said
phosphate binding compound with an appropriate wavelength where
said photoactivatable reactive group specifically forms a covalent
bond with said phosphorylated target molecule whereby said
phosphorylated target molecule is labeled.
[0237] This method is particularly useful when a stronger
association is needed than can be obtained with the non-covalent
binding of the present ternary complex. In certain aspects, it is
desirable that after the formation of the ternary complex that the
pH be raised to about 7.0 instead of an acidic pH. This method
facilitates that process due to the stable formation of the
covalent bond between the chelating moiety and the target molecule.
In addition, a covalent bond is also desirable for isolation
purposes wherein perturbation of the system may lead to unstable
ternary complexes. Preferred compounds for covalently labeling
phosphorylated target molecules includes Compounds 34, 36, 39, 42
and 44.
[0238] Unexpectedly, phosphorylated target molecules, typically
peptides, can also be isolated from a complex solution by taking
advantage of the insoluble nature of the ternary complex when
higher concentrations of the more hydrophobic phosphate-binding
compounds, such as Compound 5, are used in a moderately acidic
environment with essentially equimolar metal ion
concentrations.
[0239] Methods of the present invention for isolating
phosphorylated target molecules from solution comprise the
following steps: [0240] i) contacting the sample with a binding
solution of the present invention; [0241] ii) incubating the sample
of step i) and the binding solution for sufficient time to allow
said compound to associate with the phosphorylated target molecule
to form a ternary complex; and, [0242] iii) separating said complex
from said sample, whereby said phosphorylated target molecules are
isolated.
[0243] Hydrophobic phosphate-binding compounds of the invention
when present in a binding solution at a concentration up to a
hundred times higher than a binding solution for detection purposes
typically form insoluble aggregates when the ternary complex forms.
This property of the certain hydrophobic phosphate-binding
compounds was taken advantage of to develop a method for isolation
of phosphopeptides. Thus, when a binding solution comprising
certain hydrophobic phosphate-binding compounds is incubated with a
sample in a way to facilitate formation of the ternary complex, the
complex can be precipitated out of solution by centrifugation
(Example 13). Therefore, typically the binding mixture and sample
solution is vortexed, or mixed in a manner well known to those
skilled in the art, to simultaneously facilitate binding (formation
of aggregates) and prevent precipitation of the ternary complexes.
Following formation of the ternary complex, the solution is treated
in such a way as to isolate the precipitated complexes, wherein a
preferred method is centrifugation. The resulting pellet comprises
phosphorylated target molecules that can be further analyzed, by
methods such as MS. This method takes advantage of the affinity
"pull-down" of phosphopeptides or phosphoproteins from a complex
solution (e.g., a cell extract protein digest), whereby at an
acidic pH phosphate-binding compounds can complex with metal
(typically gallium) ions and the phosphopeptides or phosphoproteins
to form a precipitate. In addition, for the methods used to
precipitate phosphorylated target molecules from solution, aluminum
ions and ferric chloride comprising iron ions can be also used for
the formation of the ternary complex.
[0244] The present invention also contemplates further isolation,
after aggregated, of the phosphorylated target molecules, wherein
the phosphate-binding compounds are removed from the phosphorylated
target compounds, resulting in a solution free of phosphate-binding
compounds. This is accomplished when the phosphate-binding
compounds optionally comprises a tag label such as a hapten,
wherein the tag label functions as a handle by which the
phosphate-binding compounds can be pulled away from the
phosphorylated target molecules. A preferred tag label is biotin
wherein a matrix comprising biotin-binding proteins would be used
as the medium to separate the phosphate-binding compounds from the
phosphorylated target molecules. Specifically, the resulting
precipitation pellet is resuspended in a solvent that disassociates
the metal ion, phosphate-binding compound and phosphorylated target
molecule complex, such as a basic solution, about pH 7-10, or
through use of a chelator such as EDTA or EGTA. The solution is
then added to a matrix, such as a column containing Sepharose beads
bound to a biotin-binding protein, wherein the phosphate-binding
compounds comprising biotin bind to the beads and the
phosphorylated target molecules pass through the column. The
resulting eluant contains phosphorylated target molecules free from
phosphate-binding compounds that may be desirable for certain
applications. Alternatively, the dissociated mixture of
phosphate-binding compounds, metal ions and phosphorylated target
molecules can be incubated with beads comprising biotin-binding
proteins as a slurry, wherein removal of the beads by gravity, such
as by size exclusion or centrifugation, results in a solution of
phosphorylated target molecules without phosphate-binding
compounds. Preferred compounds for the formation of a precipitable
ternary complex include compounds 2, 5, 9, 12, 20, 21 and 22.
[0245] In some cases, phosphate-binding compounds can be removed
from phosphorylated target molecules without the need for affinity
purification. In this way, the aggregated ternary complex is
contacted with an organic extraction buffer (Example 27). Mixing of
the pellet with an organic solvent, such as acetonitrile,
chloroform and water results in the phosphorylated peptide entering
in the aqueous phase and the phosphate-binding compound dissolving
in the organic phase.
[0246] The isolated phosphorylated target molecules can be analyzed
by a number of methods, including but not limited to, gel
electrophoresis, MALDI-TOF MS, or LC-MS/MS. Additionally, as
described below, the phosphopeptides can be derivatized using
.beta.-elimination, with subsequent addition of nucleophiles to aid
in identification of the site of phosphorylation.
[0247] In addition to isolation of phosphorylated target molecules
from a complex sample in solution, the present invention also
contemplates the isolation of target molecules by capturing the
phosphorylated target molecules using immobilized phosphate-binding
compounds (Example 15, 25 and 26). This can be done in a number of
ways and the method is exemplified using an affinity column,
ferrofluid beads and membranes; however, the methods illustrated
are not intended to be a limitation of the method.
[0248] The methods of the present invention for isolating
phosphorylated target molecules from solution using immobilized
phosphate-binding compounds typically comprise the following steps:
[0249] i) charging a matrix comprising an immobilized
phosphate-binding compound, wherein a metal-chelating moiety
comprises Formula IV, with a salt comprising metal ions; [0250] ii)
equilibrating the matrix with a moderately acidic binding buffer,
[0251] iii) adding the sample to the matrix, wherein the
phosphorylated target molecules are bound to the matrix of step
ii); and [0252] iv) eluting the phosphorylated target molecules
from the matrix, whereby said phosphorylated target molecules are
isolated.
[0253] The matrix can be any matrix known to one skilled in the
art, including polymeric membranes, polymeric particles such as
agarose, latex, magnetic or Sepharose beads, and glass, such as
slides, beads or optical fibers. The beads can be present in slurry
or as a packed column through which the sample passes and the
membranes capture the phosphorylated target molecules. An example
of such a column is an affinity matrix comprising phosphate-binding
compounds bound to agarose (for instance, Compounds 13 and 14) or a
resin (immobilized affinity column (IMAC)). Other compounds that
find use in this method include, among others, Compounds 15, 20, 21
and 22.
[0254] Unlike the precipitation method, where an affinity column or
organic extraction buffer can be used to remove phosphate-binding
compounds from the isolated phosphorylated target molecules, the
matrix in this method can optionally comprise just the
metal-chelating moiety component of the phosphate-binding compound,
which is subsequently bound with metal (preferably gallium) ions
following the addition of the metal salt. However, a
phosphate-binding compound represented by formula (A)m(L)n(B) can
form the matrix wherein A is a reactive group that is used to
attach B by way of L to the matrix material. Thus, the matrix is
charged with the metal ion, prior to addition of the sample. The
matrix is then equilibrated with a moderately acidic binding
buffer; alternatively, the metal ion and acidic binding buffer
would be present in one solution. The acidic binding buffer
typically uses the same components as the binding solution. A
sample in an acidic binding buffer is then added to the mixture,
where phosphorylated target molecules will bind the metal ions
complexed to the metal-chelating moiety. Isolation of the
phosphorylated target molecules is accomplished by an addition of a
solution, which dissociates the ternary complex of the
phosphate-binding compound (metal-chelating moiety), metal ion and
phosphorylated target molecules. Preferably, the elution solution
comprises a base and a basic pH-buffering agent. Useful bases
include, without limitation, barium hydroxide, sodium hydroxide and
ammonium hydroxide. Alternatively, basic amine solutions are also
useful elution agents. Any base that is compatible with the sample
and metal ion phosphorylated target molecule complex that
dissociates the complex is preferred. In addition, organic solvents
such as acetonitrile is useful in eluting phosphorylated target
molecules from the phosphate-binding compound matrix, and may be
preferable, depending on the subsequent analysis of the
phosphorylated target molecules, such as with MS.
[0255] As many phosphorylated target molecules often exist only in
low abundance, the isolation methods of the present invention are
especially useful for the purification and enrichment of such
phosphorylated target molecules. These methods are useful for
purifying phosphorylated peptides from crude peptide mixtures,
which is advantageous for methods that subsequently analyze the
peptides by MALDI, MS or nanoelectrospray tandem mass spectrometry
(MS/MS). It is contemplated that a wide variety of methods can be
used to prepare samples purified and/or enriched by the affinity
matrix or separated from a complex solution. For example, dried
separated phosphopeptides can be resuspended in water for LC-MS
analysis.
[0256] The IMAC of the present invention is also readily adaptable
to microfluidics applications, such as the CD technology developed
by Gyros AB (Uppsula, Sweden), wherein high-throughput screening of
samples for proteomic analysis, such as peptide mapping with
MALDI-TOF, can be accomplished. Briefly, the Gyros AB technology
comprises a CD microlaboratory with hundreds of microstructures
(columns), wherein samples are run through the columns based on
centrifugation speeds and the eluted sample is analyzed on the CD,
permitting the entire process from a protein digest to MS analysis
to be conducted on the CD. The columns can be packed with particles
that comprise BAPTA compounds (Compounds 13 or 14); samples can be
then run through the columns and either analyzed for phosphorylated
peptide concentration, by fluorescent or chemiluminescent signal,
or applied to a matrix on the CD for MALDI-TOF analysis. Thus, the
methods of the present invention are amenable to microfluidics for
high-throughput screening of samples, which is advantageous for
proteomic studies.
[0257] Thus, the invention provides analytical reagents and methods
for use with mass spectrometry-based methods for the rapid, and
quantitative analysis of phosphoproteins or phosphopeptides in a
mixture. The reagents and methods can be applied to the detection
and identification of proteins in sample mixtures of proteins,
where the peptides isolated by the method are characteristic of the
presence of a protein in the mixture. Isolated peptides or proteins
can be characterized by mass spectrometric (MS) techniques, and by
application of sequence database searching techniques for
identifying the protein from which the sequenced peptide
originates.
[0258] The following references are examples of mass spectrometric
techniques for protein identification, and can be used with the
materials and methods of the present invention: Ideker et al.,
"Integrated Genomic and Proteomic Analyses of a Systematically
Perturbed Metabolic Network," Science 292:929-934 (2001); Gygi
& Aebersold, "Measuring Gene Expression by Quantitative
Proteome Analysis," Curr. Opin. Biotechnol. 11:396-401 (2000);
Goodlett et al., "Protein Identification with a Single Accurate
Mass of a Cysteine-containing Peptide and Constrained Database
Searching," Anal. Chem. 72:1112-8 (2000); Goodlett et al.,
"Quantitative In Vitro Kinase Reaction as a Guide for
Phosphoprotein Analysis by Mass Spectrometry," Rapid. Commun. Mass.
Spectrom. 14:344-8 (2000); McLachlin & Chait, "Analysis of
Phosphorylated Proteins and Peptides by Mass Spectrometry," Current
Opin. Chem. Biol. 5:591-602 (2001); Aebersold & Goodlett, "Mass
Spectrometry in Proteomics," Chem. Rev. 101:269-295 (2001); Vener
et al., "Mass Spectrometric Resolution of Reversible Protein
Phosphorylation in Photosynthetic Membranes of Arabidopsis
thaliana," J. Biol. Chem. 276:6959-66 (2001); Zhou et al., Nature
Biotechnol. 19:375-8 (2001). Those of skill in the art will
recognize these currently available mass spectrometry methods as
compatible with the materials and methods of the present invention.
However, the present invention also contemplates that the materials
and methods can be used with mass spectrometry techniques yet to
become available that achieve the same or similar results. In
addition, it is contemplated by the present invention that, prior
to detection, phosphopeptides can be subjected to reverse phase,
normal phase or ion-exchange columns to remove undesired materials
from the phosphopeptide sample.
[0259] The present invention also contemplates alternative methods
of detection, purification and/or enrichment. For example, the
materials and methods of the present invention may be used with
Luminex technology, which involves the labeling of latex microbeads
with two fluorophores (U.S. Pat. No. 5,981,180 and U.S. Pat. No.
6,268,222). Using precise ratios of the two fluorophores, many
different bead sets can be created, each one being unique and
distinguishable in a laser beam, based on the color code that
results from the ratio of the two dyes. Instead of a capture
antibody for a specific molecule coupled to a specific bead set,
the metal-chelators of the present invention may be used, i.e.,
phosphate-binding compound and metal ion complex or biotin-binding
protein that would bind biotin-labeled phosphate-binding compounds.
However, antibodies that bind the sample could be used and the
phosphorylated target molecule could be detected with the binding
solution of the present invention. For example, after an analyte is
bound to the metal-chelator complex on the bead, a detector
antibody coupled to phycoerythrin may be used as a reporter. The
end result is an antibody/metal-chelator sandwich assay on the
color-coded microbead. The beads and the reporter molecule may be
read on a Luminex 100 instrument using a dual laser system as they
pass through a flow cell. One laser detects the beads (the color
code for an assay) and the other laser detects the reporter
signals. Thus, it is contemplated by the present invention that
instead of a detector antibody, the metal-chelating complex may be
used in accordance with the bead detection for the separation and
detection of phosphorylated target molecules.
[0260] In the alternative, magnetic bead separation for automated
bead and particle capture systems, for example, LifeSep magnetic
beads by Dexter Magnetic Technologies or Captivate ferrofluid
(Molecular Probes, Inc), may be used with the materials and methods
of the present invention (U.S. Pat. No. 6,413,420; U.S. Ser. No.
08/868,598; US Application 20020117451; U.S. Pat. No. 4,339,337;
and U.S. Pat. No. 5,834,121) or ferrofluid beads (Example 27).
Magnetic separation works by means of specific affinity coatings
attached to tiny magnetic beads, such as the phosphate-binding
compounds. Beads are mixed with a sample containing phosphorylated
target molecules such that the phosphorylated target molecules have
the opportunity to bind tightly to the metal ion/phosphate-binding
compound on the bead. Once attached, the bead and the ternary
complex can be separated using a strong magnetic field. Depending
on the process, the phosphorylated target molecule may either be
left bound to the bead or released by washing in a suitable solvent
or a basic buffer. Thus, efficient and rapid isolation is possible
and, therefore, it is contemplated by the present invention, that
the phosphate-binding compound/metal ion complex may be used with
well-known methods of magnetic bead separation.
[0261] Thus, a wide variety of materials and methods are provided
for the separation, purification and enrichment of phosphorylated
target molecules, including the novel use of an immobilized
affinity matrix.
[0262] The present invention provides compounds and methods for the
differential isolation and identification of phosphorylated serine,
threonine or tyrosine amino acids. The materials and methods
described above for the labeling and isolation of phosphorylated
target molecules, absent mass spectrometry or other similar
techniques, are generally used for detecting protein
phosphorylation, but do not give information on the specific
location of the phosphate on the protein or polypeptide. The
present invention contemplates further analyzing isolated
phosphorylated proteins or peptides obtained by immobilized
affinity matrix or precipitation methods described above to
differentially identify phosphorylated peptides. Isolated
phosphorylated proteins are subjected to proteolytic digestion,
followed by acid hydrolysis or alkaline hydrolysis and
analyzed.
[0263] A base such as barium hydroxide or sodium hydroxide
catalyzes the dephosphorylation of the peptides, forming activated
dehydroalanine derivatives, which are vulnerable to attack by amine
or thiol-containing compounds, resulting in the formation of stable
derivatives of the original phosphopeptide. These derivatives are
more hydrophobic and are therefore more amenable to identification
by HPLC, mass spectrometry, or by Edman sequencing. Under the
conditions used, phosphoserine residues undergo elimination and
addition, phosphothreonine residues undergo elimination but not
addition and phosphotyrosine residues are unaltered by the
treatment. Thus, differential identification can be accomplished
based on this knowledge. In Edman degradation, during the acid or
base delivery the phosphate is .beta.-eliminated and the resulting
dehydro-amino acids rapidly form a dithiothreitol (DTT) adduct. See
Meyer et al., FASEB J. 7:776 (1993). In contrast, O-Hex-N-Ac on
serine and threonine is stable in Edman degradation. See Gooley
& Williams, Nature 358:557 (1997). Thus, the present invention
may be used to differentiate between serine or threonine
phosphorylation and glycosylation.
[0264] Edman degradation is thus an effective method for
quantitating serine and threonine, following .beta.-elimination and
derivatization. See Yan et al., "Protein Phosphorylation:
Technologies for the Identification of Phosphoamino Acids", J.
Chromatogr. 808:23-41 (1998)). These modified products also survive
acid hydrolysis, and can be quantitated by reversed-phase HPLC
analysis. See, e.g., Meyer et al., J. Chromatogr. 397:113 (1987)
and Holmes, FEBS Lett. 215:21 (1987). Using a similar approach,
characterization by capillary zone electrophoresis and
laser-induced fluorescence has also been used to quantitate the
phosphoserine content of peptides and proteins. See, Fadden &
Haystead, Anal. Biochem. 225:81 (1995).
[0265] Nanoelectrospray MS/MS is used for phosphopeptide sequencing
for exact determination of phosphorylation sites. See Stensballe et
al., "Characterization of Phosphoproteins From Electrophoretic Gels
by Nanoscale Fe(III) Affinity Chromatography With Off-Line Mass
Spectrometry Analysis," Proteomics 1:207-222 (2001). In-gel
digestions can be achieved as described in Shevchenko et al., Anal.
Chem. 68:850-58 (1996) and Jensen et al., Meth. Mol. Biol.
112:513-30 (1998). The present invention also contemplates that the
materials and methods can be used with mass spectrometry techniques
yet to become available that achieve the same results.
[0266] The sequence of phosphopeptides and the identification of
the site(s) of phosphorylation can also be determined by a
combination of tandem mass spectrometry and computer-assisted
database search programs, such as SEQUEST (Trademark, University of
Washington, Seattle Wash.) (McCormack et al., "Direct Analysis and
Identification of Proteins in Mixtures by LC/MS/1\4S and Database
Searching at the Low-Femtomole Level," Anal. Chem. 69:767-776
(1996); Eng et al., "An Approach to Correlate Tandem Mass Spectral
Data of Peptides with Amino Acid Sequences in a Protein Database,"
J. Amer. Soc. Mass. Spectrom. 5:976-989 (1994); U.S. Pat. No.
5,538,897. While a variety of MS methods are available and may be
used in these methods, MALDI/MS and Electrospray Ionization MS
(ESI/MS) methods are typically used.
Sample Preparation
[0267] The sample is defined to include any material that may
contain phosphorylated target molecules, substrates that interact
with kinases and phosphatases, substances that interact with kinase
and phosphatase substrates and any substance that binds
phosphorylated target molecules. Typically the sample is biological
in origin and comprises tissue, a cell or a population of cells,
cell extracts, cell homogenates, purified or reconstituted
proteins, recombinant proteins, fusion proteins, bodily and other
biological fluids, viruses or viral particles, prions, subcellular
components, or synthesized peptides or proteins. Possible sources
of cellular material used to prepare the sample of the invention
include, without limitation, plants, animals, fungi, bacteria,
archae, or cell lines derived from such organisms. The sample can
be a biological fluid such as whole blood, plasma, serum, nasal
secretions, sputum, saliva, urine, sweat, transdermal exudates,
cerebrospinal fluid, or the like. Alternatively, the sample may be
whole organs, tissue or cells from an animal. Examples of sources
of such samples include muscle, eye, skin, gonads, lymph nodes,
heart, brain, lung, liver, kidney, spleen, solid tumors,
macrophages, mesothelium, and the like.
[0268] Prior to combination with the binding solution of the
present invention, the sample is prepared in a way that makes the
phosphorylated target molecules or enzyme substrates in the sample
accessible to the phosphate-binding compounds. Alternatively, the
sample may comprise enzymes or binding proteins that interact with
phosphorylated target molecules. Typically, the samples used in the
invention comprise tissue, cells, cell extracts, cell homogenates,
purified or reconstituted proteins, peptides, recombinant proteins,
biological fluids, lipids, amino acids, nucleic acids and
carbohydrates or synthesized proteins. However, the desired target
(target molecule comprising exposed phosphate groups) may require
purification or separation prior to addition of the binding
solution due to the presence of other discrete biological
components. The desired phosphorylated target molecules and other
discrete biological components can be optionally separated from
each other or from other components in the sample by mobility
(e.g., electrophoretic gel or capillary) by size (e.g.,
centrifugation, pelleting or density gradient), or by binding
affinity (e.g., to a filter membrane or affinity resin) in the
course of the present methods. For example, when the sample is to
be separated on an SDS-polyacrylamide gel, the sample is first
equilibrated in an appropriate buffer, such as an SDS-sample buffer
containing Tris, glycerol, DTT, SDS, and bromophenol blue. For
certain aspects of the invention it is preferred that the
phosphorylated target molecules not be separated before
analysis.
[0269] When starting with a sample source that is not appropriate
for separation, e.g., whole cells or tissue homogenate, the sample
needs to first be prepared, using techniques well known to those
skilled in the art. Preparation of the sample will depend on how
the phosphorylated target molecules are contained in the sample
(see e.g., Current Protocols in Molecular Biology; Herbert,
Electrophoresis 20:660-663 (1999)). For example, an optional way of
preparing samples for 2-D gel electrophoresis followed by labeling
with the compositions and methods of the present invention includes
lysing cells using a lysis buffer that ensures that the proteome,
in addition to post-translational modifications, of a sample remain
in their in vivo state throughout the entire procedure. Examples of
such buffers include ones derived from a
urea/NP-40/2-mercaptoethanol mixture. Therefore, the lysis buffer
might additionally contain phosphatase inhibitors such as sodium
orthovanadate, sodium fluoride or .beta.-glycerophosphate in
addition to a protease inhibitor cocktail.
[0270] Typically the phosphorylated peptides and proteins in the
sample have a molecular weight greater than about 500 daltons. More
typically the phosphorylated peptides and proteins are more than
800 daltons. In one aspect of the invention, the phosphorylated
proteins comprise a mixture of phosphorylated proteins with
different molecular weights that fall within a range of molecular
weights, wherein the phosphorylated proteins are used as molecular
weight standards so that labeled phosphorylated proteins or
peptides can be accurately analyzed. Samples comprising
phosphorylated peptides subjected to the methods of the present
invention can be generated from natural or synthetic samples and
may be the result of chemical, physical or enzymatic digestion of
phosphorylated protein samples. Proteins can be digested using any
appropriate enzymatic method, such as trypsin digestion. Peptides
in the digest may be preferably sized to facilitate peptide
sequencing using tandem mass spectrometric methods, and are
typically in the size range from about 10 to about 50 amino acids
in length. Alternatively, these peptides can also function as
phosphatase substrates in a method of the invention to identify
such enzymes and to measure their quantity and/or enzymatic
activity.
[0271] Samples comprising phospholipids, wherein the phospholipids
are the target molecules, are prepared with modifications compared
to samples comprising phosphoproteins prior to applying to solid or
semi-solid matrix due to their hydrophobic nature. Most samples
typically require some sort of extraction treatment prior to
binding with the compositions and methods of the present invention.
Where the phosphorylated target molecule of interest come from
tissue samples or samples from organisms having cell walls,
mechanical or chemical disruption may be required. Suitable means
are well known in the art and include, but are not limited to, the
use of a tissue homogenizer or a French pressure cell in
conjunction with, for example, organic solvent extractions. Methods
of cell disruption and fractionation are described in Ausubel et
al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons
1997). Samples may be extracted with solvents possessing varying
hydrophobic properties, and the optimal solvent is contingent upon
the nature of the phosphorylated target molecule of interest.
Extraction techniques conventional in the art that result in a
sample suitable for detection are contemplated by the present
invention.
[0272] The following references describe various extraction
techniques: Dole et al., J. Clin. Invest. 35:150-54 (1956); Dole et
al., J. Biol. Chem. 235:2595-99 (1960); Bligh et al., Canadian J.
Biochem. Physiol. 37:914-17 (1959); Folch et al., J. Biochem.
226:497-509 (1957). The Dole et al. references describe an
extraction method that involves extractions of the sample with an
isopropyl alcohol/heptane/sulfuric acid mixture, followed by
several heptane extractions. The organic phase is dried with
nitrogen for use in subsequent steps. The Folch et al. reference
describes the extraction of lipids from biological tissue
homogenates or body fluids. Samples are extracted with
chloroform/methanol, filtered and reverse-extracted with 0.1 M KCl.
The Bligh et al. reference describes the organic extraction of
lipids from biological tissue homogenates or fluids. Samples are
extracted with methanol/chloroform and chloroform, and then
filtered and reverse-extracted with water.
[0273] Typically, the phosphorylated target molecules (proteins,
peptides, carbohydrates or lipids) are present on or in a solid or
semi-solid matrix. In one aspect of the invention, this matrix
comprises an electrophoresis medium, such as a polyacrylamide gel,
agarose gel, linear polyacrylamide solution, polyvinyl alcohol gel
or a hydrogel. The solid or semi-solid matrix can also comprise a
membrane, such as a filter membrane, a nitrocellulose,
poly(vinylidene difluoride) (PVDF) membrane, or nylon membrane
wherein the phosphorylated target molecules are immobilized on the
membrane by blotting, spotting, electroblotting (tank and
semi-dry), capillary blotting or other methods of application well
known to those skilled in the art. In accordance with the present
invention, a solid and semisolid matrix also includes a glass
slide, a plastic matrix (e.g., multi-well plate or pin), a glass or
polymeric bead or fiber or a semiconductor material. The
phosphorylated target molecules may be arrayed on the support in a
regular pattern or randomly. A preferred array of the present
invention is a hydrogel glass slide support, wherein the
phosphorylated target molecules of the sample are arrayed in a
regular pattern. The present invention contemplates that the
phosphorylated target molecules can be phosphorylated after
immobilization on a matrix material, wherein an enzyme substrate is
immobilized and the appropriate enzyme and phosphate is incubated
with the immobilized substrate. For certain aspects of the
invention it is preferred that the phosphorylated target molecules
be free from a solid or semi-solid matrix, i.e. not immobilized and
present in an aqueous solution as solubilized molecules.
Illumination
[0274] In a typical detection method, at any time after or during
binding with the phosphate-binding compounds of the present
invention, the sample is visualized whereby the phosphorylated
target molecule is detected. Visualization can comprise different
methods and is dependent on the chemical moiety A that is
covalently attached to the metal-chelating moiety of the
phosphate-binding compound. When the chemical moiety A is a label,
visualization typically comprises illumination with a wavelength of
light capable of exciting the reagent to produce a detectable
optical response, as defined above, and observed with a means for
detecting the optical response. Equipment that is useful for
illuminating the phosphate-binding compounds of the invention
includes, but is not limited to, hand-held ultraviolet lamps,
mercury arc lamps, xenon lamps, lasers and laser diodes. These
illumination sources are optionally integrated into laser scanners,
fluorescence-based microplate readers, standard or
minifluorometers, or chromatographic detectors. The degree and/or
location of binding, compared with a standard or expected response,
indicates whether and to what degree the sample possesses a given
characteristic, i.e., phosphorylated target molecules.
[0275] The optical response is optionally detected by visual
inspection, or by use of any of the following devices: CCD cameras,
video cameras, photographic film, laser-scanning devices,
fluorometers, photodiodes, quantum counters, epifluorescence
microscopes, scanning microscopes, fluorescence-based microplate
readers, or by a means for amplifying the signal such as
photomultiplier tubes.
[0276] The detectable optical response can be quantified and used
to measure the degree of phosphorylation of the phosphorylated
target molecule in the sample mixture. Quantification is typically
performed by comparison of the optical response to a prepared
standard or to a calibration curve. Typically, the measured optical
response is compared with that obtained from a standard dilution of
a known concentration of the phosphorylated target molecule in an
electrophoretic gel, HydroGel or on a membrane. Generally, a
standard curve must be prepared whenever an accurate measurement is
desired. Alternatively, the standard curve is generated by
comparison with a reference dye or dyed particle that has been
standardized versus the reagent-target conjugate desired.
[0277] Alternatively, stained electrophoretic gels are used to
analyze the composition of complex sample mixtures and additionally
to determine the relative amount of a particular phosphorylated
target molecule in samples. This can be accomplished, for instance,
in conjunction with determination of the number of phosphate groups
on a molecule and a total protein stain to differentiate between an
increase in the amount of protein versus an increase in phosphate
groups on a particular protein or peptide.
[0278] Inductively coupled plasma mass spectrometry (ICP-MS) is a
useful technology for the trace elemental analysis of
environmental, biological, and pharmaceutical samples. Recently,
the feasibility of directly measuring phosphorous as m/z 31 signal
liberated from .beta.-casein using laser ablation ICP-MS has been
demonstrated on electroblot membranes (Marshall, P., et al. Analyst
127: 459-461 (2002)). Though 16 pmole of the pentaphosphorylated
protein was detectable on blots, the technique was not successfully
performed on polyacrylamide gels due to very high background
signal. This was undoubtedly due to the presence of isobaric
species and overlap from adjacent species generated from the
polyacrylamide gel matrix and electrophoresis buffer components.
The detection of low concentrations of phosphorous presents several
analytical challenges for ICP-MS due to its poor ionization in the
argon ICP and the presence of interfering polyatomic species
directly at mass 31 (.sup.15N.sup.16O and .sup.14N.sup.16O.sup.1H)
and indirectly at mass 32 (.sup.16O.sub.2 and .sup.32S). ICP-MS
could be used to detect phosphoproteins stained with the methods of
this invention. The detection procedure is envisioned to involve
the following steps. First, proteins separated by gel
electrophoresis are fixed to remove the SDS. A typical fixative
would be 40% methanol/10% acetic acid. Next, gels would be stained
for phosphoproteins using the methods of the invention. Next, the
gels would be washed to remove excess stain. The more prominent
phosphoproteins could be visualized by fluorescence imaging at this
point and background staining can be minimized by inspection and
adjustment of wash times as appropriate. Gels are then dried down
and the gel is subjected to laser ablation ICP-MS by methods
similar to those described in Marshall et al, 2002. Sampling can be
performed by single or multi-spot analysis, straight-line scans or
rastering. In the case of rastering, virtual gels can be
constructed from the data obtained as described by Loo R R, et al.,
Anal Chem. 73:4063-70 (2001)). Using the ruthenium-containing
SYPRO.RTM. Ruby dye staining technology, gallium (aluminum or iron)
signal from the phosphoprotein stain, as well as ruthenium for the
total protein stain could be independently quantified.
[0279] Thus, it is contemplated by the present invention that a
wide variety of instrumentation may be used to detect the
phosphorylated target molecules, e.g., electrospray ionization
(ESI) tandem mass spectrometry (MS/MS). A series of different
techniques, including automated high performance liquid
chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS, and solid phase
extraction (SPE)-capillary zone electrophoresis (CZE)-MS/MS, are
described in Figeys et al., Electrophoresis 19:1811-1818
(1998).
[0280] When measuring fluorescence polarization, many forms of
automation may be used and are known by those skilled in the art.
As one example, any standard fluorometer equipped for polarization
experiments or measurements may be used in practicing this
embodiment of the invention to both irradiate the mixture and
measure the polarization. Wavelengths suitable to excite the
fluorophore depend on the nature of the fluorophore, as described
above. Typically, one uses cut-off filters to define a wavelength
range, which is determined by the excitation and emission
wavelengths of the fluorophore. For example, for
fluorescein-labeled peptides, one would typically use an excitation
cutoff filter of 485 nm. Standard fluorometers can be used, or, for
example, a fluorescence-based plate reader. Thus, in addition, one
of skill in the art of automation may use various instruments to
measure fluorescence polarization in accordance with the materials
and methods of the invention.
[0281] The use of
1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic
acid (TF-BAPTA) or any of the fluoride-containing phosphate-binding
molecules described in this invention with gallium for binding to
the phosphorylated target molecule could be detected using
19.sub.F-NMR spectroscopy (Doughty D A, Tomutsa L. Magn Reson
Imaging 1996; 14(7-8):869-73). Additionally, radioactive gallium-67
(half-life: 78 hr), gallium-68 (half-life: 1.13 hr) or gallium-72
(half-life: 14.1 hr) could be employed with any of the
phosphate-binding compounds of the invention to generate a
detectable signal by autoradiography or scintigraphy.
[0282] As described above, while a wide variety of methods of
detection are used with the present invention, a preferred method
includes the use of fluorescence. Fluorescence from the
phosphate-binding compound metal (preferably gallium) ion complex
simultaneously binding to the phosphorylated target molecule can be
visualized with a variety of imaging techniques, including ordinary
light or fluorescence microscopy.
Kits
[0283] Suitable kits for labeling, isolating and identifying
enzymes that interact with phosphorylated target molecules also
form part of the invention. Such kits can be prepared from readily
available materials and reagents and can come in a variety of
embodiments. The contents of the kit will depend on the design of
the assay protocol or reagent for detection or measurement. All
kits will contain instructions, appropriate reagents and
phosphate-binding compounds, separation media, and solid supports,
as needed. Typically, instructions include a tangible expression
describing the reagent concentration or at least one assay method
parameter such as the relative amounts of reagent and sample to be
added together, maintenance time periods for reagent/sample
admixtures, temperature, buffer conditions and the like to allow
the user to carry out any one of the methods or preparations
described above.
[0284] A kit for binding phosphorylated target molecules comprises
a binding reagent that is typically prepared in solution, wherein
the binding solution is identical to what was described above.
Optionally, the kit would comprise any one of the following;
molecular weight markers for both phosphorylated and
non-phosphorylated target molecules, a total protein stain and a
staining solution for glycoproteins. When the kit is used to detect
phosphorylated proteins or peptides in a gel or on a blot,
molecular weight markers are typically part of the kit.
Alternatively, when the kit is used to stain phosphorylated target
molecules in a solution or on an array, molecular weight markers
would typically not be part of the kit.
[0285] Another kit in the present invention finds use in
identifying kinases or phosphatases or measuring their activity
and/or evaluating the effect of inhibitors and activators on these
enzymes. Typically this kit would comprise appropriate substrate
immobilized on matrix material, binding solution and appropriate
controls. Alternatively the phosphate-binding compounds would be
immobilized on the matrix and the end user would supply both
substrate and enzyme. This kit would comprise a solution containing
a metal ion salt and an acid or appropriate buffer that when added
to the immobilized phosphate-binding compounds would form the
binding solution of the present invention.
[0286] Another kit of the present invention finds use in isolating
phosphorylated proteins or peptides from a complex sample mixture,
wherein the ternary complex is pulled out of solution. The kit
would optionally comprise a binding solution, elution buffer and
optionally a protein or hapten-binding support, wherein the support
could be a multiwell plate, agarose resin, polymeric microbeads or
magnetic beads, containing an appropriate affinity reagent, e.g. a
biotin- or hapten-binding protein, an antibody, a lectin, a
protein-binding nucleic acid or other biopolymer covalently
attached to the support. The kit would further optionally contain
one or more of a spin column, standard peptide mixture and
nucleophilic derivatization compound.
[0287] Another kit of the invention that finds use in isolating
phosphorylated proteins or peptides from a complex sample mixture
comprises a matrix containing phosphate-binding compounds that are
covalently attached to the matrix, typically in the form of a
column. The kit would typically comprise, in addition to the
phosphate-binding compounds immobilized on the matrix, a metal
salt, a wash buffer, a moderately acidic binding buffer, and an
elution buffer. The metal salt is preferably gallium chloride and
the elution buffer preferably comprises barium hydroxide.
[0288] Those skilled in the art will appreciate that a wide variety
of additional kits and kit components can be prepared according to
the present invention, depending upon the intended user of the kit,
and the particular needs of the user.
Applications
[0289] The present invention is useful for a wide variety of
applications in a wide variety of areas including, but not limited
to, basic research applications, high-throughput screening,
proteomics, microarray technology, diagnostics, and medical
therapeutics. Those skilled in the art will appreciate that the
invention can be used in a wide variety of assay formats in a wide
variety of diagnostic applications. The foregoing description seeks
merely to illustrate the many applications of the materials and
methods of the present invention, and does not seek to limit the
metes and bounds of the invention as described in the above
sections.
[0290] The materials and methods of the present invention are
useful for a number of applications. The present invention may be
used to generate data that are used as a reference point for a
human patient or animal sample for the diagnosis of disease,
progression of disease, and/or predisposition for disease. By way
of example, if a disease is associated with changes in protein
composition in certain cells, e.g., protein phosphorylation in
different organ systems, cell sources or tissue types, a patient
sample may be used to generate a protein profile according to the
materials and methods of the invention, and compared with profiles
of corresponding samples of normal or non-diseased samples and/or
diseased origin to determine the presence or absence of,
progression of, and/or predisposition to the particular disease in
question. It is contemplated by the present invention that many
diseases may be diagnosed with data or images generated by the
materials and methods of the present invention, including diseases
for which particular aberrations in protein expression are either
known or not known. Such disease states include, but are not
limited to, metabolic diseases that are associated with the lack of
certain enzymes, proliferative diseases that are associated with
aberrant expression of certain genes, e.g., oncogenes or tumor
suppressors, or developmental diseases that are associated with
aberrant gene expression. Thus, if it is known that a given disease
of interest is associated with certain changes of a particular type
of cell, tissue, cell source, or organ system, a human patient or
animal may be diagnosed simply based on its individual expression
profile generated by 2-D gel electrophoresis or another appropriate
separation and analysis technique such as bead-based analysis
technology developed by Luminex, or others, or evaluations done on
microarrays in accordance with any aspect of the present invention.
In another aspect, expression profiles generated by one of these
methods may be used to analyze a diseased organ, tissue or cell
type and compared with the corresponding profile counterpart
obtained from a non-diseased sample.
[0291] Moreover, the information generated by the materials and
methods of the present invention may be used to "backtrack" or
identify and/or associate novel or known genes and their
corresponding products that are involved in the manifestation of,
progression of, or predisposition to a disease of interest, and
with the development of symptoms of a particular disease, by
generating the amino acid sequence of a phosphoprotein or
phosphopeptide of interest based on the materials and methods of
the present invention. For example, ESTs are partial nucleotide
sequences obtained from cDNA derived from mRNA from any given cell
line. Thus, the present invention may be used to generate amino
acid sequence data, and from the amino acid sequence data,
extrapolate potential DNA sequences that can be used to search EST
databases. For example, MS/MS sequence data in the form of a
peptide sequence tag, may be used to query EST databases if a
protein is not identified by searching the conventional full length
sequence databases. If an EST is retrieved, then the corresponding
DNA clone can be ordered and sequenced. The apoptotic protease
FLICE/Caspase-8 and the trinucleotide repeat binding protein
p20-CGGBP was identified and cloned by this approach. See Muzio et
al. "FLICE, a Novel FADD-homologous ICE/CED-3-like Protease, is
Recruited to the CD95 (Fas/APO-1) Death-Inducing Signaling
Complex," Cell 85:817-827 (1996) and Deissler et al., "Rapid
Protein Sequencing by Tandem Mass Spectrometry and cDNA Cloning of
p20-CGGBP. A Novel Protein that Binds to the Unstable Triplet
Repeat 5'-d(CGG)n-3' in the Human FMR1 Gene," J. Biol. Chem.
272:16761-16768 (1997). During analysis of protein components
isolated from the human spliceosome, a relatively large number of
ESTs were retrieved by MS/MS data. In the alternative, it may be
necessary to generate amino acid sequence data for sequence
homology searching, e.g., by BLAST algorithm searching. If the
sequence is significantly related to a characterized protein from
another species, then its function may be directly deduced. If no
related proteins exist, however, then the amino acid sequence data
may be used to design oligonucleotide probes for cloning of the
cognant gene. Complete sequence determination of the protein can
then be performed at the DNA level by established genetic and
molecular biology techniques.
[0292] The phosphorylated target molecules can be from many
different sources, including cell types, cell conditions, genetic
background, states of perturbation or of different developmental
states. Cell sources for analysis may be transgenic or
non-transgenic, transfected or non-transfected, virus- or
prion-infected or non-infected. "Perturbation" refers to
experimental manipulation of the sources, i.e., cells, such as
treatment with a particular compound or drug compared to
non-treatment of a drug. Alternatively, it can refer to treatment
with a particular compound or drug compared to treatment of a
source or cell with a different dosage of a particular compound or
drug.
[0293] For example, cells can be subjected to a candidate drug
regimen to generate a phosphoprotein expression profile in
accordance with the present invention. The images of 2-D gels
generated in accordance with the present invention may be stored on
a database, and the database may be later queried for a cell source
representing a different treatment, e.g., protein expression
profiles generated by a response to a different drug or where no
drug is present, or where the candidate drug is used in a different
way. Moreover, the candidate drug may bind specifically to a
particular protein, permitting analysis of cells or other sources,
which express that protein. The database query may derive
information about cell sources that express a particular
protein.
[0294] Thus, the materials and methods of the present invention
could be used to gain valuable information of the effects of
various drugs and compounds on the cellular phosphoprotein status.
For example, it was demonstrated that the compounds FK-506,
cyclosporin and rapamycin, used to suppress tissue rejection,
inhibit certain protein phosphatases. Schreiber et al., Cell
70:365-68 (1992). A database of lymphoid proteins detected by 2-D
polyacrylamide gel electrophoresis has also been generated. The
database contains 2-D patterns and derived information pertaining
to polypeptide constituents of unstimulated and stimulated mature T
cells and immature thymocytes, cultured T cells and cell lines that
have been manipulated by transfection with a variety of constructs
or by treatment with specific agents, single cell-derived T and B
cell clones, cells obtained from patients with lymphoproliferative
disorders and leukemia, and a variety of other relevant cell
populations. See Hanash & Teichroew, "Mining the Human
Proteome: Experience with the Human Lymphoid Protein Database,"
Electrophoresis 19:2004-2009 (1998). Thus, in accordance with the
present invention, cells treated with a suspected drug compound can
be compared to untreated cells to generate a 2-D gel
electrophoresis profile. Furthermore, it may be observed, for
example, that certain drug compounds induce the activation of
different sets of kinases or phosphatases. Such evidence could lead
to the elucidation of the mechanism by which many drug compounds
work and manifest their effects.
[0295] A 2-D gel electrophoresis study was performed to generate a
phosphoprotein profile in cultures that were subjected to the
effect of oxygen/glucose deprivation. The results suggested that
this model could be a good method to observe the development of the
tissue and its response to an ischaemic lesion. See Tavares et al.,
"Profile of Phosphoprotein Labeling in Organotypic Slice Cultures
of Rat Hippocampus," Neurochemistry 12:2705-2709 (2001).
[0296] The materials and methods of the present invention can also
be used to study biological phenomena, such as, for example, signal
transduction, mitosis, cell proliferation, cell motility, cell
shape, gene regulation, and many other cellular processes. The
mechanism of action of kinases and phosphatases and the
physiological relevance of site-specific phosphorylation of
substrate proteins can be explored with the materials and methods
of the present invention. The materials and methods of the present
invention offer the advantage of high-resolution 2-D gel
electrophoresis to simultaneously resolve hundreds of cellular
polypeptides. Using the materials and methods of the present
invention, the potential for the identification of proteins and the
expression of their genes at various stages of cell growth,
differentiation, or disease, is extensive. Thus, the invention
provides methods and materials for the detection and quantitation
of phosphorylation of specific cellular proteins that may provide
insight into the mechanisms by which phosphorylation is employed
for the regulation in cells.
[0297] It is well known that the critical events in the cell cycle
are controlled by a complex interplay of kinases and phosphatases.
Thus, the status of phosphorylation of different protein isoforms
during different phases of the life cycle is important to
researchers. Thus, in accordance with the materials and methods of
the present invention, the phosphorylation of different proteins
related to the stage of the cell cycle related to the activity of
certain kinases or phosphatases may be explored using the materials
and methods of the present invention. By way of example, a global
analysis of phosphoproteins in cells can be used to analyze the
primary signals of, for example, mitogenesis in selected cells, or
in G1 or S phase cells. Thus, the materials and methods of the
present invention may be useful in investigating the
phosphorylation status of various proteins during the cell
cycle.
[0298] Those of skill in the art will recognize that a database can
be generated using the materials and methods of the present
invention to produce a record that may show the correlation between
gene expression at the RNA and protein level to the function of the
cell. For examples, in situations where the cells under study are
obtained in both cancerous and normal conditions, comparison of the
relative gene expression can be used to identify genes that can
serve either as diagnostic markers of pathology or as sites for the
pharmacological intervention or treatment of, for example, cancer.
Similarly, other diseases can be analyzed merely by substituting
the source of cells for analysis.
[0299] Thus, the present invention may be used to generate a
comprehensive phosphoprotein expression profile from any cell type
or biological fluid of interest. A cell type of interest may be any
cell, or portion thereof with genetic material. A reference cell
can be of any cell type in which the difference in protein
expression patterns and levels is desired to be measured.
Preferably, the cells are maintained as similar to their native
state as possible and culture techniques, incubation times etc.,
are performed identically between the two to minimize any
non-naturally occurring differences. For example, development of a
comprehensive protein profile of pre-cancerous, and/or malignant
test cells and a normal reference cell can be achieved according to
the invention. Such expression profiles can be used to characterize
molecular events, for example, related to tumor development and the
cellular mechanisms involved.
[0300] In accordance with the present invention, a cell of interest
and a reference cell could be obtained from the same patient to get
an individual phosphoprotein expression profile that can be used to
diagnose or treat that patient for those diseases that involve
protein phosphorylation. For example, when a tumor is excised, a
margin of non-transformed cells is typically removed as well.
Phosphoprotein expression profiles can help to ensure that the
cells removed all had similar profiles to normal cells rather than
the metastatic cells from the same patient for those cancers that
involve, or are thought to involve, protein phosphorylation.
[0301] One example of cell lines that may be analyzed using the
materials and methods of the present invention includes human tumor
cell lines. For example, human tumor cell lines representing a
broad spectrum of human tumors and exhibiting acceptable properties
and growth characteristics may be grown according to standard
methods for cell line expansion, cryopreservation and/or
characterization for use with the present invention. If
phosphorylation is implicated in cellular aging, the materials and
methods of the present invention may be used to analyze test and
reference cells, i.e., to develop phosphoprotein expression
profiles associated with aging, such as different stages of
ontogenesis, for example, protein profiles of embryonic
liver-derived hematopoietic stem cells. Thus, the invention
contemplates a comparison of any diseased state to a normal
reference state.
[0302] In addition, studying the effects of various ligands added
to cells can assess the effects of various agonists on the
reversible phosphorylation on multiple cellular proteins. Thus, for
example, the in vivo substrates of a kinase of interest could be
determined by treating cells with suspected substrates and
comparing the resulting gel images of 2-D separated proteins with
untreated controls. As an increasing number of cytokines are being
discovered and characterized, many or all of which will activate
protein kinases or phosphatases as they manifest their effects on
target cells, the materials and methods of the present invention
may be especially useful for exploring such mechanisms. For
example, the identity of some of these proteins may suggest assays
to be formulated for the location and characterization of kinases
and phosphatases induced by lymphokines or cytokines and lead to a
better understanding of autoimmune diseases. Methods for
identifying phosphoproteins upregulated in response to the
cytokines IL-2 or IFN-.gamma. were described using both silver
staining and Western blotting for protein detection and
identification. The silver-stained profile served as a
"fingerprint" for phosphorylation events that occur in response to
cytokine treatment. See Stancato & Petricoin III,
"Fingerprinting of Signal Transduction Pathways Using a Combination
of Anti-Phosphotyrosine Immunoprecipitations and Two-Dimensional
Polyacrylamide Gel Electrophoresis," Electrophoresis 22:2120-2124
(2001).
[0303] The materials and methods of the present invention can also
be used to map kinase and phosphatase substrates in vitro. For
example the identification of substrates for various kinases can be
determined by processing extracts from cells and allowing a
purified kinase to phosphorylate its substrate proteins. One
skilled in the art could compare all the cytosolic proteins as
candidate substrates for the kinase under investigation to identify
major substrates for a kinase of interest. Similar to in vitro
assays for kinases, it is possible to use the advantages offered by
2-D separation and assays on microarrays, in multiwell plates, in
microfluidics devices, on microbeads and using other
high-throughput assay technologies and the invention to isolate and
characterize the phosphatases that catalyze the removal of
phosphate from phosphorylated substrates. Thus, the activity of
kinases and phosphatases responsible for phosphorylating and
dephosphorylating individual proteins can be analyzed. See, e.g.,
Fruehling & Longnecker, "In Vitro Assays for the Detection of
Protein Tyrosine Phosphorylation and Protein Tyrosine Kinase
Activities," Methods in Mol. Biol. 174(Ch. 36):337-343 (2001).
[0304] The applications described herein are provided merely to
illustrate a wide variety of potential uses of the invention, and
are in no way intended to limit the scope of the invention. A
detailed description of the invention having been provided above,
the following examples are given for the purpose of illustrating
the invention and shall not be construed as being a limitation on
the scope of the invention or claims.
EXAMPLES
[0305] Generally, the nomenclature as used herein, and the
laboratory procedures in cell culture, molecular genetics, and
protein chemistry described below are those well known and commonly
employed in the art. Generally, enzymatic reactions and
purification steps are performed according to the manufacturer's
specifications. Units, prefixes, and symbols may be denoted in
their SI accepted form. Numeric ranges are inclusive of the number
defining the range and include each integer within the defined
range.
Example 1
Determination of BAPTA Selectivity for Gallium and Gallium Ions for
Phosphorylated Target Molecules and a Screening Method for
Phosphate-Binding Compounds
[0306] (A) BAPTA with Trivalent Gallium Ions Selectively Detects
Phosphoproteins.
[0307] A comprehensive search of metal-chelating compounds was
performed to identify fluorescent reagents that when combined with
a gallium salt (gallium chloride) would selectively detect
phosphorylated target molecules (particularly phosphopeptides and
phosphoproteins) in a mixture of phosphorylated and
nonphosphorylated target molecules. The compounds were tested in a
fluorescence spectrophotometer for their ability to bind gallium
(III) ion and selectively detect the phosphoprotein ovalbumin.
Binding to gallium (III) ion was determined by a fluorescence
increase of the same compound in the presence of up to 5 .mu.M
gallium chloride in 75 mM NaOAc (pH 4.0) and 140 mM NaCl. Ovalbumin
detection was also judged by a fluorescence increase; however, the
compounds were placed in a solution containing 75 mM NaOAc (pH
4.0), 140 mM NaCl, 1-4 .mu.M ovalbumin, and 0.5 .mu.M gallium
chloride. Selectivity of phosphoprotein detection was evaluated by
virtual elimination of the fluorescence increase in the presence of
the same solution lacking gallium chloride. Using compound 1, a
variety of metal ions, including iron and gallium, were screened to
determine which ion(s) were best suited for phosphoprotein
detection. Metal ions were assayed for binding to compound 1,
phosphoprotein detection, and general protein staining by
monitoring a fluorescence increase at 530 nm in 75 mM NaOAc (pH
4.0), 140 mM NaCl, 0.5-5 .mu.M metal ion, with or without 4 .mu.M
ovalbumin or 1 .mu.M lysozyme. Only trivalent cations that bound to
compound 1 resulted in a fluorescence increase at 530 nm and only
gallium (III) ion was capable of selectively indicating
phosphoproteins when bound to compound 1. Therefore, gallium (III)
ion is the most preferred metal ion for phosphoprotein detection.
This methodology was extrapolated to identify other compounds
wherein a different dye attached to the metal-chelating moiety.
(B) Differential Binding Affinity of Compound 1 for Phosphate
Compounds.
[0308] Compound 1 complexed with gallium (III) ion has differential
affinities for various phosphate substrates in 75 mM NaOAc (pH 4.0)
and 140 mM NaCl. Some of the phosphate-containing compounds studied
were inorganic phosphate, phosphate attached to a protein, a
peptide or an amino acid, pyrophosphate, ATP, and DNA. The
affinities for these phosphate-based substrates for the Compound
1/gallium (III) ion reagent were determined to be .about.50 .mu.M
for inorganic phosphate and phosphate attached to a protein, a
peptide or an amino acid, .about.200 nM for pyrophosphate and ATP,
and no binding was detected for DNA. Compare these values to the
affinity of compound 1 for gallium (III) ion of 2.5 .mu.M. Most
known phosphate compounds should fall into one of these three
categories with respect to how it will bind to BAPTA gallium (III)
ion; 1) single phosphate group (i.e., inorganic phosphate or
phosphate on a protein), 2) multiple linked phosphate group (i.e.,
pyrophosphate or ATP), or 3) bridging phosphate group (i.e. nucleic
acids).
(C) Compound 4 Displays Dual-Emission Wavelengths Upon
Simultaneously Binding to Gallium (III) Ion and Phosphate.
[0309] Concentrations of 0.1-1.0 .mu.M of compound 4 in a solution
of 75 mM NaOAc (pH 4.0) and 140 mM NaCl display a single emission
peak centered at 410 nm (excitation 350 nm). Addition of 10 nM to 1
mM gallium chloride results in a decrease in the 410 nm emission
and a concomitant increase in emission at 490 nm, with an
isosbestic point of 475 nm. The half-maximal response for this
transition from the blue to green emitting state occurs at
approximately 1.8 .mu.M gallium chloride. Therefore, 0.1 .mu.M
compound 4 with 1.7 .mu.M gallium chloride in 75 mM NaOAc (pH 4.0)
and 140 mM NaCl display both the 410 nm and 490 nm emission peaks.
The addition of phosphate can alter the equilibrium between the
emission peaks in favor of the longer wavelength 490 nm peak.
(D) Screening for Phosphate-Binding Compounds that Simultaneously
Bind Gallium and Immobilized Phosphorylated Target Molecules.
[0310] A panel of test proteins was loaded on a denaturing SDS
polyacrylamide gel, separated by electrophoresis, and the gels were
fixed with 45% methanol, 5% acetic acid. Typically the test gels
contained 500-600 ng each of myosin, .beta.-galactosidase,
phosphorylase b, ovalbumin (2 phosphates), carbonic anhydrase,
soybean trypsin inhibitor, lysozyme, aprotinin,
.alpha..sub.2-macroglobulin, phosphorylase b, glucose oxidase,
bovine serum albumin, .alpha..sub.1 acid glycoprotein, carbonic
anhydrase, avidin, and lysozyme. The gels also contained a 4-fold
dilution series of .alpha.-casein (8 phosphates), 500 ng to 2 ng
loaded. Thus the gels contained a range of proteins with different
physicochemical properties, such as proteins with hydrophobic
binding pockets (e.g. BSA), glycosylated proteins (e.g.
.alpha..sub.2-macroglobulin, glucose oxidase and avidin), acidic
proteins (e.g. soybean trypsin inhibitor), basic proteins (e.g.
lysozyme and aprotonin), and two different phosphoproteins
(ovalbumin, .alpha.-casein). The dilution series of .alpha.-casein
yielded an estimate of phosphoprotein staining sensitivity. A
selection of phosphate-binding compounds comprising different dye
labels and different chelating moieties was initially screened in
minimal binding buffers of pH 3.0 to 7.0, with a variety of metal
ions, in the presence or absence of metal ion. Dye and metal ion
concentrations ranged from 0.1 to 10 .mu.M, typically 0.3 to 3
.mu.M, and most frequently at 1.0 .mu.M. Binding conditions that
produced preferential staining of phosphoproteins typically were at
pH 3.0 to 5.5, in the presence of certain trivalent metal ions.
Under these conditions, optimal preferential phosphoprotein
staining was obtained with certain dye labels and the BAPTA
chelating moiety with an equimolar concentration of Ga.sup.3+.
Further evaluation of the successful dyes revealed that the pH
optimum was 4.0, and that addition of salt (e.g. 250-750 mM NaCl)
improved staining specificity chiefly by decreasing intensity of
staining of non-phosphoproteins. A broadened screen of dyes was
undertaken with 1 .mu.M candidate dye, 1 .mu.M of Ga.sup.3+ in 50
mM sodium acetate, pH 4.0, 500 mM sodium chloride.
(E) Binding Solution Formulation
[0311] The binding solution comprises a phosphate-binding compound
with a metal ion in molar ratios of 1:2 to 2:1 and a buffer at
about pH 3.0 to 6.0. Typically, the binding solution comprises a pH
3.0 to 5.5 buffer (50 to 100 mM), salt (e.g. 100 to 1000 mM NaCl,
or 100 to 300 mM MgCl.sub.2) and equimolar concentrations of
Ga.sup.3+ and of a phosphate-binding compound (e.g Compound 2),
typically 1 to 10 .mu.M each for detection purposes. Concentrations
of the metal ion and phosphate-binding compound are typically at
least 100 times higher concentration for isolation purposes than is
present in the binding solution for detection purposes (see,
Example 13). An optimal binding solution for a gel stain was
prepared as follows: 500 .mu.g of compound 2 was dissolved in 873
.mu.l water for a 1 mM stock solution. Five g of GaCl.sub.3 were
dissolved in 28.4 mL water for a 1 M solution, from which 1 mL was
combined with 9 mL water to make a 0.1 M solution, from which 10
.mu.L was added to 990 .mu.L water for a 1 mM stock solution. One
liter of a 1 M stock solution of sodium acetate, pH 4.0 was
prepared by dissolving 136 g of sodium acetate trihydrate in ca.
800 ml water, adjusting pH to 4.0 by adding ca. 23.5 mL 12 M HCl
and bringing volume to 1 liter. One liter of a 4 M stock solution
of sodium chloride was prepared by dissolving 233.8 NaCl in ca. 800
mL water and bringing the volume to 1 liter with water. The sodium
acetate and sodium chloride stock solutions were filtered through a
0.45 .mu.M filter. For 100 mL of binding solution, 5 ml of 1 M
sodium acetate, pH 4.0, 12.5 ml of 4 M sodium chloride, and 20 mL
of 1,2 propanediol were combined with water to a final volume of
100 mL, to which was added while stirring 100 .mu.L of 1 mM
GaCl.sub.3 and 100 .mu.l of 1 mM Compound 2 to obtain a final
binding solution of 1 .mu.M Compound 2,1 .mu.M Ga.sup.3+, 20%
1,2-propane diol, 500 mM NaCl, 50 mM sodium acetate, pH 4.0.
Example 2
Detection of Phosphoproteins in SDS-Polyacrylamide Gels
[0312] Phosphoproteins were separated by SDS-polyacrylamide gel
electrophoresis utilizing a 4% T, 2.6% C stacking gel, pH 6.8 and
13% T, 2.6% C separating gel, and pH 8.8, according to standard
procedures. % T is the total monomer concentration
(acrylamide+crosslinker) expressed in grams per 100 mL and % C is
the percentage crosslinker (e.g., N,N'-methylene-bis-acrylamide,
N,N'-diacryloylpiperazine or other suitable agent). The separating
gels were 8 cm wide by 5 cm high and 0.75 cm in thickness. After
electrophoresis, the gels were fixed by immersing them in 100 mL
45% methanol and 5% acetic acid for 90 minutes. The gels were
washed twice in water for a total of 30 minutes. The gels were then
added to a binding solution of the invention (Example 1E) and
incubated for 120 minutes at room temperature with gentle orbital
shaking, typically 50 rpm. The binding buffer contained 50 mM NaOAc
(pH 4.0), 250 mM sodium chloride, 20% v/v 1,2-propanediol, 1 .mu.M
gallium chloride. To prepare the binding solution, 120 .mu.L of a 1
mM stock solution of Compound 2 and 120 .mu.L of a 1 mM stock
solution of gallium chloride were added to 1080 .mu.L water. This
mixture was then added to 59 mL of the binding buffer to yield the
binding solution. Alternatively, the phosphate-binding compound and
the gallium chloride can be added separately, directly to 60 mL of
the binding diluent. Binding solutions that utilize other
phosphate-binding compounds of the present invention can be
prepared and similarly tested for gel staining. After incubation in
binding solution, the gel was washed with 75 mL of 50 mM NaOAc (pH
4.0) and subjected to two washes of 30 minutes each.
[0313] For Compound 2 and other dyes that can be excited at 532 nm,
images were acquired on a Fujifilm FLA 3000 laser scanner using 532
nm excitation and 580 nm bandpass emission filters. For fluorescent
phosphate-binding compounds that absorb in the ultraviolet or at
visible wavelengths below 532 nm, excitation was performed using
300 nm and detection was via Roche Lumi-Imager or Fujifilm FLA 3000
laser scanner using 473 nm excitation and 580 nm bandpass emission.
The data were displayed using Image Gauge Analysis software. Images
of phosphoproteins were displayed as dark bands. Proteins not
containing phosphate were not labeled or were very lightly stained
relative to the phosphoproteins. When gels were labeled as above
but with gallium chloride omitted from the binding solution,
phosphoproteins were not selectively stained, and could not be
distinguished from background or had very light nonspecific
staining. Gels were washed overnight with 50 mM NaOAc (pH 4.0) and
images were acquired as above. The background and nonspecific
staining was further reduced relative to phosphoprotein staining.
Replacement of gallium chloride by other gallium salts gave
comparable results with all indicators tested; however, replacement
by other metals, including Fe.sup.3+ and Al.sup.3+ typically gave
inferior results in staining of phosphoproteins.
[0314] Fixation of the gels in methanol/acetic acid can be done
overnight or the gels can be left in fixative for several days.
Other salts can be used instead of sodium chloride, including
magnesium chloride, magnesium sulfate, and ammonium sulfate. Sodium
chloride concentration is preferably between 100 mM to 1000 mM. If
salt is not included in the binding solution, nonspecific staining
of nonphosphoproteins is increased. Nonspecific staining is reduced
to low levels by extensive washing with .about.50 mM NaOAc (pH
4.0). Buffers other than NaOAc may be used, including formate and
2-(N-morpholino)ethanesulfonic acid. If 1,2-propanediol is omitted,
the background staining of the gel is increased but phosphoproteins
are still selectively stained. The most effective pH ranges of the
acidic buffers are in the range of 3.0 to 6.0.
Example 3
Serial Dichromatic Detection of Phosphoproteins and Total Protein
in SDS Polyacrylamide Gels
[0315] After detection of the phosphoproteins as in Example 2, the
gel was incubated overnight with 60 ml SYPRO.RTM. Ruby protein gel
stain (Molecular Probes, Eugene, Oreg.) with gentle orbital
shaking, typically 50 rpm. The gel was then incubated in 7% acetic
acid, 10% methanol for 30 minutes, also at 50 rpm. The orange
signal from the phosphorylated and non-phosphorylated proteins was
collected with a standard CCD camera-based imaging system with 300
nm UV light excitation and a 600 nm bandpass filter.
Example 4
Detection of Phosphopeptides in a Polyacrylamide Gel
[0316] Peptides generated by a trypsin digestion of bovine milk
.beta.-casein were separated by electrophoresis in a Novex.RTM.
Tricine gel (16% polyacrylamide, Invitrogen.TM. life technologies).
After electrophoresis the gel was fixed for 1 hour in 100 mL 40%
methanol, 10% acetic acid, and then fixed for 1 hour in 100 mL of
40% methanol, 0.82 M NaOAc, 0.5% glutaraldehyde. The gel was washed
with three changes of water, and then incubated for 100 minutes in
30 mL staining solution containing 50 mM NaOAc, pH 4.0, 500 mM
sodium chloride, 1 .mu.M compound 2, 1 .mu.M gallium chloride. The
gel was then washed with three changes of 50 mM NaOAc in 75
minutes. Images were acquired on a Fujifilm FLA 3000 laser based
scanner with 532 nm excitation and 580 nm bandpass emission filter
and data displayed using the Image Gauge Analysis software. The two
known phosphopeptides that result from a trypsin digest of
.beta.-casein were visible as prominent bands on the gel. The gel
was then stained with 60 mL SYPRO.RTM. Ruby protein gel stain by
incubating the gel overnight in the stain, and then incubating the
gel in 7% acetic acid, 10% methanol for 30 minutes.
Example 5
Detection of Phosphoproteins in Isoelectric Focusing Gels
[0317] Isoelectric focusing (IEF) can be performed utilizing a
variety of pre-cast and laboratory prepared gels that employ
different chemistries to generate a pH gradient. In this instance,
Ampholine PAG plates were run horizontally for 1500 volt-hours
using a Multiphor II electrophoresis unit (Amersham-Pharmacia
Biotech, Uppsala, Sweden) per the manufacturer's instructions. The
gels were fixed in 100 mL of 45% methanol, 5% acetic acid
overnight. The gels were then washed with several changes of equal
volumes of water, and incubated for 130 minutes in 50 mL of
staining solution containing 50 mM 2-(N-morpholino)ethanesulfonic
acid (pH 3.0), 1000 mM NaCl, 1 .mu.M compound 2, and 1 .mu.M
gallium chloride. The gels were washed with 50 mL of 50 mM
2-(N-morpholino)ethanesulfonic acid (pH 3.0), 1 M NaCl twice for 30
minutes per wash, and then in 50 mM 2-(N-morpholino)ethanesulfonic
acid (pH 3.0). Images were acquired as described in Example 2.
Example 6
Detection of Phosphoproteins in Two-Dimensional Gels
[0318] A human MRC-5 lung fibroblast cell lysate protein mixture
(150 .mu.g) was diluted into urea buffer (7 M urea, 2 M thiourea,
2% CHAPS, 1% Zwittergent 3-10, 0.8% carrier ampholytes (3-10), 65
mM DTT) and applied on a first dimension IPG strip (3-10 nonlinear,
18 cm). After overnight rehydration, the strips were covered with
mineral oil and the proteins were focused for 75,000 volts total.
IPG strips were then laid on top of 1 mm thick, 20 cm.times.20 cm,
12.5% T, 2.6% C polyacrylamide gels containing 375 mM Tris base, pH
8.8 and SDS-polyacrylamide gel electrophoresis was performed
according to standard procedures, except that the cathode electrode
buffer was 50 mM Tris, 384 mM glycine, 4% SDS, pH 8.8 while the
anode electrode buffer was 25 mM Tris, 192 mM glycine, 2% SDS, pH
8.8. After the second dimension electrophoresis, gels were fixed in
750 mL 45% methanol, 5% acetic acid for 20 hours. Gels were washed
twice, 75 minutes per wash, with water and then put in 500 ml
staining solution. The staining solution contained 50 mM NaOAc, pH
4.0, 250 mM sodium chloride, 20% v/v 1,2-propanediol, 1 .mu.M
compound 2, 1 .mu.M gallium chloride. 500 .mu.L of compound 2, in
stock solution at 1 mM and 500 .mu.L of gallium chloride, in stock
solution at 1 mM were added to 9 mL water. This mixture was then
added to 490 mL of the staining buffer. The gel was incubated for 8
hours in the binding solution; the solution was decanted and the
gels were washed with 3 changes of 800 mL 50 mM NaOAc, pH 4.0, 30
to 40 minutes per wash, and then washed overnight in 1 liter 50 mM
NaOAc, pH 4.0. Images were acquired on a Fujifilm FLA 3000 laser
scanner with 532 nm excitation and 580 nm bandpass emission filter
and data displayed using Image Gauge Analysis software. Images of
phosphoproteins were displayed as dark spots. Proteins not
containing phosphate were not stained or were very lightly stained
relative to the phosphoproteins. When gels were stained as above
but with GaCl.sub.3 omitted from the staining solution
phosphoproteins were not selectively stained, and could not be
distinguished from background or light nonspecific staining. In
addition, staining of phosphoproteins resulted in a trail of spots
that correlated with different percentage of phosphorylation of the
same protein, i.e., the protein had the same molecular weight but
the charge was different due to the addition or removal of a
phosphate group. Thus, 2-D gel analysis is a useful tool for
identify phosphoproteins using methods of the present invention and
allows for identification of changes in phosphorylation of a single
protein.
Example 7
Serial Dichromatic Detection of Phosphoproteins and Total Protein
in 2-D Gels
[0319] Electrophoresis and phosphoprotein detection was performed
as in Example 6. After detection of the phosphoproteins, the gel
was stained with 500 mL SYPRO.RTM. Ruby protein gel stain by
incubating the gel overnight in the stain, and then washing the gel
in 7% acetic acid, 10% methanol for two changes, at 30 minutes each
wash. Images were acquired as described in Example 2.
Alternatively, the orange signal from the phosphorylated and
nonphosphorylated proteins is collected with a standard CCD
camera-based imaging system with 300 nm UV light excitation and a
600 nm bandpass filter.
Example 8
Detection of Phosphoproteins Electroblotted to PVDF or
Nitrocellulose Membranes
[0320] Proteins of interest were separated by SDS-polyacrylamide
electrophoresis and transferred to PVDF membrane using standard
procedures, and the membrane was allowed to air dry. The PVDF
membrane was quickly dipped in 100% methanol, washed with a
solution of 40% methanol, 5% acetic acid for 15 minutes, and with
two changes of water for 10 minutes each. The blot was then added
to a binding solution and incubated for 80 minutes at room
temperature with gentle orbital shaking. The binding solution
contained 50 mM NaOAc, pH 4.0, 500 mM sodium chloride, 1 .mu.M
Compound 1 or Compound 4, and 1 .mu.M gallium chloride. Typically,
60 .mu.L of the phosphate-binding compound, in stock solution at 1
mM and 60 .mu.L of gallium chloride, in stock solution at 1 mM were
added to 540 .mu.L water. This mixture was then added to 29.5 mL of
the staining buffer. Alternatively the phosphate-binding compounds
and the gallium chloride may be added separately, directly to 30 mL
of the staining diluent. After incubation in staining solution, the
gel was washed with 50 mL of 50 mM NaOAc; pH 4.0, 2 washes of 30 to
50 minutes each.
[0321] Images were acquired with a standard CCD camera imaging
system (BioRad FluorS Max) with a reflective 300 nm UV light
source, and a 465 nm bandpass emission filter for Compound 4.
Proteins not containing phosphate were not labeled or were very
lightly stained relative to the phosphoproteins. When the blot was
stained as above but with GaCl.sub.3 omitted from the staining
solution, phosphoproteins were not selectively stained, and could
not be distinguished from background or light nonspecific staining.
For imaging with Compound 1, the wet blots were placed face down in
the Fujifilm FLA 3000 laser scanner with a 473 nm excitation laser
and 520 nm bandpass emission filter, and data displayed using the
Image Gauge Analysis software.
Example 9
Dichromatic Detection of Phosphoproteins and Total Protein
Electroblotted to PVDF Membrane
[0322] Serial dichromatic detection of phosphoprotein and total
protein on PVDF membrane was accomplished by post-staining the blot
labeled and imaged to detect phosphoprotein as in Example 8 (above)
with SYPRO.RTM. Ruby protein blot stain to detect total protein.
The blot was floated face down on a solution of 10% methanol, 7%
acetic acid for 15 minutes followed by face staining with
SYPRO.RTM. Ruby dye for 15 minutes. The blot was washed face down
on water, 3 changes in 10 minutes. The membrane was allowed to air
dry. The fluorescent signal from total proteins was acquired with a
standard CCD camera imaging system (BioRad FluorS Max) with a
reflective 300 nm UV light source and a 610 nm longpass filter.
[0323] Dichromatic staining was achieved by image acquisition with
a standard CCD camera imaging system (BioRad FluorS Max) with a
reflective 300 nm UV light source and a 465 nm bandpass emission
filter as in Example 8. The signal from the phosphoprotein stained
with Compound 4/Ga (III) could be distinguished from the signal
from total protein stained with SYPRO.RTM. Ruby, not detected with
the 465 nm bandpass filter.
[0324] For Compound 1, SYPRO.RTM. Ruby staining and image
acquisition as above reveals fluorescent signal from total protein,
revealing the phosphoproteins as a subset when the SYPRO.RTM. Ruby
image is compared to the fluorescent image obtained as in Example
6, above.
Example 10
Detection of Phosphatase Activity
[0325] Phosphoproteins and non-phosphorylated proteins were
incubated with commercially available calf intestinal alkaline
phosphatase at 37.degree. C. for 30 minutes under standard
conditions. Control digests were done under the same conditions
with no enzyme. Suitable test proteins include bovine
.alpha.-casein, ovalbumin, and pepsin as phosphoproteins; and
bovine serum albumin, chicken egg white lysozyme, and soybean
trypsin inhibitor as non-phosphorylated proteins. Electrophoresis
was performed as per Example 2, with control (undigested) and
phosphatase-treated samples loaded pairwise, 1250 ng protein per
lane. Phosphoprotein detection was performed as per Example 2
above, with images taken 90 minutes after labeling and again after
overnight washing. An additional gel was labeled as per Example 2
but with no gallium chloride in the binding solution. For the gel
labeled with the full binding solution, comparisons of the control,
undigested sample proteins showed that the phosphoproteins appeared
as dark bands according to the software display and the
nonphosphoproteins were not labeled or were only very lightly
stained. For the gel labeled with the formulation lacking gallium
chloride, phosphoproteins showed the same degree of no labeling or
only very light staining as the nonphosphoproteins, and this level
of signal was the same as the nonphosphoproteins in the gels
labeled with the full formulation including gallium chloride.
Comparison of the pairwise phosphoproteins in the fully labeled gel
showed that the signal from the alkaline phosphatase-treated sample
was significantly less than the signal from the undigested control.
The very light signal from the nonphosphoproteins, if detectable,
was virtually the same for the control and enzyme-treated
samples.
[0326] After detection of the phosphoproteins, the gel was stained
for total protein with SYPRO.RTM. Ruby protein gel stain as per
Example 2 and images of SYPRO.RTM. Ruby staining were acquired as
per Examples 3 and 7. The signal for total protein staining was
similar for the pairwise control and digested samples for both
gels, indicating that the reduced signal from alkaline
phosphatase-treated phosphoprotein samples was not due to protein
degradation.
Example 11
Detecting Kinase Activity
[0327] Bovine muscle myosin light chain was incubated with
commercially available cloned calmodulin-dependent protein kinase
II (New England BioLabs) according to the manufacturer's
instructions, with 100 mM adenosine triphosphate (ATP) and the
supplied buffer components. A parallel, control incubation was done
with no enzyme. A sample of each reaction mixture was loaded in
adjacent lanes and analyzed by electrophoresis as in Example 2. The
gels were fixed in 100 mL of 45% methanol, 5% acetic acid for 60
minutes. The gels were then washed with several changes of water.
One gel was incubated for 110 minutes in 30 mL of binding solution
containing 50 mM 2-(N-morpholino)ethanesulfonic acid, pH 3.0, 1000
mM NaCl, 1 .mu.M compound 2, 1 .mu.M gallium chloride. The other
gel was incubated in an identical solution, minus gallium chloride.
The gels were washed with 50 mL 50 mM
2-(N-morpholino)ethanesulfonic acid, pH 3.0, 1000 mM NaCl twice for
30 minutes per wash, and then in 50 mM 50 mM
2-(N-morpholino)ethanesulfonic acid, pH 3.0. Image acquisition for
phosphoprotein detection was done as in Example 2 and serial
dichromatic detection of phosphoproteins and total protein was done
as in Example 3.
[0328] Staining for total protein revealed identical profiles of 3
major bands in both lanes. Staining for phosphoprotein revealed one
major band in both lanes, with the signal from the band in the lane
corresponding to the reaction containing the enzyme 3.4-fold
greater than the no-enzyme control.
Example 12
TRAIL/Apo2L Detection
[0329] To determine the cell signaling factors involved in
TRAIL/Apo2L mediated apoptosis, a proteomics approach involving 2-D
gel electrophoresis and mass spectrometry is used. This approach
involves comparing 2-D gels of colon cancer cells (Colo205) treated
and not treated with a soluble fragment of TRAIL/Apo2L (amino acids
114-281) for various lengths of time ranging from several seconds
to several hours. To assist in comparison of 2-D gels, compound 7
bound to gallium ions is used in conjunction with the SYPRO.RTM.
Ruby total protein stain. Since cell signaling often involves
protein phosphorylation, the use of compound 7 highlights spots
likely to be involved in death receptor signaling or apoptotic
signaling. Protein spots that are significantly different between
the TRAIL/Apo2L treated and untreated Colo205 cells are identified
by subsequent mass spectrometry analysis.
Example 13
Precipitation of Phosphopeptide
[0330] Mixtures of two non-phosphorylated peptides (Angiotensin I
and II) and two phosphorylated peptides (pT/pY and RII) were
combined (5 .mu.L each) in a final volume of 100 .mu.L containing
100 mM NaOAc, pH 4.0, 0.2 mM GaCl.sub.3 and 0.1 mM compound 9. The
mixtures were vigorously vortexed for 1 hour at room temperature
and then centrifuged in a microfuge at full speed for 5 minutes.
The supernatants were removed and stored. The pellets were
resuspended by triturating with a micropipet tip in 100 .mu.L wash
buffer (100 mM NaOAc, pH 4.0, 0.2 mM GaCl.sub.3). The samples were
again centrifuged for 5 minutes and the supernatant wash components
were saved for analysis. The pellets were dissolved in 100 .mu.L
50% acetonitrile, 0.1% TFA for further analysis by HPLC or MALDI
mass spectrometry. Pellets can also be dissolved in various
different basic solutions of choice.
[0331] If phosphate-binding compound removal is required after
precipitation, extraction with chloroform can be used. Also, any
biotinylated phosphate-binding compound, such as compound 9, can be
used in the precipitation procedure. After separation of the
pellet, phosphopeptides from the phosphate-binding compound/gallium
complex using organic or base treatment, the phosphate-binding
compound can be removed using an immobilized streptavidin support
(e.g., streptavidin-agarose or streptavidin magnetic beads.)
Example 14
Detection of a Phosphopeptide in Solution by Fluorescence
Polarization using Compound 2-Ga.sup.3+
[0332] To demonstrate complexation of Compound 2-Ga.sup.3+ to
phosphoproteins and phosphopeptides, fluorescence polarization of
free dye was examined and compared to the complex (compound
2-Ga.sup.3+) in the presence of a phosphorylated and
non-phosphorylated protein and peptide.
[0333] First, an assay was conducted with a (1) phosphoprotein
(ovalbumin) and a (2) control non-phosphorylated protein
(lysozyme). A modified binding solution containing 1.0 .mu.M
Compound 2 was incubated in 50 mM 2-(N-morpholino)ethanesulfonic
acid (pH 3.0 to 3.5), 500 mM NaCl at room temperature in parallel
to solutions containing, in addition, (a) 1 .mu.M gallium chloride,
(b) 100 .mu.M lysozyme plus 1 .mu.M gallium chloride, or (c) and
100 .mu.M ovalbumin plus 1 .mu.M gallium chloride. The fluorescence
polarization of the resulting solutions was then measured in a
fluorescence spectrophotometer with excitation at 530 nm and
emission at 545 to 700 nm. The integrated polarized emission
spectra yielded anisotropy "r values" of: r=0.10+/-0.003+/-0.02
(Compound 2 plus gallium); r=0.10+/-0.002 (Compound 2 plus lysozyme
non-phosphorylated control); r=0.34+/-0.002 (Compound 2 plus
gallium plus ovalbumin), indicating phosphorylation-dependent
binding of the Compound 2 to this phosphoprotein (see FIG. 10A)
[0334] Second, an assay was conducted with a (1) phosphopeptide,
(2) non-phosphorylated peptide and (3) control with no peptide.
Phosphorylated and non-phosphorylated delta sleeping inducing
peptide DSIP (Typ-Ala-Gly-Gly-Asp-Ala-Ser (PO.sub.3)-Gly-Glu) were
purchased from SynPep Corporation (Dublin, Calif.) Ovalbumin (Cat.
A-7641) and lysozyme (Cat. L-7651) were purchased from Sigma
Chemical Company (St. Louis, Mo.). For the assay 100 .mu.M of each
peptide and a peptide-free control in binding solution (50 mM NaOAc
pH 4.0, 500 mM NaCl, 1 .mu.M Compound 2 and 1 .mu.M GaCl.sub.3)
were incubated for 30 minutes at room temperature. The fluorescence
polarization and anisotropy measurements were made using an
Aminco-Bowman Series-2 Spectrometer (Spectronic Instruments, Inc.,
Rochester, N.Y.) using wavelength settings excitation 555.+-.4 nm
and emission wavelength setting 580 nm.+-.4 nm. Alternatively,
fluorescence was measured with the Wallac 1420 Multilabel Counter
(PerkinElmer Life Sciences) using wavelength settings excitation
535.+-.17.5 nm and emission wavelength setting 590 nm.+-.17.5 nm.
The binding solution alone and binding solution in the presence of
non-phosphorylated peptide demonstrates very similar fluorescence
polarization and anisotropies. However, in the presence of the
phosphopeptide there is a significant increase in the fluorescence
polarization and the anisotropy values. This result demonstrates
specific binding of the phosphopeptide to the Compound 2-Ga.sup.3+
complex in solution but not the non-phosphorylated peptide. See
FIG. 10B.
[0335] This assay also provides a method for screening compounds
that will bind trivalent gallium ions and label phosphorylated
peptides and for solution based kinase assays.
Example 15
Isolation and Characterization of Phosphopeptides from Complex
Protein Digests with a Matrix-Immobilized Phosphate-binding
compound
[0336] A phosphate-binding compound-agarose column (compound 13 or
14) (typically 200 .mu.L of medium) was charged with 0.1 M
GaCl.sub.3 and washed with de-ionized H.sub.2O until the pH of the
flow-through material approached 7.0. The column was then
equilibrated with 5 column volumes of binding buffer (100 mM NaOAc
buffer (pH 3.0)). The phosphopeptide mixture was vacuum dried in
the SpeedVac (Savant) or similar instrument and dissolved in
binding buffer. If the final pH of the peptide mixture is not 3.0,
then it can be adjusted with 1-10 M acetic acid as appropriate. The
protein digest (1-5 mg/mL) was applied in 1 column volume or less
(but no less than half the column volume) and followed with 2
column volumes of binding buffer. Flow-through (FT) fractions were
combined and stored for further analysis. The column was washed
with 2 column volumes of 100 mM NaOAc (pH 7.0), 500 mM NaCl, 10%
acetonitrile followed by 1 column volume of NaOAc (pH 7.0). The FT
fractions were combined and stored for further analysis. Bound
peptides were eluted with 3 separate column volumes of saturated
Ba(OH).sub.2 that are collected in a single tube. The pH of the
resulting elution fraction was greater than pH 11.0, and when it
was not, it was immediately adjusted with saturated Ba(OH).sub.2.
The elution fraction was incubated for 90 minutes at 30.degree. C.
After incubation, the sample was divided into 2 portions, one of
which was neutralized to pH 5.0-7.0 with glacial acetic acid and
stored frozen. One-half volume of de-ionized water is added to the
other tube followed by the addition of a concentrated nucleophilic
thiol or amine (methylamine, cystamine or
.beta.-mercaptoethylamine) to achieve a final concentration of
0.1-0.5 M in a volume not exceeding 1/6 of the starting
sample/H.sub.2O volume. The reaction mix was incubated for an
additional 60 minutes at 30.degree. C., then neutralized to pH
5.0-7.0 with glacial acetic acid. For MALDI-TOF mass spectrometry
analysis, peptides were purified from samples using C18 ZipTips
(Millipore) using standard protocols, vacuum dried in a SpeedVac
dryer and dissolved in 50% acetonitrile and 0.1% TFA. An equal
volume of 10 mg/mL MALDI matrix (.alpha.-cyano-5-hydroxycinnamic
acid) in the same solvent was added. The solution was mixed
thoroughly and 1 .mu.L was spotted onto the MALDI target.
[0337] Differential mass weight analyses of both peptide fractions
resulted in the determination of the number of phosphorylation
sites on the peptides, as well as the nature of the phosphoamino
acids. Under the conditions used, only phosphoserine residues
undergo elimination and nucleophilic addition (loss of phosphoric
acid -98 amu, +mass weight of nucleophilic addition reagent).
Phosphothreonine residues undergo elimination only (loss of
phosphoric acid only, -98 amu) and phosphotyrosine residues remain
unchanged, as phosphotyrosine is stable in strong base.
Example 16
Quantitating the Number of Phosphates on Ovalbumin
[0338] Solutions of 1 .mu.M and 4 .mu.M ovalbumin were incubated in
75 mM NaOAc (pH 4.0), 140 mM NaCl, 0.1 .mu.M Compound 4, and 1.7
.mu.M gallium chloride at room temperature for 5-10 minutes. The
fluorescence intensity of the resulting solution was then measured
at 410 nm in a fluorescence spectrophotometer and compared to a
standard phosphate calibration curve to determine the number of
phosphates on ovalbumin. The standard phosphate calibration curve
was produced by equilibrating known concentrations (1, 2, 4, 6, 8,
and 10 .mu.M) of a 19 amino acid phosphoserine-containing peptide
in 75 mM NaOAc (pH 4.0), 140 mM NaCl, 0.1 .mu.M compound 4, and 1.7
.mu.M gallium chloride and measuring the fluorescence intensity at
410 nm. Next the fluorescence intensity was graphed versus the
known concentration of phosphopeptide. The fluorescence intensity
from the solution containing ovalbumin was compared to the standard
curve to reveal .about.2 .mu.M and .about.8 .mu.M phosphate.
Finally, accounting for the protein's concentration resulted in the
determination of two phosphate groups per molecule of
ovalbumin.
Example 17
Phospholipid Detection
[0339] To test the detection of phospholipids with the present
invention, different phospholipids were spotted onto a
nitrocellulose membrane. The phospholipids were obtained from
Echelon Research Labs in a format called a PIP Array.TM., which
contains 8 different phosphoinositides (Ptdlns) at 7 different
concentrations. PIP Arrays.TM. were used for determining the
sensitivity limits of the invention for detecting
phospholipids.
PIP Array.TM.
[0340] 1. Ptdlns 100 50 25 12.5 6.3 3.2 1.6 pmol [0341] 2. Ptdlns
(3) P [0342] 3. Ptdlns (4) P [0343] 4. Ptdlns (5) P [0344] 5.
Ptdlns (3,5) P2 [0345] 6. Ptdlns (4,5) P2 [0346] 7. Ptdlns (3,4) P2
[0347] 8. Ptdlns (3,4,5,) P3
[0348] One PIP Array.TM. was washed in 50 mM NaOAc (pH 4.0) for 15
min. After the wash, the PIP Array.TM. was incubated in 50 mM NaOAc
(pH 4.0), 20% 1,2-propanediol, 500 mM NaCl, 1 .mu.M compound 1, and
1 .mu.M GaCl.sub.3 for 1 hour, by incubating the array at 100-150
RPM on an orbital shaker. After incubating the PIP Array.TM. the
array was washed 3 times in 50 mM NaOAc (pH 4.0) for 15 minutes
each at 100-150 RPM on an orbital shaker to remove unbound dye and
reduce the background fluorescence. An image of the PIP Array.TM.
was generated using a laser based scanner (Fuji FLA 3000) with an
excitation wavelength of 473 nm and an emission filter of 520 nm.
Of the eight phosphoinositides, four gave a strong positive signal.
These included phosphatidic acid, phosphoinositide (4,5) P.sub.2,
phosphoinositide (3,4) P.sub.2 and phosphoinositide (3,4,5)
P.sub.3. The strongest signal was obtained with phosphoinositide
(3,4) P.sub.2 followed by phosphoinositide (4,5) P.sub.2 and then
phosphoinositide (3,4,5) P.sub.3.
Example 18
Phosphoprotein Detection on Microarrays
[0349] Four specific, purified proteins including .beta.-casein,
ovalbumin, pepsin and bovine serum albumin were arrayed from a
source plate (384 well plate) at a concentration of 0.975
.mu.g/mL-0.5 mg/mL in water, onto HydroGel coated slides (Perkin
Elmer), using the BioChip Arrayer.TM. (Packard Instrument Co.,
Meriden, Conn.). The BioChip Arrayer.TM. utilizes a PiezoTip.TM.
Dispenser consisting of 4 glass capillaries. Proteins were
dispensed from the PiezoTip.TM. by droplets 333 pL in volume to
create array spots 175 microns in diameter with a 500 micron
horizontal and vertical pitch (pitch=center to center spacing of
spots). Proteins were arrayed in duplicate in four rows, with 10
dilution points, resulting in an array of 160 spots. The resulting
concentration range of the array was 166.5 pg/spot -0.325 pg/spot.
For detection of phosphoproteins, slides were incubated for 1 hour
on a rotator in 1 .mu.M of Compound 2, in buffer containing 0.5 M
NaCl, 20% 1,2-propanediol, 1 .mu.M GaCl.sub.3, and 0.05 M NaOAc, pH
4.0. Slides were then washed for 1 hour on a rotator in 0.05 M
NaOAc, pH 4.0, containing 10% methanol followed by a 15 minute
water wash. Slides were then spun briefly in a microarray
high-speed centrifuge affixed with a rotor with a slide holder
(Telechem) at .about.6000 rpm to remove excess liquid. After the
slides were dry, the arrays were imaged using the ScanArray.RTM.
5000 XL Microarray Analysis System (Packard Instrument Co.,
Meriden, Conn.) using the 543.5 nm laser and either 570 nm or 592
nm emission filter. Phosphate content per protein was determined to
be as follows: .beta.-casein, five phosphates; ovalbumin, two
phosphates; pepsin, one phosphate; and BSA, no phosphates.
Example 19
Phosphopeptide Detection on Microarrays
[0350] Two peptides, Kemptide and pDSIP, were arrayed on to
HydroGel coated slides (Perkin Elmer) from a source plate
(384-well) with a concentration of 0.03125 to 2 mg/mL peptide in
water. The amino acid sequence of Kemptide is
Leu-Arg-Arg-Ala-Ser-Leu-Gly (MW 771.9). The amino acid sequence of
pDSIP is Trp-Ala-Gly-Gly-Asp-Ala-Ser(PO.sub.3H)-Gly-Glu (MW 929.5).
Arrays were spotted using a manual glass slide arrayer (V & P
Scientific, San Diego, Calif.) fixed with 4 rows of 8 pins (32
total), .about.500 micron diameter spot size, 1.125 micron
horizontal pitch and 750 micron vertical pitch (pitch=center to
center spacing of spots). The hand arrayer collected 6 nL of
peptide from the source plate and transferred .about.6 nL to the
array surface by direct contact. The resultant peptide
concentration was 0.18 to 12 ng/spot. Peptides were arrayed in
replicates of 6, resulting in an array of 84 spots. For specific
detection of pDSIP, the phosphopeptide, slides were incubated for 1
hour on a rotator in 1 .mu.M dye of compound 2 in buffer containing
0.5 M NaCl, 20% 1,2-propanediol, 1 .mu.M GaCl.sub.3, and 0.05 M
NaOAc, pH 4.0. Slides were then washed for 1 hour on a rotator in
0.05 M NaOAc, pH 4.0, containing 10% methanol followed by a
15-minute water wash. Slides were then spun briefly in a microarray
high-speed centrifuge affixed with a rotor with a slide holder
(Telechem) at .about.6000 rpm to remove excess liquid. After the
slides were dry, the arrays were imaged using the ScanArray.RTM.
5000 XL Microarray Analysis System (Packard Instrument Co.,
Meriden, Conn.) using the 543.5 nm laser and either 570 nm or 592
nm emission filter.
Example 20
Detection of Immobilized Kinase Substrates in Microarray Format;
Selective Detection of Glycogen Synthase 1-10
[0351] Two specific peptides, Abl peptide and glycogen synthase
1-10, were arrayed from a source plate (384-well plate)
concentration of 0.03-2 mg/mL in water, onto HydroGel coated slides
(Perkin Elmer). Abl peptide (New England Biolabs) is a substrate
for Abl tyrosine kinase and its amino acid sequence is
E-A-I-Y-A-A-P-F-A-K-K-K (MW 1336). Glycogen synthase 1-10
(Calbiochem) is a substrate for Calcium-Calmodulin-Dependent
protein Kinase II and its amino acid sequence is
P-L-S-R-T-L-S-V-S-S (MW 1045.2). Arrays were spotted using a manual
glass slide arrayer (V&P Scientific, San Diego, Calif.) fixed
with 4 rows of 8 pins (32 total), .about.500 micron diameter spot
size, 1.125 micron horizontal pitch and 750 micron vertical pitch
(pitch=center-to-center spacing of spots). The handarrayer
collected 6 nL of peptide from the source plate and transferred
.about.6 nL to the hydrogel coated slide by direct contact. The
resultant peptide concentration is 0.18 to 12 ng/spot. Peptides
were arrayed in replicates of 6, resulting in array of 96 spots (12
spots, of which were 0 ng/spot). Slides were left overnight after
arraying in a humidity chamber. Slides were then blocked for 1 hour
in 100 mM HEPES, 1% BSA while rotating (Barnstead/Thermolyne
Labquake rotisserie). After blocking, the slides were spun briefly
in a small microarray high-speed (max .about.6000 rpm) centrifuge
affixed with a rotor with a slide holder (Telechem) to remove
excess liquid. Next, kinase reactions were performed by attaching a
Grace Biolabs Hybriwell.TM. hybridization sealing system
(40.times.22.times.0.25 mm) to the hydrogel coated slide to enclose
the area containing the hydrogel polyacrylamide pad. The reaction
was carried out in an 80 .mu.L reaction volume containing 20,000
U/mL or 1600 units enzyme (Calmodulin-Dependent protein Kinase II,
NEB) using buffer, CaCl.sub.2, calmodulin, and ATP supplied with
the enzyme. 1.times. CamKII buffer included 50 mM Tris-HCl, 10 mM
MgCl.sub.2, 2 mM dithiothreitol, 0.1 mM Na.sub.2EDTA, pH 7.5.
CaCl.sub.2, calmodulin and ATP working concentrations were 2 mM,
1.2 .mu.M and 0.10 mM. The reaction solution with enzyme was
pipetted into the Hybriwell.TM. through 1 of 2 ports on the seal
cover. Ports were then sealed with seal-tabs, placed in a
CMT-hybridization chamber (VWR Scientific) and incubated on a
nutator (Clay Adams) in a 37.degree. C. incubator. The kinase
reaction was carried out for 3 hours. After incubation, the slides
were removed from the hybridization chamber and washed 2 times for
5 minutes in 10% SDS followed by 5-7 times for 5 minutes in water
while rotating. Slides were then transferred immediately to binding
solution comprising 1 .mu.M of compound 2 in 50 mM NaOAc, pH 4.0;
500 mM NaCl; 20% 1,2-propanediol; and 1 .mu.M GaCl.sub.3 for 45
minutes while rotating. Slides were then washed 3 times for 15
minutes each time in 50 mM NaOAc, pH 4.0, 10% methanol followed by
a 15-minute water wash. Slides were then dried and imaged using the
Scan Array.RTM. 5000 XL Microarray Analysis System (Packard
Instrument Co., Meriden, Conn.) using the 543.5 nm laser and either
570 nm or 592 nm emission filters. Calmodulin-dependent kinase II
specifically phosphorylates glycogen synthase 1-10 peptide. Using
the 543.5 nm excitation and 570 nm emission filter, glycogen
synthase peptide is the only fluorescently labeled peptide on the
array. Sensitivity of detection after kinase reaction is at least
0.375 ng or 0.35 pmol.
Example 21
Detection of Immobilized Kinase Substrates in Microarray Format;
Specific Detection of Abl Peptide Substrate
[0352] The following experiment was performed essentially as
described in Example 20 with the following differences. Two
specific peptides, Abl peptide and Kemptide, were arrayed from a
source plate (384-well plate) concentration of 0.03 to 2 mg/mL in
water, onto hydrogel-coated slides (Perkin Elmer). Kemptide (New
England Biolabs) is a substrate for cAMP-dependent Protein Kinase
(PKA) catalytic subunit and its amino acid sequence is
L-R-R-A-S-L-G (MW 771). Arrays were spotted as described in Example
20 and kinase reactions performed as described previously. The
reaction was carried out in a 80 .mu.L reaction volume containing
3,750 U/mL or 300 units enzyme (Abl Protein Tyrosine Kinase, NEB)
using buffer and ATP supplied with the enzyme. 1.times. Abl buffer
included 50 mM Tris-HCl, 10 mM MgCl.sub.2, 1 mM EGTA, 2 mM
dithiothreitol, 0.01% Brij 35, pH 7.5. Labeling of slides and
imaging were performed as previously described. Using the 543.5 nm
excitation and 570 nm emission filter, Abl peptide substrate is the
only fluorescently labeled peptide on the array. Sensitivity of
detection after kinase reaction is at least 0.18 ng or 0.14
pmol.
Example 22
Ratiometric Analysis of Phosphorylated Target molecules using a
Binding solution of the Present Invention and the Total Protein
Stain SYPRO.RTM. Ruby Gel Stain
[0353] SDS-polyacrylamide gels were loaded with serial dilutions of
phosphoproteins and non-phosphoproteins and gels stained as
described in Example 2. The gels were illuminated and fluorescence
signal quantified (intensity) for each protein concentration and
each stain (phosphoprotein and total protein). The ratio of these
fluorescence intensities was then graphed, phosphoprotein and total
protein, against the amount of protein loaded in the well (ng). The
fluorescence intensity for each stain was next plotted against the
protein concentration, this graph allows for the calculation of a
constant number for each of the two stains, the Y-intercept value.
The Y-intercept value is then subtracted from the fluorescence
intensity values and the resulting rations (phosphoprotein to total
protein) are again graphed against the protein concentration. This
resulting graph produces numbers wherein stained phosphoproteins
have ratio values 50-100 times greater than nonphosphorylated
proteins, thus nonspecific staining and low abundance
phosphoproteins can be distinguished from non-phosphorylated
proteins. See, FIG. 10.
Example 23
Incorporation of ATP-.gamma.-S During Phosphorylation of
Immobilized Peptides on Microarrays and Subsequent Detection of the
Phosphorothioate Group with a Binding Solution of the Present
Invention
[0354] Three specific peptides including Abl peptide, Kemptide and
Glycogen Synthase 1-10 were arrayed from a source plate (384-well
plate) concentration of 0.03 .mu.g/mL to 0.5 mg/mL in water, onto
hydrogel-coated slides (Perkin Elmer). Proteins were spotted using
the BioChip Arrayer.TM. (Packard Instrument Co., Meriden, Conn.)
that utilizes a PiezoTip.TM. Dispenser consisting of 4 glass
capillaries. Proteins were dispensed from the PiezoTip.TM. by
droplets 333 pL in volume to create array spots 175 microns in
diameter with a 500 micron horizontal and vertical pitch
(pitch=center to center spacing of spots). Each peptide was arrayed
in quadruplicate from a 2-fold dilution series consisting of 15
dilution points, resulting in an array of 240 spots (including 60
water spots=0 pg/spot protein). The resultant peptide concentration
was 0.01 pg/spot to 166.5 pg/spot. Slides were left overnight after
arraying in a humidity chamber. Slides were then blocked for 1 hour
in 100 mM HEPES, 1% BSA, pH 7.5, while rotating
(Barnstead/Thermolyne Labquake rotisserie). After blocking, the
slides were spun briefly in a small microarray high-speed (max
.about.6000 rpm) centrifuge affixed with a rotor with a slide
holder (Telechem) to remove excess liquid. Next, kinase reactions
were performed by attaching a Grace Biolabs Hybriwell.TM.
hybridization sealing system (40.times.22.times.0.25 mm) to the
hydrogel-coated slide to enclose the area containing the hydrogel
acrylamide pad. The reaction was carried out in a 80 .mu.L reaction
volume containing 3750 U/mL or 300 units enzyme (Abl Protein
Tyrosine Kinase, NEB) using buffer supplied with the enzyme.
1.times. Abl buffer included 50 mM Tris-HCl, 10 mM MgCl.sub.2, 1 mM
EGTA, 2 mM dithiothreitol, 0.01% Brij 35, pH 7.5. ATP-.gamma.-S
(Sigma Chemical Company) was substituted in place of ATP at a
working concentration of 0.10 mM. A control reaction was run
simultaneously on a second slide using ATP itself, supplied with
the enzyme, at a working concentration of 0.10 mM. The reaction
solution containing the enzyme was pipetted into the Hybriwell.TM.
through 1 of 2 ports on the seal cover. Ports were then sealed with
seal-tabs, placed in a CMT-hybridization chamber (VWR Scientific)
and incubated on a nutator (Clay Adams) in a 37.degree. C.
incubator. The kinase reaction was carried out for 3 hours. After
incubation, the slides were removed from the hybridization chamber
and washed 2 times for 5 minutes in 10% SDS followed by 7 times for
5 minutes in water while rotating. Slides were transferred
immediately to 1 .mu.M compound 2 in 50 mM NaOAc, pH 4.0, 500 mM
NaCl, 20% 1,2-propanediol, 1 .mu.M GaCl.sub.3 for 45 minutes while
rotating. Slides were washed 3 times for 15 minutes each time in 50
mM NaOAc, pH 4.0, 4% acetonitrile, followed by a 15 minute water
wash. Slides were dried and imaged using the Scan Array.RTM. 5000
XL Microarray Analysis System (Packard Instrument Co., Meriden,
Conn.) using the 543.5 nm laser and either 570 nm or 592 nm
emission filters. Abl Protein Tyrosine Kinase phosphorylates the
tyrosine residue of the Abl peptide substrate with the
phosphorothioate (or phosphate as in control). Using the 543.5 nm
excitation and 570 nm emission filter, Abl peptide substrate is the
only fluorescently labeled peptide on the array. Sensitivity of
detection after kinase reaction was 2.6 pg of the peptide labeled
using ATP-.gamma.-S. By contrast, 0.325 pg of Abl peptide, in the
control reaction using ATP, was labeled and detected with binding
solution containing Compound 2.
Example 24
Isolation of Phosphopeptides Via Immobilized Streptavidin or
Phosphate-Binding Compound on Membranes
[0355] Immunodyne membranes, purchased from Pall, were labeled by
adding enough 5-10 mg/mL streptavidin or BAPTA-amine to wet the
membrane (approximately 15 .mu.L per cm.sup.2). After air-drying
for 15-30 minutes the membranes were washed 3 times for 5 minutes
with 50% Acetonitrile/0.1% TFA, then twice for 5 minutes with
water. Experiments were performed using 6 mm diameter circles of
membrane. Streptavidin-labeled membranes were charged by soaking in
200 .mu.L of 40 .mu.M Compound 9/250 .mu.M GaCl.sub.3, and the
BAPTA labeled membranes were charged by soaking in 200 .mu.L 250 uM
GaCl.sub.3. After washing once with 10% acetonitrile/400 mM NaCl,
100 mM NaOAc, pH 4, then twice with water, the membranes were
soaked in 100 .mu.L of peptide mixture containing 2 .mu.g each of 2
non-phosphopeptides and 3 phosphopeptides (approximately 6
nanomoles of total peptide) for 15 min. The membranes were washed
once with 10% acetonitrile/400 mM NaCl/100 mM NaOAc, pH 4, then
twice with 100 mM NaOAc, pH 4, for 5 min per wash. Finally the
membranes were eluted with 100 .mu.L of 50% acetonitrile/0.1% TFA.
The eluates and peptide supernatants (peptide solution after
incubation of membranes) were prepared for MALDI analysis.
Example 25
Isolation of Phosphopeptides with Immobilized Streptavidin on
Ferrofluid Beads
[0356] A. Isolation of Phosphopeptides Using Streptavidin
Ferrofluid Magnetic Particles in Conjunction with BAPTA-Biotin
Dyes.
[0357] Streptavidin ferrofluid magnetic particles were used in
conjunction with BAPTA-biotin compounds to isolate phosphopeptides
from a complex mixture. The magnetic particles were labeled with
Compound 9 and two versions of rhodamine-biotin dyes (Compound 15
and Compound 25) due to the binding between biotin and
streptavidin. After washing away phosphate-binding compounds, the
particles were charged with 0.1 M GaCl.sub.3, then washed with 0.1
M NaOAc, pH 4.0. Fifty .mu.L of the magnetic particle slurry (4
mg/mL) was added to the phosphopeptide mix in a total volume of 100
.mu.L. The particles were incubated with gentle vortexing for 20
minutes and the particles were isolated using a magnetic separator.
The supernatants were removed and the beads were washed 2.times.
with 100 .mu.L 100 mM NaOAc, pH 4.0. The phosphopeptides were
eluted with 100 .mu.L 50% acetonitrile, 0.1% TFA. The isolated
peptides were analyzed by MALDI and demonstrated that only
phosphorylated peptides were isolated.
B. Quantitative Binding of Phosphopeptides Using Streptavidin
Ferrofluid Particles
[0358] Quantitative binding of phosphopeptides was accomplished
using labeled ferrofluid particles. Streptavidin ferrofluid
particles were labeled with Compound 9 until saturated. After
washing away unbound dye, the particles were charged with 0.1 M
GaCl.sub.3, then washed with 0.1 M NaOAc, pH 4.0. 100 .mu.L of
magnetic particles (4 mg/mL) was added with standard peptide mix
containing 550 picomoles of phosphopeptides. The mixture was
incubated for 20 min while gently vortexing. The particles were
isolated using a magnetic separator and the resulting supernatant
was removed. The beads were washed 2.times. with 100 .mu.L 100 mM
NaOAc, pH 4.0 and the phosphopeptides were eluted with 0.15 M
ammonium hydroxide. Greater than 95% of the phosphopeptides (550
pmoles) were isolated.
C. Ferrofluid Particle Isolation of Phosphopeptides Coupled with
Base Elimination/Addition
[0359] Streptavidin ferrofluid (StFF) particles labeled with
Compound 9 were used for phosphopeptide isolation and coupled with
base elimination/addition. Phosphopeptides were isolated on StFF
and the phosphopeptides eluted with 50 .mu.L of 0.15 M
Ba(OH).sub.2. Subsequently, 3 .mu.L methylamine was added to the
eluate and peptides were incubated at 37.degree. C. for 1 hour. The
reaction was neutralized with glacial acetic acid to pH 4.0 and the
eluted peptides were desalted with ZipTips. The use of
streptavidin-labeled ferrofluid beads allows for a strong
interaction between avidin and biotin that facilitates isolation of
larger phosphorylated target molecules.
Example 26
Precipitation of Phosphopeptides with DTPA Compounds
[0360] Phosphate-binding compounds comprising DTPA (Compounds 20,
21 and 22) were used in a precipitation reaction for the isolation
of phosphopeptides from complex solutions. Precipitation reactions
contained 100 .mu.M Compound 20, 21 or 22, 200 .mu.M GaCl.sub.3,
100 mM NaOAc, pH 4.0 and 5 .mu.L of an 8-peptide mix (250 ng/.mu.L
each). The samples were vigorously vortexed for 20 minutes then
centrifuged at 14,000 rpm for 5 minutes. The supernatants were
removed and stored and the pellets were washed 2.times. in 100 mM
NaOAc, pH 4.0 by resuspension with a pipet tip and re-centrifuging.
The final pellets were dissolved in 100 .mu.L of 50% acetonitrile,
0.1% TFA. Supernatants and pellet fractions were diluted 1:100 in
50% acetonitrile, 0.1% TFA and then mixed 1:1 with MALDI matrix and
then spotted onto a MALDI target. The MALDI analyses demonstrated
the selective isolation of phosphorylated peptides.
Example 27
[0361] Serial detection of total phosphopeptide content with a
binding solution of the present invention followed by specific
detection of phosphotyrosine residues in peptides using a
phosphotyrosine specific monoclonal antibody and (A) Alexa
Fluor.RTM. 647 goat anti-mouse or (B) Zenon.TM. One Alexa
Fluor.RTM. 647 mouse IgG labeling reagent, both in microarray
format.
[0362] (A) Three pairs of peptides, the phosphorylated and the
non-phosphorylated forms of Kemptide, pp60 c-src and DSIP, were
arrayed on to HydroGel coated slides (Perkin Elmer) from a source
plate (384-well) with a concentration of 0.95 .mu.g/ml -0.5 mg/ml
peptide in water, using the BioChip Arrayer.TM. (Perkin Elmer) as
described in Example 20. For specific detection of the
phosphopeptides on the array, the slides were incubated for 45
minutes on a rotisserie in a binding solution comprising 1 .mu.M
Compound 2, in buffer containing 0.5 M NaCl, 20% 1,2 propanediol, 1
.mu.M GaCl.sub.3, and 0.05 M sodium acetate, pH 4.0. Slides were
then washed three times for 15 minutes on a rotisserie in 0.05 M
sodium acetate, pH 4.0, containing 4% acetonitrile followed by a 15
minute water wash. Slides were then spun briefly in a microarray
high-speed centrifuge affixed with a rotor with a slide holder
(Telechem) at .about.6000 rpm to remove excess liquid. After slides
were dry, the arrays were imaged using the ScanArray.RTM. 5000 XL
Microarray Analysis System (Packard Instrument Co., Meriden, Conn.)
using the 543.5 nm laser and 570 nm emission filter. The binding
solution specifically labeled 1.3-2.6 pg pDSIP, 2.6-5.2 pg
pKemptide and 10.4-20.8 pg pp60 c-src (pY). Following
phosphopeptide detection, the slides were immediately placed in
blocking buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 0.2%
Tween-20, 0.25% MOWIOL-488, 0.5% BSA and incubated while rotating
for 3-5 hours. Slides were then transferred to blocking buffer
(described above) containing a 1:1000 dilution (final concentration
of 1.5 .mu.g/mL) of phosphotyrosine monoclonal antibody (supplied
at 1.5 mg/mL; P-Tyr-100; Cell Signaling Tech.) and incubated
overnight at 4.degree. C. while rotating. After overnight
incubation with the primary antibody, the slides were washed three
times for 10 minutes in blocking buffer and then incubated for 45
minutes, while rotating, in blocking buffer containing a 1:5000
dilution (final concentration of 0.4 .mu.g/mL) of Alexa Fluor.RTM.
647 goat anti-mouse (supplied at 2 mg/mL). Finally, slides were
washed two times for 10 minutes in blocking buffer followed by two
5 minute washes in 50 mM Tris, pH 7.5, 150 mM NaCl and spun briefly
in a microarray high-speed centrifuge. After the slides were dry,
the arrays were imaged using the ScanArray.RTM. 5000 XL Microarray
Analysis System (Packard Instrument Co., Meriden, Conn.) using two
protocols with a 543.5 nm laser/570 nm emission filter set and a
632.8 nm laser/670 nm emission filter set. Using the 543.5 nm
excitation and 570 nm emission filter, there was no signal
detected. Using the 632.8 nm excitation and 670 nm emission filter,
pp60 c-src (pY) was specifically detected to a sensitivity of 5.2
pg.
[0363] (B) After overnight incubation with the primary antibody,
the slides were washed three times for 10 minutes in blocking
buffer and then incubated for 45 minutes, while rotating, in
blocking buffer containing a 1:100 dilution (final concentration of
2 .mu.g/mL) of Zenon.TM. One Alexa Fluor.RTM. 647 mouse IgG
labeling reagent (supplied at 200 .mu.g/mL). Finally, slides were
washed once for 5 minutes in blocking buffer, once for 5 minutes in
50 mM Tris, pH 7.5, 150 mM NaCl and spun briefly in a microarray
high-speed centrifuge. After the slides were dry, the arrays were
imaged using the ScanArray.RTM. 5000 XL Microarray Analysis System
(Packard Instrument Co., Meriden, Conn.) using two protocols with a
543.5 nm laser/570 nm emission filter set and a 632.8 nm laser/670
nm emission filter set. Using the 543.5 nm excitation and 570 nm
emission filter, there was no signal detected. Using the 632.8 nm
excitation and 670 nm emission filter, pp60 c-src (pY) was
specifically detected to a sensitivity of 0.65 pg.
Example 28
Size Exclusion Column (SEC) and Reverse Phase (RP)HPLC Analysis of
Phosphopeptides Using a Binding Solution of the Present
Invention
[0364] 10-40 .mu.L of sample containing 2-60 .mu.M phosphopeptide
(or a control of 100 .mu.m non-phosphopeptide), 20 .mu.M Compound
2, 40 .mu.M GaCl.sub.3, 100 mM sodium acetate pH 4 and 0-20% ethyl
alcohol or isopropyl alcohol was injected onto a size exclusion
column (Superdex 30, 10.times.300 mm or Superdex Peptide
3.2.times.300 mm). Mobile phase was 50 mM sodium acetate pH 4, 500
mM NaCl plus 20% ethyl alcohol or isopropyl alcohol. The runtime
was 45 min. UV and fluorescence signal was monitored at 214 nm and
555ex/580em, respectively.
[0365] SEC results demonstrated an enhanced fluorescent signal in
the presence of a binding solution of the present invention and a
phosphopeptide (60 .mu.M) compared to a non-phosphopeptide.
[0366] The same sample was also analyzed by reverse phase HPLC with
a Vydac 238TP52, 2.1.times.250 mm C.sub.18 column. Solvent A=100 mM
sodium acetate pH 4, 0-20% ethyl alcohol. Solvent B=100 mM sodium
acetate pH 4, 20% ethyl alcohol, 60% methanol. A gradient
separation was performed, 0-55% solvent B over 30 min at 0.2
mL/min. UV (214 nm) and fluorescence (ex 555/em 580) signals were
monitored.
[0367] RP results demonstrated an enhanced fluorescent signal in
the presence of a binding solution of the present invention and a
solution without gallium chloride. Samples were analyzed containing
a mono-phosphotyrosine, mono-phosphothreonine and
mono-phosphoserine containing peptide, with comparable results.
Example 29
Detection of Phosphopeptides on Streptavidin-Polystyrene Beads
Using a Binding Solution of the Present Invention
[0368] Streptavidin-polystyrene beads (4.0-4.9 .mu.M) were charged
with either one of two biotinylated synthetic peptides, a
phosphopeptide or a non-phosphopeptide. The phosphopeptide had a
molecular weight of 1812 g/mol and the amino acid sequence was
biotinyl-.epsilon.-aminocaproyl-Glu-Pro-Gln-Tyr(PO.sub.3H.sub.2)-Glu-Glu--
Ile-Pro-Ile-Tyr-Leu-OH. The non-phosphopeptide had a molecular
weight of 2342.55 g/mol and the amino acid sequence was
biotinyl-Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp--
Phe-NH.sub.2. Beads were charged in 100 mM Tris, 100 mM NaCl, pH
7.5 and washed several times in the same buffer, following
charging, before staining. Both sets of beads were then stained
with 1 .mu.M Compound 2 in buffer containing 0.05 M sodium acetate,
pH 4.0, 1 .mu.M GaCl.sub.3, 0.5 M NaCl, and 20% 1,2 propanediol for
45 minutes. Following staining, beads were washed in 0.05 M sodium
acetate, pH 4.0, 4% acetonitrile and mixed in different ratios of
phosphopeptide-charged beads with non-phosphopeptide charged beads.
All steps were performed with rotation and rigorous sonication and
vortexing. The mixed bead populations were then imaged using a
Nikon Eclipse 800 Epi-Fluorescent Microscope using Omega Optical,
Inc. filter set XF102-2 (Exciter: 560AF55; Dichroic: 595DRLP;
Emitter: 645AF75). The fluorescent signal of the phosphopeptide
charged beads was found to be 6-fold higher, on average, than the
non-phosphopeptide charged beads.
Example 30
Detection of Kinase-Mediated Phosphorylation of Peptide Substrates
Bound to Streptavidin-Polystyrene Particles Using a Binding
Solution of the Present Invention
[0369] Streptavidin-polystyrene beads (4.0-4.9 .mu.M) were charged
with a biotinylated synthetic peptide called crosstide. Crosstide
is a peptide substrate for the serine/threonine kinase Akt/Protein
Kinase B and is a 1808 g/mol peptide with the following amino acid
sequence, Gly-Arg-Pro-Arg-Thr-Ser-Ser-Phe-Ala-Glu-Gly. Beads were
charged in 100 mM Tris, 100 mM NaCl, pH 7.5 and washed several
times in the same buffer, following charging, before staining. The
crosstide peptide on the streptavidin polystyrene particle was then
phosphorylated using 500 ng of Akt/PKB kinase in 40 .mu.L of 15 mM
MOPS, pH 7.2, 18.75 mM .beta.-glycerol phosphate, 3.75 mM EGTA,
0.75 mM sodium orthovanadate, 0.75 mM DTT supplemented with 200
.mu.M ATP. A control reaction was performed in which all reaction
components were added, including ATP, except the kinase enzyme.
Phosphorylation was carried out for 60 minutes at 30.degree. C.
with continuous rotation and stopped by incubating beads and kinase
at 100.degree. C. for 5 minutes. Beads were then washed again by
incubating in 100 mM Tris, 100 mM NaCl, pH 7.5 followed by staining
with 1 .mu.M Compound 2 in buffer containing 0.05 M sodium acetate,
pH 4.0, 1 .mu.M GaCl.sub.3, 0.5 M NaCl, and 20% 1,2 propanediol for
45 minutes. Following staining, beads were washed in 0.05 M sodium
acetate, pH 4.0, 4% acetonitrile and imaged using a Nikon Eclipse
800 Epi-Fluorescent Microscope using Omega Optical, Inc. filter set
XF102-2 (Exciter: 560AF55; Dichroic: 595DRLP; Emitter: 645AF75).
The fluorescent signal of the peptide charged beads exposed to
Akt/PKB kinase was found to be 2.2-fold higher, with no overlap in
standard deviation, than the control peptide charged beads not
exposed to enzyme.
Example 31
Synthesis of Compound 2
[0370] A solution of 3-dimethylaminophenol (0.47 g, 3.5 mmol) and
5-fluoro-5'-formyl BAPTA tetramethyl ester (1.00 g, 1.7 mmol) in 20
mL propionic acid was heated at 110.degree. C. for 2 hours, cooled
and poured into 120 mL aqueous NaOAc. The resulting purple gum was
rinsed with water, dissolved in ethyl acetate, and evaporated to
give 1.20 g dihydro-Rhod-5F tetramethyl ester as a red foam.
[0371] To dihydro-Rhod-5F tetramethyl ester (1.2 g, 1.5 mmol) in
1:1 chloroform/methanol (40 mL) was added chloranil (0.51 g, 2.0
mmol). The solution was stirred overnight at room temperature then
evaporated. The residue was purified by flash chromatography using
chloroform/methanol/acetic acid (50:5:1) as eluant to give 0.54 g
of Rhod-5F tetramethyl ester as a red foam.
[0372] To Rhod-5F tetramethyl ester (0.48 g, 0.55 mmol) in dioxane
(25 mL) was added 1 M KOH (4.4 mL, 4.4 mmol). The solution was
stirred overnight, then evaporated. The residue was dissolved in 10
mL water and 50 mL of 5% HCl was added. A precipitate was filtered
and dried to give 275 mg of Rhod-5F free acid as a red powder. This
product was converted to the potassium salt with aqueous KOH,
followed by column chromatography with water on Sephadex LH-20 to
give tripotassium salt Compound 2 as a red powder.
Example 32
Synthesis of Compound 5 (Rhodamine BAPTA compound)
[0373] 8-Hydroxyjulolidine (0.76 g, 4.1 mmol), 5-fluoro-5'-formyl
BAPTA tetramethyl ester (1.16 g, 2.0 mmol) and p-TsOH (20 mg) in 20
mL propionic acid were heated at 60.degree. C. overnight, then
cooled and poured into 150 mL aqueous 3 M NaOAc. A purple powder
was collected by filtration, rinsed with water, and dried to give
2.15 g of dihydro-X-Rhod-5F tetramethyl ester as a purple
powder.
[0374] To dihydro-X-Rhod-5F tetramethyl ester (2.1 g, 2.4 mmol) in
1:1 chloroform/methanol (80 mL) was added chloranil (1.45 g, 5.9
mmol). The solution was stirred 4 hours at room temperature then
evaporated. The residue was purified by flash chromatography using
chloroform/methanol/acetic acid (50:5:1) to give 3.0 g of X-Rhod-5F
tetramethyl ester as a red foam.
[0375] To X-Rhod-5F tetramethyl ester (3.0 g, 2.9 mmol) in dioxane
(25 mL) and methanol (25 mL) was added 1 M KOH (30 mL, 30 mmol).
The solution was stirred overnight then evaporated. The residue was
dissolved in 10 mL water and this added to 50 mL of 5% HCl. A
precipitate was filtered and dried to give 500 mg of Compound 5
free acid as a purple powder. 100 mg of the free acid was converted
to the potassium salt with aqueous KOH, followed by chromatography
with water on Sephadex LH-20 to give 40 mg of Compound 5 as its
potassium salt, a purple powder.
Example 33
Synthesis of Quinazolinone-Labeled BAPTA (Q-BAPTA) Compounds
(Compounds 7 and 23)
Preparation of 5-Fluoro-Q-BAPTA (Compound 7)
[0376] a catalytic quantity of p-toluenesulfonic acid (TsOH) was
added to a solution of anthranilamide (29 mg, 0.21 mmol) and
5'-fluoro-5-formyl-4-hydroxy-BAPTA tetramethylester (128 mg, 0.21
mmol) in 10 mL dichloroethane/5 mL ethanol. The solution was
refluxed overnight then cooled. Chloranil (57 mg, 0.23 mmol) was
added. After 2 hours, the solution was evaporated and the residue
was purified by flash chromatography using 5% methanol/chloroform
to yield 50 mg of the tetramethylester of Compound 7 as a
light-amber immobile oil; m/z 711 (710 calc for
C.sub.34H.sub.34N.sub.4O.sub.12F).
[0377] To a green solution of the tetramethylester of compound 7
(50 mg, 0.07 mmol) in 1:1 dioxane:methanol (5 mL), was added 1 M
aqueous KOH (0.56 mL, 0.56 mmol). The yellow solution was stirred
overnight then evaporated. The residue was purified with water on
Sephadex LH-20, generating 53 mg of compound 7 as its potassium
salt as a yellow powder; m/z (positive mode) 655 (651 calculated
for C.sub.30H.sub.23N.sub.4O.sub.12F).
Preparation of 5,6-Difluoro-Q-BAPTA (Compound 23)
[0378] 5,6-Difluoro-4'-hydroxy-5'-formyl BAPTA tetramethylester
(0.100 g, 0.163 mmol) and anthranilamide (0.022 g, 0.162 mmol) were
dissolved in a mixture of methylene chloride (10 mL) and ethanol (5
mL). TsOH (5 mg) was added and the reaction mixture was refluxed
for 3 hrs. Chloranil (0.044 g, 0.18 mmol) was added to the
solution. The mixture was refluxed for 2 more hours and evaporated.
The crude product was purified by preparative TLC using 2:1
chloroform-ethyl acetate as eluant. The main component
(R.sub.f=0.5) was isolated with ethyl acetate, which solution was
evaporated to give 5,6-difluoro-Q-BAPTA tetramethylester as a
colorless powder (0.029 g, 24%).
[0379] 5,6-Difluoro-Q-BAPTA tetramethylester (0.027 g, 0.037 mmol)
was dissolved in a mixture of 1 mL of methanol and 1 mL of dioxane.
1 M KOH (1 mL) was added to the solution and the reaction mixture
was kept overnight at room temperature. Volatiles were evaporated,
the crude product was redissolved in water and purified on a
Sephadex LH-20 column, eluting with water. The product was
lyophilized to give 0.021 g of 5,6-difluoro-Q-BAPTA potassium salt
(Compound 23) as a yellow powder (R.dbd.CH.sub.2CO.sub.2K).
##STR00023##
Example 34
Synthesis of (borapolyazaindacene BAPTA) Compounds (Compound 8 and
24)
Preparation of BODIPY FL Dye BAPTA-5F (Compound 8)
[0380] To a cold solution of 5-fluoro-BAPTA tetramethylester (1.00
g, 1.82 mmol) in 9 mL acetic anhydride was added 70% nitric acid
(0.15 mL, 2.3 mmol). After 10 minutes, the reaction solution was
poured into 30 mL aqueous NaOAc then saturated aqueous sodium
bicarbonate was added. The mixture was extracted with chloroform
(2.times.30 mL). The extract was washed with brine, dried over
sodium sulfate, and concentrated to an amber residue. This was
purified by flash chromatography using ethyl acetate/hexanes to
give 0.43 g of 5-nitro-5'-fluoro-BAPTA, tetramethylester as a
yellow powder.
[0381] To 5-nitro-5'-fluoro-BAPTA, tetramethylester (0.43 g, 0.72
mmol) in 1:1 methanol/dioxane (10 mL) was added 1 M KOH (5.8 mL,
5.8 mmol). The solution was stirred overnight then evaporated. The
residue was dissolved in 10 mL water, and the pH lowered to 2 with
aqueous HCl. A precipitate was collected and dried to give 0.31 g
of 5-nitro-5'-fluoro-BAPTA free acid as a yellow powder.
[0382] A solution of 5-nitro-5'-fluoro-BAPTA free acid (0.31 g,
0.58 mmol) in 30 mL methanol was shaken over 10% Pd/carbon (0.15 g)
under 38 psi hydrogen gas for 6 hours, then filtered and evaporated
to give 0.26 g of 5-amino-5'-fluoro-BAPTA free acid as a colorless
powder.
[0383] BODIPY FL dye free acid (Molecular Probes, Inc. D-2183, 27
mg, 0.09 mmol) in 5 mL anhydrous THF was treated with an oxalyl
chloride (0.20 mmol) and diisopropylethylamine (DIEA, 0.20 mmol)
under argon. After 15 minutes, the solution was evaporated. The
residue was dissolved in 3 mL anhydrous dioxane, and this solution
was slowly added to a solution of 5-amino-5'-fluoro-BAPTA free acid
(50 mg, 0.10 mmol) in 5 mL water that had been pH-adjusted to
pH=9.5 with sodium carbonate. This solution was stirred overnight
then evaporated to near dryness. This solution was purified with
water elution on Sephadex LH-20 to yield 41 mg of BODIPY FL Dye
BAPTA-5F (Compound 8), sodium salt as an orange powder.
Preparation of BODIPY FL Dye-EDA-BAPTA (Compound 24)
[0384] To a solution of 5-amino-BAPTA free acid (853 mg, 1.74 mmol)
in water (50 mL) and con. HCl (1.0 mL), thiophosgen (10 mL) in
chloroform (50 mL) was added and vigorously stirred at rt for 8 h.
The organic solvent was evaporated, and the precipitate was
collected by a centrifugal. The dried precipitate was redissolved
in THF (20 mL), and precipitated with hexanes (200 mL). The
precipitate was collected by a centrifugal and dried to give
5-isothiocyanato-BAPTA free acid (640 mg).
[0385] The pH of a solution of BODIPY FL ethylenediamine
hydrochloride salt (15 mg, 0.04 mmol, Molecular Probes) in 3 mL
water was raised to 7.6 by dropwise addition of aqueous sodium
bicarbonate. A solution of 5-isothiocyanato-BAPTA free acid (22 mg,
0.04 mmol) in 2 mL dioxane was added. The pH was raised to 9.5 with
aqueous sodium carbonate, and the orange solution was stirred at
room temperature overnight. The solution was evaporated to 2 mL,
and the this solution was purified on Sephadex LH-20 using water
for elution to give 17 mg of BODIPY FL-EDA-BAPTA sodium salt
(Compound 24) as a fine orange powder after lyophilization
(R.dbd.CH.sub.2CO.sub.2Na).
##STR00024##
Example 35
Synthesis of Biotinylated BAPTA Compounds (Compounds 9, 12 and
18)
Preparation of Biotin-BAPTA-5F (Compound 12)
[0386] A solution of 5-nitro-5'-fluoro-BAPTA, tetramethylester was
reduced by catalytic hydrogenation over 10% Pd/C in ethyl acetate.
To the resulting 5-amino-5'-fluoro-BAPTA, tetramethylester (0.10 g,
0.18 mmol) in anhydrous dichloromethane/THF (4:1, 5 mL) was added
glutaric anhydride (40 mg, 0.36 mmol) and catalytic DMAP. The
solution was stirred overnight then evaporated. The residue was
purified by flash chromatography using 10% methanol/chloroform to
give 0.13 g of the glutaramide of 5-amino-5'-fluoro-BAPTA,
tetramethylbester as an oil.
[0387] To the glutaramide of 5-amino-5'-fluoro-BAPTA,
tetramethylester (0.18 mmol) in 5 mL anhydrous THF and 5 mL
anhydrous acetonitrile was added N-hydroxysuccinimidyluronium
tetrafluoroborate (108 mg, 0.36 mmol). After two hours a solution
of biotin ethylenediamine hydrobromide (66 mg, 0.18 mmol, Molecular
Probes) and DIEA (0.05 mL) in 2 mL anhydrous DMF was added. After
stirring overnight, the volatiles were evaporated. The residue was
triturated with water (15 mL), and the resulting precipitate was
collected, rinsed with water, and dried to give 0.10 g of
biotin-BAPTA-5F tetramethylester as a gray powder.
[0388] To biotin-BAPTA-5F tetramethylester (0.10 g, 0.11 mmol) in
1:1 methanol/dioxane (4 mL) was added 1 M KOH (1.0 mL, 1.0 mmol).
The solution was stirred overnight then evaporated. The residue was
purified on Sephadex LH-20 using water, which gave biotin-BAPTA-5F
(Compound 12) potassium salt as a colorless powder after
lyophilization.
Preparation of Rhod-biocytin (Compound 18)
[0389] To a 0.5 M solution of 4-(succinimidyloxycarbonyl)-rhod
tetramethyl ester in anhydrous THF was added 1.1 equivalent of
N-t-BOC-ethylenediamine and 1.1 equivalent of DIEA. The resulting
solution was stirred for 30 minutes then evaporated. The residue
was purified by flash chromatography using
chloroform/methanol/acetic acid. The purified carbonate was
dissolved in dichloromethane and treated with trifluoroacetic acid
(20 equivalents). This solution was stirred 30 minutes, then
evaporated and dried to give the ethylenediamine carboxamide of
4-carboxy-rhod tetra methyl ester.
[0390] To a 0.5 M solution of the ethylenediamine carboxamide of
4-carboxy-rhod tetramethyl ester in DMF was added N-t-BOC-biocytin
succinimidyl ester (1.5 equivalent, described in Wilbur et al.,
Bioconjugate Chemistry, 11: 584-98 (2000)) and DIEA (1.5
equivalent). The resulting solution was stirred at room temperature
until the TLC indicated consumption of the fluorescent starting
material. The volatiles were removed in vacuo, and the residue was
purified by flash chromatography using chloroform/methanol/acetic
acid to give N-t-BOC-rhod-biocytin tetramethyl ester.
[0391] A 0.5 M solution of N-t-BOC-rhod-biocytin tetramethyl ester
in 1:1 methanol/dioxane was treated with 12 equivalents of 1 M KOH.
The resulting solution was stirred overnight at room temperature
then evaporated to dryness. The residue was purified on Sephadex
LH-20 using water to give Compound 18 as a red powder after
lyophilization (R.dbd.CH.sub.2CO.sub.2K).
##STR00025##
Preparation of Rhod-4-biotin-BAPTA (Compound 9)
[0392] A suspension of (2'-nitrophenoxy)-2-chloroethane (20.15 g,
0.10 mol), methyl (4-hydroxy-3-nitro)benzoate (21.67 g, 0.11 mol),
and K.sub.2CO.sub.3 (27.60 g, 0.20 mol) was stirred at 130.degree.
C. for 16 h, cooled to room temperature, and poured into ice water
(1.2 L). The precipitate was filtered, washed with H.sub.2O and
dried to give 32.00 g of
(4'-methoxycarbonyl-2'-nitrophenoxy)-2-(2''-nitrophenoxy)ethane as
a yellow solid.
4'-Methoxycarbonyl-2'-nitrophenoxy)-2-(2''-nitrophenoxy)ethane
(20.0 g, 55.2 mmol) was hydrogenated over 10% Pd/C (3.0 g) in DMF
(300 mL) at 40 psi for 5 h. The mixture was filtered from catalyst
through Celite. The filtrate was evaporated and ether (100 mL) was
added. The product was filtered and washed with ether (2.times.25
mL) to give 13.2 g of
1'-amino-4'-methoxycarbonylphenoxy)-2-(2''-aminophenoxy)ethane as
an off-white solid.
[0393] A mixture of
2'-amino-4'-methoxycarbonylphenoxy)-2-(2''-aminophenoxy)ethane
(13.20 g, 44 mmol), methanol (50 mL), dioxane (50 mL), and 1 M KOH
(100 mL, 100 mmol) was stirred at 65.degree. C. for 5 h, then
overnight at room temperature. The mixture was evaporated and the
residue was suspended in H.sub.2O (500 mL). Aqueous 1 M HCl was
added to pH 5.0. The precipitated product was filtered, washed with
H.sub.2O, and dried on a filter for 4 h, then washed with ether
(3.times.25 mL) to give 12.5 g of
2'-amino-4'-carboxy-1'-phenoxy)-2-(2''-aminophenoxy)ethane as an
off-white solid.
[0394] Diphenyldiazomethane was prepared by vigorously stirring
benzophenone hydrazone (6.66 g, 34 mmol) and yellow HgO (17.60 g,
80 mmol) in hexanes (200 mL) for 3 h. The mixture was filtered from
inorganics, evaporated and the residue was dissolved in acetone (50
mL). This solution was added to a suspension of
2'-amino-4'-carboxy-1'-phenoxy)-2-(2''-aminophenoxy)ethane (5.76 g,
20 mmol) in acetone. The mixture was stirred for 16 h at 35.degree.
C., then the excess of diphenyldiazomethane was decomposed with
AcOH (2 mL) over 2 h. The mixture was evaporated, and the crude
product was purified by flash chromatography on SiO.sub.2 using
CHCl.sub.3 as eluant to give 6.80 g of
2'-amino-4'-diphenylmethoxycarbonylphenoxy)-2-(2''-aminophenoxy)etha-
ne as an off-white solid.
[0395] A mixture of
2'-amino-4'-diphenylmethoxycarbonylphenoxy)-2-(2''-aminophenoxy)ethane
(8.28 g, 18.24 mmol), DIEA (16.3 mL, 94 mmol), methyl bromoacetate
(35.3 mL, 376 mmol), and NaI (1.50 g, 10 mmol) in MeCN (400 mL) was
refluxed under stirring for 70 h, cooled to room temperature and
evaporated. The residue was dissolved in CHCl.sub.3 (500 mL),
washed with 1% AcOH (3.times.200 mL), H.sub.2O (200 mL), sat. NaCl
(200 mL), filtered and evaporated. The residue was purified by
flash chromatography on SiO.sub.2 using a gradient of 30-40% EtOAc
in hexanes as eluant to give 10.01 g of
4-diphenylmethoxycarbonyl-BAPTA tetramethyl ester as a white
solid.
[0396] To a solution of Vilsmeier reagent made from POCl.sub.3 (5
mL, 50 mmol) in DMF (35 mL) was added a solution of
4-diphenylmethoxycarbonyl-BAPTA tetramethyl ester (3.71 g, 5 mmol)
in DMF (15 mL). The mixture was stirred at 40.degree. C. for 24 h,
then another portion of Vilsmeier reagent (25 mmol) was introduced
and the mixture was stirred at 40.degree. C. for 70 h. The mixture
was cooled to room temperature and quickly poured into an ice-sat.
K.sub.2CO.sub.3 mixture (1200 mL). After 1 h, the precipitate was
filtered, washed with H.sub.2O and dried to give 3.78 g of
4-diphenylmethoxycarbonyl-5'-formyl-BAPTA tetramethyl ester as a
colorless solid.
[0397] A mixture of 4-diphenylmethoxycarbonyl-5'-formyl-BAPTA
tetramethyl ester (2.90 g, 3.8 mmol), m-dimethylaminophenol (1.21
g, 8.8 mmol), and TsOH (100 mg,) in propionic acid (40 mL) was
stirred at 68.degree. C. for 20 h, then cooled to room temperature
and poured into 3 M NaOAc (600 mL). After 1 h, the precipitate was
filtered, washed with water, and dried to give 3.70 g of
4-diphenylmethoxycarbonyl-dihydrorhod tetramethyl ester as a
purple-red solid.
[0398] A mixture of 4-diphenylmethoxycarbonyl-dihydrorhod
tetramethylester (2.050 g, 2.0 mmol) and powdered chloranil (0.492
g, 2.0 mmol) in CHCl.sub.3 and MeOH (40 mL of each) was stirred for
2 h, filtered and evaporated. The residue was purified by flash
chromatography on SiO.sub.2 using a gradient 5-6.5% MeOH in
CHCl.sub.3/1% AcOH as eluant to give a crude product, which was
re-dissolved in CHCl.sub.3, filtered from SiO.sub.2, and evaporated
to give 0.533 g of 4-diphenylmethoxycarbonyl-rhod tetramethyl ester
as a dark-purple solid.
[0399] To 4-diphenylmethoxycarbonyl-rhod tetramethyl ester (51 mg,
0.05 mmol) in dioxane (2 mL) and MeOH (1 mL) was added 1 M KOH to
give pH 12.0. The mixture was stirred for 20 h, then the pH
adjusted to 9.0 with 0.1 M HCl. The mixture was evaporated and the
residue purified on Sephadex LH-20 using H.sub.2O as eluant. The
product was lyophilized to give 26 mg of 4-carboxy-rhod
tetrapotassium salt as a red-purple solid.
[0400] To 4-diphenylmethoxycarbonyl-rhod tetramethyl ester (102 mg,
0.1 mmol) in CHCl.sub.3 (10 mL) was added TFA (10 mL) and the
resulting mixture was stirred for 1 h then evaporated and
co-evaporated with CHCl.sub.3 (3.times.10 mL). Ether (10 mL) was
added and the precipitate was filtered and washed with ether
(3.times.10 mL) to give 82 mg of 4-carboxy-rhod tetramethyl ester
as a dark purple solid.
[0401] To 4-carboxy-rhod tetramethyl ester (80 mg, 0.093 mmol) in
DMF (2 mL) was added DIEA (0.35 mL, 2 mmol) and dry
O-trifluoroacetyl-N-hydroxysuccinimide (TFA-SE, 225 mg, 1 mmol).
The mixture was stirred for 2 h, then more TFA-SE (113 mg, 0.5
mmol) was introduced and the mixture stirred for another 16 h. The
mixture was diluted with CHCl.sub.3 (50 mL), washed with 1% AcOH
(3.times.20 mL), H.sub.2O (25 mL), sat. NaCl (50 mL), filtered and
evaporated. Ether (25 mL) was added and the precipitated product
was filtered and washed with ether to give 86 mg of
4-(succinimidyloxycarbonyl)-rhod tetramethyl ester as a dark-purple
solid.
[0402] To biotin cadaverine (34 mg, 0.077 mmol, Molecular Probes,
Inc.) in DMF (1 mL) and DIEA (0.055 mL, 0.40 mmol) was added a
solution of 4-(succinimidyloxycarbonyl)-rhod tetramethyl ester (36
mg, 0.038 mmol). The mixture was stirred for 3 h, diluted with
CHCl.sub.3 (200 mL), washed with 1% AcOH (3.times.150 mL), H.sub.2O
(100 mL), sat. NaCl (200 mL), filtered and evaporated. The residue
was purified on two preparative TLC SiO.sub.2 plates, using 12%
MeOH and 2.5% AcOH in CHCl.sub.3 as eluant to give 38 mg of
4-(N-(5''-biotinylaminopentyl)aminocarbonyl)-rhod tetramethyl
ester.
[0403] To 4-(N-(5''-biotinylaminopentyl)aminocarbonyl)-rhod
tetramethyl ester (30 mg, 0.025 mmol) in MeOH (2 mL) and H.sub.2O
(1 mL) was added 1 M KOH to give pH 12.0. The mixture was stirred
for 20 h then adjusted to pH 8.5 with 0.1 M HCl. The mixture was
evaporated and the residue purified on Sephadex LH-20 using
H.sub.2O as eluant. The product was lyophilized to give 30 mg of
Compound 9 (4-(N-(5''-biotinylaminopentyl)aminocarbonyl)-rhod
tetrapotassium salt) as an orange-red solid.
Example 36
Synthesis of 4-(4'-(Aminophenyl)-2-ethylamino)carbonylmethyl-rhod
tripotassium Salt (Compound 10)
[0404] A suspension of (2'-nitrophenoxy)-2-chloroethane (5.87 g, 29
mmol), methyl 4-hydroxy-3-nitrophenyl acetate (6.15 g, 29 mmol),
and K.sub.2CO.sub.3 (8.28 g, 60 mmol) was stirred at 120.degree. C.
for 16 h, cooled to room temperature, and poured into ice water
(0.6 L). The precipitate was filtered, washed with H.sub.2O and
dried to give 4.49 g of
(4'-methoxycarbonylmethyl-2'-nitrophenoxy)-2-(2''-nitrophenoxy)ethane
as a yellow solid.
[0405]
4'-(Methoxycarbonylmethyl-2'-nitrophenoxy)-2-(2''-nitrophenoxy)etha-
ne (9.6 g, 25.5 mmol) was hydrogenated over 10% Pd/C (1.0 g) in DMF
(250 mL) at 40 psi for 16 h. The mixture was filtered from catalyst
through Celite. The filtrate was evaporated and the residue was
purified by flash chromatography on SiO.sub.2 using a gradient of
25-35% EtOAc in hexanes to give 5.53 g of
(2'-amino-4'-methoxycarbonylmethylphenoxy)-2-(2''-aminophenoxy)ethane
as an off-white solid.
[0406] A mixture of
(2'-amino-4'-methoxycarbonylmethylphenoxy)-2-(2''-aminophenoxy)ethane
(5.50 g, 17.4 mmol), methanol (40 mL), dioxane (40 mL), and 1 M KOH
(35 mL, 35 mmol) was stirred at 45.degree. C. for 1 h, then
overnight at room temperature. The mixture was evaporated and the
residue was suspended in H.sub.2O (100 mL). Aqueous 1 M HCl was
added to pH 3.0. Precipitated product was filtered, washed with
H.sub.2O, and dried to give 4.59 g of
(2'-amino-4'-carboxymethyl-1'-phenoxy)-2-(2''-aminophenoxy)ethane
as an off-white solid.
[0407] Diphenyldiazomethane was prepared by vigorously stirring
benzophenone hydrazone (2.94 g, 15 mmol) and yellow HgO (8.80 g, 40
mmol) in hexanes (70 mL) for 5 h. The mixture was filtered from
inorganics, and the filtrate was evaporated and the residue was
redissolved in acetone (20 mL). This solution was added to the
solution of the
2'-amino-4'-carboxymethyl-1'-phenoxy)-2-(2''-aminophenoxy)ethane
(3.02 g, 10 mmol) in acetone (120 mL). The resulting mixture was
stirred for 16 h at 35.degree. C., then the excess of
diphenyldiazomethane was decomposed with AcOH (0.5 mL) over 2 h.
The mixture was evaporated, and the crude product was purified by
flash chromatography on SiO.sub.2 using 1% MeOH in CHCl.sub.3 as
eluant to give 4.44 g of
(2'-amino-4'-diphenylmethoxycarbonylmethylphenoxy)-2-(2''-aminophenoxy)et-
hane as an off-white solid.
[0408] A mixture of
2'-amino-4'-diphenylmethoxycarbonylmethylphenoxy)-2-(2''-aminophenoxy)eth-
ane (2.12 g, 4.5 mmol), DIEA (4.0 mL, 23.5 mmol), methyl
bromoacetate (8.8 mL, 94 mmol), and NaI (0.50 g, 4.7 mmol) in MeCN
(90 mL) was refluxed for 70 h, cooled to room temperature and
evaporated. The residue was dissolved in CHCl.sub.3 (500 mL),
washed with 1% AcOH (3.times.200 mL), H.sub.2O (200 mL), sat. NaCl
(200 mL), filtered and evaporated. The residue was purified by
flash chromatography on a SiO.sub.2 column using a gradient of
30-40% EtOAc in hexanes as eluant to give 2.82 g of
4-diphenylmethoxycarbonylmethyl-BAPTA tetramethyl ester as a
colorless solid.
[0409] To a solution of Vilsmeier reagent made from POCl.sub.3 (1.5
mL, 30 mmol) in DMF (10 mL) was added a solution of
4-(diphenylmethoxycarbonylmethyl)-BAPTA tetramethyl ester (3.78 g,
5 mmol) in DMF (5 mL). The mixture was stirred for 24 h then
quickly poured into an ice-sat. K.sub.2CO.sub.3 mixture (500 mL).
The mixture was extracted with CHCl.sub.3, dried over MgSO.sub.4
and evaporated. The mixture of products was separated on SiO.sub.2
using a gradient of 30-40% EtOAc in hexanes to give 1.65 g of
aldehyde 4-(diphenylmethoxycarbonylmethyl)-5'-formyl-BAPTA
tetramethyl ester as a colorless solid.
[0410] A mixture of
4-(diphenylmethoxycarbonylmethyl)-5'-formyl-BAPTA tetramethyl ester
(784 mg, 1.0 mmol), m-dimethylaminophenol (301 mg, 2.2 mmol), and
TsOH (20 mg, catalyst) in propionic acid (10 mL) was stirred at
65.degree. C. for 20 h, then cooled to room temperature and poured
into 3 M NaOAc (150 mL). After 1 h, the precipitate was filtered,
washed with water, and dried to give 450 mg of
4-(diphenylmethoxycarbonylmethyl)-dihydrorhod tetramethyl ester as
a purple-red solid.
[0411] A mixture of 4-(diphenylmethoxycarbonylmethyl)-dihydrorhod
tetramethyl ester (420 mg, 0.43 mmol) and powdered chloranil (122
mg, 0.5 mmol) in CHCl.sub.3 and MeOH (20 mL of each) was stirred
for 3 h, filtered and evaporated. The residue was purified by flash
chromatography on SiO.sub.2 using a gradient of 5-7% MeOH in
CHCl.sub.3/0.5% AcOH as eluant to give a crude product, which was
redissolved in CHCl.sub.3, filtered from SiO.sub.2, and evaporated
to give 275 mg of
4-(diphenylmethoxycarbonylmethyl-5'-tetramethyl)-rhod tetramethyl
ester as a dark-purple solid.
[0412] To a solution of
4-(diphenylmethoxycarbonylmethyl-5'-tetramethyl)-rhod tetramethyl
ester (250 mg, 0.25 mmol) in CHCl.sub.3 (20 mL) was added TFA (20
mL) and the resulting mixture was stirred for 1 h, then evaporated
and co-evaporated with CHCl.sub.3 (3.times.30 mL). Ether (30 ml)
was added to the residue and the precipitate was filtered and
washed with ether (3.times.10 mL) to give 200 mg of
4-carboxymethyl-rhod tetramethyl ester as a dark-purple solid.
[0413] To 4-carboxymethyl-rhod tetramethyl ester (128 mg, 0.15
mmol) in DMF (5 mL) and DIEA (0.40 mL, 2.2 mmol) was added dry
TFA-SE (338 mg, 1.5 mmol). The mixture was stirred for 16 h, then a
solution of 4-aminophenylethylamine (0.4 mL, 4 mmol) and DIEA (0.4
mL, 2.2. mmol) was introduced. The mixture was stirred for 2 h,
diluted with CHCl.sub.3 (500 mL), washed with 1% AcOH (3.times.100
mL), sat. NaCl (2.times.200 mL), filtered and evaporated. Ether (25
mL) was added to the residue, and the precipitated product was
filtered and washed with ether to give 126 mg of
4-(4'-(aminophenyl)-2-ethylamino)carbonylmethyl-rhod tetramethyl
ester as a dark-red solid.
[0414] To 4-(4'-(aminophenyl)-2-ethylamino)carbonylmethyl-rhod
tetramethyl ester (100 mg, 0.1 mmol) in dioxane (2 mL), MeOH (2 mL)
and H.sub.2O (1 mL) was added 1 M KOH to give pH 12.0. The mixture
was stirred for 50 h then the pH was adjusted to 9.0 with 0.1 M
HCl. The mixture was evaporated and the residue was purified on
Sephadex LH-20 using H.sub.2O as eluant and the product lyophilized
to give 21 mg of Compound 10 as an orange-red solid.
Example 37
Synthesis of BAPTA-Agarose Compounds (Compounds 13 and 14)
Preparation of BAPTA-Agarose (compound 13)
[0415] A solution of 5-isothiocyanato-BAPTA free acid (65 mg, 0.12
mmol, U.S. Pat. No. 5,453,517) in 3 mL anhydrous DMF was added to a
slurry of amino agarose (50% aqueous slurry, 16 .mu.mol amine/mL, 6
mL, 96 .mu.mole amine, Pierce) that had been diluted with 15 mL
DMF. The pH was raised to 10 with DIEA (1.5 mL). The resulting
light-brown mixture was stirred at room temperature for 48 hours
then centrifuged. The BAPTA-agarose (compound 13) pellet was rinsed
with acetone (2.times.) and water (2.times.) then suspended in
water.
Preparation of BAPTA-5F-Agarose (Compound 14)
[0416] A solution of 5-amino-5'-fluoro-BAPTA free acid (0.26 g,
0.51 mmol) in 12 mL aqueous HCl was diluted with 12 mL chloroform
then treated with thiophosgene (3 mL). The orange mixture was
stirred at room temperature overnight then evaporated. The mixture
was centrifuged, yielding a brown gum that was dried then dissolved
in 2 mL anhydrous THF. Addition of 20 mL ethyl acetate gave a
precipitate, which was isolated by centrifugation to give
5-fluoro-5'-isothiocyanato-BAPTA free acid as a light gray-brown
powder.
[0417] 5-Fluoro-5'-isothiocyanato-BAPTA free acid (25 mg, 0.05
mmol) in 1 mL anhydrous DMF was added to 2 mL of a 50% aqueous
slurry of amino agarose that had been diluted with 5 mL DMF. The pH
was raised to 10 with a few drops of DIEA. The light-brown mixture
was stirred at room temperature for 48 hours then centrifuged.
BAPTA-5F-agarose (Compound 14) pellet was rinsed with acetone
(2.times.) and water (2.times.) then suspended in water.
Example 38
Synthesis of Compound 15 (TAMRA-Biotin BAPTA Compound)
[0418] A solution of BAPTA-4-isothiocyanate free acid (18 mg, 0.033
mmol) in 5 mL dioxane was added to a solution of
5-(and-6)-tetramethylrhodamine biocytin (Molecular Probes Inc., 29
mg, 0.033 mmol) in 4 mL water. The resulting pH (3.5) was raised to
10 with aqueous sodium carbonate. The resulting red solution was
stirred at ambient temperature overnight, the concentrated in
vacuo. The residue was purified by column chromatography on
Sephadex LH-20, using water as eluant. The product was lyophilized
to give TAMRA-biotin-BAPTA as 26 mg of red powder: LCMS m/2 726
(1452 calculated for C.sub.73H.sub.84N.sub.11O.sub.17S.sub.2).
Example 39
Synthesis of Compound 16 (Rhodamine BAPTA Compound)
[0419] 5-Formyl-5'-nitro-BAPTA tetramethyl ester (200 mg, 0.33
mmol) and 8-hydroxyjulolidine (125 mg, 0.66 mmol) in 5 mL propionic
acid was heated under nitrogen at 70.degree. C. for 1 hour, cooled
to room temperature and poured into 30 mL concentrated potassium
acetate solution. The mixture was extracted with chloroform then
washed with brine, dried over sodium sulfate, and evaporated to a
red oil that was purified by flash chromatography using ethyl
acetate/hexanes to give 0.225 g of dihydro-X-Rhod-5N tetramethyl
ester as a yellow foam.
[0420] To dihydro-X-Rhod-5N tetramethyl ester (0.12 g, 0.12 mmol)
in 1:1 chloroform/methanol (5 mL) was added chloranil (40 mg, 0.16
mmol). The solution was stirred overnight, diluted with 50 mL
chloroform, washed with brine, dried over sodium sulfate, and
evaporated. The residue was purified by flash chromatography using
15% methanol/chloroform to give 63 mg of X-Rhod-5N tetramethyl
ester as a purple powder.
[0421] To X-Rhod-5N tetramethyl ester (0.11 g, 0.11 mmol) in 5 mL
methanol was added 2 M KOH (0.6 mL, 1.2 mmol). The solution was
stirred at room temperature overnight, then evaporated. The residue
was dissolved in water (5 mL) and the pH lowered to 2 with 2 M HCl.
A precipitate was collected by centrifugation, dissolved in fresh
aqueous KOH and precipitated with aqueous HCl. This procedure was
repeated five times to give 90 mg of Compound 16 free acid as a
purple powder.
Example 40
Synthesis of 4-Hydroxy-5-benzothiazolyl-BAPTA (Compound 17)
[0422] A solution of 4-hydroxy-5-formyl-5'-methyl BAPTA,
tetramethylester (0.40 g, 0.68 mmol) and 2-aminothiophenol (75 mg,
0.70 mmol) in DMSO (5 mL) was heated at reflux for 15 minutes.
After cooling the yellow solution was diluted with 50 mL water. A
yellow precipitate was filtered and dried, then purified by flash
chromatography using ethyl acetate/hexanes to give 0.22 g of
4-hydroxy-5-benzothiazolyl-BAPTA tetramethylester as a yellow
foam.
[0423] To 4-hydroxy-5-benzothiazolyl-BAPTA tetramethylester (0.21
g, 0.30 mmol) in 1:1 methanol/dioxane (10 mL) was added 1 M KOH
(3.0 mL, 3.0 mmol). The solution was stirred for 3 hours then
evaporated. The residue was purified on Sephadex LH-20 using water
as eluant to give 0.13 g of compound 17 as a yellow-green powder
(R.dbd.CH.sub.2CO.sub.2K).
##STR00026##
Example 41
Synthesis of 4'-Carboxymethyl-4-methoxy-rhod, potassium salt
(Compound 19)
[0424] A suspension of (4'-methoxy-2'-nitrophenoxy)-2-chloroethane
(11.29 g, 48.7 mmol), methyl 4-hydroxy-3-nitrophenylacetate (10.80
g, 51.2 mmol), and K.sub.2CO.sub.3 (13.80 g, 100 mmol) was stirred
at 130.degree. C. for 4 h, cooled to room temperature, and poured
into ice water (0.8 L), allowed to coagulate for 2 days. The
precipitate was filtered, washed with H.sub.2O and dried to give
15.1 g of
(4'-methoxycarbonylmethyl-2'-nitrophenoxy)-2-(4''-methoxy-2''-nitrophenox-
y)ethane as a yellow solid.
[0425]
(4'-Methoxycarbonylmethyl-2'-nitrophenoxy)-2-(4''-methoxy-2''-nitro-
phenoxy)ethane (15.0 g, 43.3 mmol) was hydrogenated over 10% Pd/C
(2.0 g) in CH.sub.2Cl.sub.2 (250 mL) at 45 psi for 16 h. The
mixture was filtered through Celite. The filtrate was evaporated
and the residue was treated with ether (200 mL). The precipitate
was filtered and washed with ether (3.times.25 mL) to give 11.21 g
of
(2'-amino-4'-methoxycarbonylmethylphenoxy)-2-(2''-amino-4''-methoxyphenox-
y)ethane as off-white solid.
[0426] A mixture of
(2'-amino-4'-methoxycarbonylmethylphenoxy)-2-(2''-amino-4''-methoxyphenox-
y)ethane (8.65 g, 25 mmol), methanol (80 mL), dioxane (80 mL), and
1 M KOH (50 mL, 50 mmol) was stirred at 60.degree. C. for 1 h, then
overnight at room temperature. The mixture was evaporated and the
residue was suspended in H.sub.2O (300 mL). Aqueous 1 M HCl was
added to pH 4.0. The precipitate was filtered, washed with
H.sub.2O, and dried to give 6.84 g of
(2'-amino-4'-carboxymethyl-1'-phenoxy)-2-(2''-amino-4''-methoxyphenoxy-
)ethane as off-white solid.
[0427] Diphenyldiazomethane was prepared by vigorously stirring
benzophenone hydrazone (5.88 g, 30 mmol) and yellow HgO (17.60 g,
80 mmol) in hexanes (150 mL) for 6 h. The mixture was filtered from
inorganics, filtrate was evaporated and the residue was
re-dissolved in acetone (40 mL). This solution was added to the
solution of
(2'-amino-4'-carboxymethyl-1'-phenoxy)-2-(2''-amino-4''-methoxyphenoxy)et-
hane acid (6.64 g, 20 mmol) in acetone (200 mL). The resulting
mixture was stirred for 48 h at 35.degree. C., evaporated and the
residue was suspended in CHCl.sub.3. To the suspension was added
AcOH (4 mL) to decompose the excess reagent and the mixture was
stirred for 2 h, then evaporated, and the crude product was
purified by flash chromatography on SiO.sub.2 using 0.5% MeOH in
CHCl.sub.3 as eluant to give
(2'-amino-4'-diphenylmethoxycarbonylmethylphenoxy)-2-(2''-amino-4''-metho-
xyphenoxy)ethane, 7.81 g (78%) as an off-white solid. A mixture of
diamine
(2'-amino-4'-diphenylmethoxycarbonylmethylphenoxy)-2-(2''-amino-4''-metho-
xyphenoxy)ethane (4.62 g, 9.3 mmol), DIEA (52 mL, 300 mmol), methyl
bromoacetate (19 mL, 200 mmol), and NaI (0.75 g, 5 mmol) in MeCN
(150 mL) was refluxed under stirring for 70 h, cooled to room
temperature and evaporated. The residue was dissolved in CHCl.sub.3
(400 mL), washed with 1% AcOH (3.times.200 mL), H.sub.2O (200 mL),
sat. NaCl (2.times.200 mL), filtered and evaporated. The residue
was purified by flash chromatography on SiO.sub.2 using a gradient
of 25-40% EtOAc in hexanes as eluant to give 3.01 g of
4-diphenylmethoxycarbonylmethyl-4'-methoxy-BAPTA tetramethyl ester
as a colorless solid.
[0428] To a solution of Vilsmeier reagent made from POCl.sub.3
(0.28 mL, 3 mmol) in DMF (2 mL) was added a solution of
4-diphenylmethoxycarbonylmethyl-4'-methoxy-BAPTA tetramethyl ester
(762 mg, 1 mmol) in DMF (2 mL). The mixture was stirred for 2 h,
then was quickly poured into an ice-sat. K.sub.2CO.sub.3 mixture
(50 mL). The mixture was extracted with CHCl.sub.3 (7.times.20 mL),
dried over MgSO.sub.4 and evaporated. The mixture of products was
separated by column chromatography on SiO.sub.2 (4.times.35 cm bed)
using a gradient of 30-45% EtOAc in hexanes to give 760 mg of
4-diphenylmethoxycarbonylmethyl-5'-formyl-4'-methoxy-BAPTA
tetramethyl ester as a colorless solid.
[0429] A mixture of
4-diphenylmethoxycarbonylmethyl-5'-formyl-4'-methoxy-BAPTA
tetramethyl ester (1.58 g, 2.0 mmol), m-dimethylaminophenol (602
mg, 4.4 mmol), and TsOH (50 mg, catalyst) in propionic acid (20 mL)
was stirred at 65.degree. C. for 20 h, then cooled to room
temperature and poured into 3 M NaOAc (300 mL). After 1 h, the
precipitated product was filtered, washed with water, and dried to
give 2.00 g of 4-diphenylmethoxycarbonylmethyl-5'dihydrorhod
tetramethyl ester as a purple-red solid.
[0430] A mixture of compound
4-diphenylmethoxycarbonylmethyl-4'-methoxy-5'-dihydrorhod
tetramethyl ester (2.00 g, 1.9 mmol) and powdered chloranil (0.50
g, 2 mmol) in CHCl.sub.3 and MeOH (50 mL of each) was stirred for 4
h, filtered and evaporated. The residue was purified by flash
chromatography on SiO.sub.2 using a gradient of 5-7% MeOH in
CHCl.sub.3/0.5% AcOH to give a crude product, which was redissolved
in CHCl.sub.3, filtered from SiO.sub.2, and evaporated to give 480
mg of 4-(diphenylmethoxycarbonylmethyl)-4'-methoxy-rhod,
tetramethyl ester as a dark-purple solid.
[0431] To a solution of
4-(diphenylmethoxycarbonylmethyl)-4'-methoxy-rhod, tetramethyl
ester (45 mg, 0.04 mmol) in dioxane (1 mL), MeOH (2 mL) and
H.sub.2O (2 mL) was added 1 M KOH to pH 12.0. The mixture was
stirred for 50 h, then pH was adjusted to 9.0 with 0.1 M HCl. The
mixture was evaporated and the residue was purified on Sephadex
LH-20 column (2.6.times.90 cm bed) using H.sub.2O as eluant and
lyophilized to give 12 mg of Compound 19 as a red solid
(R.dbd.CH.sub.2CO.sub.2K).
##STR00027##
Example 42
Synthesis of Compound 20 Containing a DTPA Metal-Chelating
Moiety
[0432] BODIPY.RTM. TR cadaverine, Molecular Probes D-6251, 10 mg,
0.019 mmol was dissolved into a mixture of
(S)-1-p-isothiocyanatobenzyldiethylenetriaminepentaacetic acid
(DTPA isothiocyanate, Molecular Probes I-24221, 10 mg, 0.019 mmol)
in 2 mL water. The pH was raised to 10 with aqueous sodium
carbonate. The resulting blue solution was stirred at room
temperature for two days, then concentrated in vacuo. The residue
was purified by column chromatography on Sephadex LH-20 using
E-pure water as eluant to give 2 mg of Compound 20 as a purple
powder.
Example 43
Synthesis of Compound 21 Containing a DTPA Metal-Chelating
Moiety
[0433] For the synthesis of carbamate 21a a solution of
penta-t-butyl 1-(S)-(p-aminobenzyl)-diethylenetriaminepentaacetate
(prepared according to the published procedure of Donald T. Corson
& Claude F. Meares. Bioconjugate Chem., 11(2): 292-299 (2000),
0.800 g, 1.03 mmol) in 20 mL of methylene chloride was added 1 mL
of pyridine followed by the addition of a solution of the acid
chloride of N--CBZ-6-aminohexanoic acid (0.290 g, 1.02 mmol) in 5
mL of methylene chloride. The reaction mixture was stirred
overnight at room temperature and concentrated in vacuo. The
residue was dissolved in 100 mL of ethyl acetate and the resulting
solution was washed with 10% HCl (2.times.30 mL), water (30 mL),
brine (30 mL) and dried over sodium sulfate. The solution was
concentrated and put on a silica gel column (packed with ethyl
acetate). The column was eluted with ethyl acetate to remove
impurities then the desired product was eluted with 10:1
chloroform-methanol. Pure fractions were combined and the solvent
evaporated to give amide 21a (0.54 g, 54%) as a viscous oil.
[0434] For the synthesis of amino acid 21b, the carbamate 21a
(0.700 g, 0.683 mmol) was dissolved in 10 mL of TFA. The reaction
mixture is kept for 3 days at room temperature. Volatiles were
evaporated and the residue was re-evaporated twice from toluene,
leaving a viscous oil. The oil was stirred with ethyl acetate until
it solidified. The resulting solid was filtered and dried to give
the amino acid 21b (0.400 g, 96%).
[0435] For the synthesis of Compound 21, the amino acid 21b (0.090
g, 0.147 mmol) was suspended in 10 mL of water. The pH was adjusted
to .about.8 using 1 M KOH. The resulting solution was added to a
solution of BODIPY.RTM. TMR-X, SE, Molecular Probes D-6117, 0.03 g,
0.049 mmol in 5 mL of DMF. The reaction mixture was stirred
overnight at room temperature. The pH was monitored and adjusted to
.about.8 during the first 2 hrs. The solution was evaporated and
the residue re-dissolved in water and purified on Sephadex LH-20
using water for elution. The combined product fractions were
concentrated to .about.3 mL then lyophilized to give 0.061 g of
Compound 21 as a red powder.
##STR00028##
Example 44
Synthesis of Compound 22 Containing a DTPA Metal-Chelating
Moiety
[0436] BODIPY.RTM. FL EDA, Molecular Probes D-2390, 7 mg, 0.019
mmol was dissolved into a mixture of DTPA isothiocyanate, Molecular
Probes I-24221, 10 mg, 0.019 mmol in 2 mL water. The pH was raised
to 10 with aqueous sodium carbonate. The resulting orange solution
was stirred at room temperature for 3.5 hours, then evaporated. The
residue was purified by on Sephadex LH-20 using water as eluant to
give 29 mg of Compound 22 as an orange powder.
Example 45
Synthesis of Compound 25
A BAPTA-Biotin
[0437] To a solution of biotin-cadaverine (21 mg, 0.047 mmol,
Molecular Probes) in 2 mL water was added 2 drops saturated sodium
carbonate solution. A solution of 5-isothiocyanato-BAPTA free acid
(25 mg, 0.047 mmol) in 3 mL dioxane was added. The reaction pH was
raised to 9.5 with more sodium carbonate solution, and the solution
was stirred overnight at ambient temperature. The volatiles were
removed in vacuo and the residue was purified by chromatography on
Sephadex LH-20 using water as eluant to give compound 25 as 40 mg
of a pale brown powder (R.dbd.CH.sub.2CO.sub.2Na).
##STR00029##
Example 46
Synthesis of Compound 26
A Rhod-BAPTA-BAPTA
[0438] To a solution of the ethylenediamine carboxamide of
4-carboxy-rhod tetramethyl ester (as in example 34, 0.04 mmol) in
1:1 water:dioxane (5 mL) was added a solution of
5-isothiocyanato-BAPTA free acid (24 mg, 0.044 mmol) in 1:1
water:dioxane (8 mL). The pH was raised to 8.5 by addition of
aqueous sodium bicarbonate. The resulting red solution was stirred
at ambient temperature overnight, then concentrated in vacuo and
purified by column chromatography on Sephadex LH-20 using water as
eluant to give the intermediate tetramethyl ester tetracarboxylate
as 11 mg of red powder. To a solution of this intermediate (0.007
mmol) in 1.4 mL water was added 1 M KOH (0.07 mmol). After 3 hours
the pH (13) was lowered to 9 with aqueous acetic acid, followed by
concentration in vacuo. The resulting residue was purified by
column chromatography on Sephadex LH-20 using water as eluant to
afford compound 26 as a red powder (R.dbd.CH.sub.2CO.sub.2K).
##STR00030##
Example 47
Synthesis of Compound 27a-i
[0439] The synthesis of 27a will serve to illustrate the synthetic
method used to make compounds 27a-27f. Oxalyl chloride (18 .mu.L,
0.20 mmol) was added to a solution of
4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic
acid (Molecular Probes B-2183, 50 mg, 0.17 mmol) in 5 mL anhydrous
THF, followed by diisopropylethylamine (DIEA, 35 .mu.L, 0.20 mmol).
The resulting solution was stirred at room temperature for 15
minutes, followed by concentration in vacuo. The resulting acid
chloride was dissolved in 3 mL dry dioxane. This solution was added
dropwise to a solution of 27' (83 mg, 0.17 mmol, X.dbd.H) in 5 mL
with stirring; the pH was maintained at 9 with sodium carbonate.
The resulting cloudy orange mixture was stirred for 1 hour,
whereupon silica gel TLC analysis indicated formation of 27a
(R.sub.f 0.40, dioxane-isopropyl alcohol-water-ammonium hydroxide
15:58:13:14). The volatiles were removed in vacuo, and the residue
purified by column chromatography on Sephadex LH-20 using water as
eluant. Pure product fractions were pooled and lyophilized to give
27a as 99 mg of a fluffy orange powder (68% yield): .sup.1H NMR
(D.sub.2O) .delta. 7.34 (s, 1H), 6.98-6.68 (m, 8H), 6.33 (d, J=4.0
Hz, 1H), 6.19 (s, 1H), 4.18 (m, 4H), 3.66 (s, 8H), 3.18 (t, 7.6 Hz,
2H), 2.72 (t, 7.6 Hz, 2H), 2.41 (s, 3H), 2.13 (s, 3H); LCMS (m/z)
765 (765 calcd for C.sub.36H.sub.38N.sub.5O.sub.11BF.sub.2).
TABLE-US-00003 ##STR00031## ##STR00032## ##STR00033## R.sup.1
R.sup.2 X 27a CH.sub.3 CH.sub.3 H 27b Ph Ph H 27c H --CH.dbd.CH-Ph
H 27d H --(CH.dbd.CH).sub.2-Ph H 27e H 2-pyrrolyl H 27f H
--CH.sub.2CH.sub.2CO.sub.2N.sub.a H 8 CH.sub.3 CH.sub.3 F
Example 48
Synthesis of Compound 28 (Dansyl BAPTA)
[0440] A solution of dansyl chloride (22 mg, 0.081 mmol) in 3 mL
dioxane was added to a solution of 5-amino-BAPTA (40 mg, 0.081
mmol) in 1:1 dioxane/water (10 mL) at pH 9 (maintained with sodium
carbonate). The resulting solution was stirred at room temperature
for 1 hour, then concentrated in vacuo. The residue was purified by
column chromatography on Sephadex LH-20 using water as eluant to
give Compound 28 as 40 mg of a buff powder.
##STR00034##
Example 49
Mass Spectrometry-Based Identification of Phosphoproteins from 2-D
gels
[0441] 2-D gels are run according to standard protocols. All steps
are performed with a volume of 500 ml/gel and 50 rpm on an orbital
shaker. To remove SDS the 2-D gels are fixed in 50% MeOH, 7% HAc
overnight with 1 change after one hour. The gels are washed the
next day for 4.times.15 min in dH20 before staining for 2.5-3 h
with the binding solution of the present invention. To remove
unspecific staining as well as to lower the background, 3 washes
for 1 h each in 50 mM sodium acetate, pH 4.0, 4% acetonitrile are
performed. This is followed by another wash in 50 mM NaAc, 15+%
1,2-Propanediol, pH 4.0 for another hour before imaging with 532 nm
laser excitation and 580 nm emission filter. The wash is continued
over night and the imaging repeated the next day.
[0442] The phosphoprotein stain is followed by SYPRO Ruby protein
gel stain over night and destaining for 2-3 h in 10% MeOH, 7% HAc
before imaging again. The gel is scanned with a 473 nm laser
excitation and 580 nm emission filter and spots are cut out. For
destaining the spots are placed into a 1.5 mL centrifuge tube and
destained with 100 .mu.L 50% MeOH, 5% HAc, 30 min, 100 .mu.L 0.1%
TFA, 30 min, 100 .mu.L 50% MeOH/5% HOAc, 30 min, and finally
dehydrated in 100 .mu.L 100% acetonitrile (ACN), 10 min. The pieces
are completely air dried before reduction and alkylation. If the
proteins were already reduced and alkylated before the 2-D gel
electrophoresis, the next steps can be omitted.
[0443] For the alkylation and reduction of cysteines, add enough 20
mM dithiothreitol (DTT) in 0.1 M NH.sub.4HCO.sub.3 in order to
completely cover the dried gel pieces (.about.50 .mu.L). It may be
necessary to add more as the gel pieces re-swell. Incubate at
56.degree. C. for 1 hr. Remove the DTT solution and add an equal
volume (50 .mu.L) of 100 mM iodoacetamide (IAAm) in 50 mM
NH.sub.4HCO.sub.3. Incubate at room temperature in the dark for 30
min., discard the supernatant and wash the gel pieces 2.times. with
100 .mu.L 0.1 M NH.sub.4HCO.sub.3 for 15 min. with occasional
vortexing to remove excess reagents. To extract any excess reagents
from the gel pieces wash with 100 .mu.L of 0.1 M
NH.sub.4HCO.sub.3/50% ACN for 15 min with occasional shaking.
Discard the supernatant and wash with 100% ACN. Discard the
supernatant and completely dry the gel pieces (air).
[0444] For in gel digestion with trypsin prepare a fresh solution
of 0.05 mg/ml modified trypsin (Promega) in 50 mM
NH.sub.4HCO.sub.3/10% ACN. Keep on ice if not to be used
immediately. Add 10 .mu.L of the fresh trypsin solution and allow
the gel pieces to soak up the trypsin solution before proceeding to
the next step, i.e. 10 min. Fully re-swell the gel pieces by adding
20 .mu.L 50 mM NH.sub.4HCO.sub.3/10% ACN (V.sub.TOT=30 .mu.L) and
incubate overnight at 37.degree. C.
[0445] To extraction the peptides, terminate digestion by adding 1
.mu.L of 10% TFA; 10 min./RT. Vortex, spin, take out the
supernatant and place in an 0.5 ml eppendorf tube. Add 50 .mu.L
0.1% TFA to the pieces and incubate 30 min. Shake, spin, combine
this supernatant with the first one. Add 50 .mu.L 60% ACN/0.1% TFA,
30 min., shake, spin, and combine with first supernatant in tube.
Dry the peptides in a Speed-Vac and dissolve again in 10 .mu.L 10%
ACN/0.1% TFA.
[0446] The peptide mix can be desalted and concentrated with a C18
ZipTip column from Millipore or spotted directly depending on the
sample concentration. Mix 0.5 .mu.L matrix (5 mg/ml .alpha.-cyano
4-hydroxycinnamix acid in 50% acetonitrile, 0.1% TFA) and 0.5 .mu.L
of sample on the target. Dry the spot and analyse in the mass
spectrometer.
Example 50
Covalent Labeling and Detection of Phosphoproteins with Compound 34
(Can also use Compound 39, Compound 36, Compound 42, and Compound
44) in Polyacrylamide Gels
[0447] Purified proteins (PeppermintStick.TM. phosphoprotein
molecular weight standards, Molecular Probes product number P33350;
alpha casein; or pepsin) were separated on 13% T, 0.8% C gels by
SDS-polyacrylamide gel electrophoresis. % T is the total monomer
concentration expressed in grams per 100 ml. and % C is the
percentage crosslinker. The 0.75 mm. thick, 6.times.10 cm. gels
were subjected to electrophoresis using the Bio-Rad mini-Protean 3
system according to standard procedures.
[0448] Following separation of the proteins on the gel, the gel was
fixed for 30 minutes in 50 ml of 50% methanol/10% acetic acid, then
overnight in 100 ml of fresh fixative to ensure complete removal of
the SDS. Next, the gel was rinsed two times, 10 minutes each, in
100 ml deionized water. The gel was then incubated for 90 minutes
in the dark in 50 ml of 50 mM sodium acetate, pH 4, 500 mM sodium
chloride, 20% acetonitrile, 1 .mu.M gallium chloride, 1 .mu.M
Compound 34 (or Compound 36, 39, 42 or 44) dye. The gel was
destained in the dark two times, 10 minutes each time, in 50 ml of
50 mM NaOAc (pH 4.0). The gel was transferred to 100 ml deionized
water, and then exposed to ultraviolet light (wavelength 254 nm) by
placing the gel directly on a UVP brand 3UV.TM. transilluminator
for 2 minutes. A parallel gel, loaded, run, fixed and rinsed as
described, was incubated for 90 minutes in a binding solution of
the present invention containing compound 2 (See, Example 2), then
destained 3.times.30 minutes in 50 ml of 50 mM NaOAc (pH 4.0). For
both gels, the red fluorescent signal produced by the dye was
visualized using the 532 nm excitation line of the SHG laser on the
Fuji FLA-3000G Fluorescence Image Analyzer (Fuji Photo, Tokyo,
Japan) and 580 band pass emission filter. Phosphoprotein bands were
visible in both gels.
[0449] Both gels were then washed in 60 ml of 0.1 M sodium
carbonate, pH11, for 1 hour to remove noncovalently bound dye.
After this base treatment, the gels were rinsed in 100 ml deionized
water. The gels were imaged as before. The gel stained with
Compound 2 had no signal left on it. The gel incubated in Compound
34 retained fluorescence localized to the phosphoprotein bands.
Example 51
A Fluorescence Quenching Assay for Measuring Phosphorylation in
Solution
[0450] Fluorescently labeled phosphotyrosine peptides were combined
with Compound 2, with or without gallium. Initially, 50 .mu.L mock
kinase reactions were prepared containing 50 .mu.M labeled peptide,
100 .mu.M ATP and 10 mM Tris buffer, pH 7.5. Subsequently, the
reactions were diluted 10 fold to a final volume of 500 .mu.L
containing 50 mM Na Acetate, pH 4.0, 15 .mu.M Compound 2, and 15
.mu.M GaCl.sub.3. 300 .mu.L of each reaction was transferred to a
96-well plate for analysis. The samples were excited at 488 nm, and
emission scans were recorded from 500-650 nm. The results are shown
in the fluorescence spectra of FIG. 12. The phosphorylated peptides
are shown as follows: A, pp60-Oregon Green 488 dye (OG); B,
p-Abl-OG dye; C, pp60-Alexa Fluor 488 dye (A488); D, pStat3-OG dye
label. Spectra with circles represent samples without GaCl.sub.3
addition, and spectra with squares represent samples with
GaCl.sub.3 addition. In all cases, when GaCl.sub.3 is added to the
reaction mixture, Oregon Green dye label and Alexa Fluor 488 dye
label emission at 520 nm is significantly quenched. The results
demonstrate that quenching of the 488 nm excitable compounds can be
used to specifically quantitate phosphorylation of labeled
compounds without interference from free ATP in solution.
Example 52
Preparation of Compound 34
[0451] To a solution of 4-(succinimidyloxycarbonyl)-rhod
tetramethyl ester (30, 0.20 g, 0.18 mmol) in 3:1 THF/MeCN (20 mL)
was added N-t-butoxycarbonylethylenediamine hydrochloride (43 mg,
22 mmol) and diisoproylethylamine (DIEA, 38 .mu.L). The resulting
solution was stirred overnight at room temperature (rt), then
concentrated in vacuo. The residue was dissolved in 40 mL
chloroform and washed with 10% citric acid (1.times.20 mL), dried
over sodium sulfate, and concentrated to give compound 31 as 0.23 g
of a red solid.
[0452] A solution of compound 31 (0.18 g, 0.18 mmol) in 20 mL
dichloromethane was treated with 5 mL trifluoroacetic acid (TFA).
After 2 h at room temperature the volatiles were removed in vacuo,
and chloroform/toluene (1:1, 10 mL) stripped from the residue,
leaving compound 32 as 0.20 g of a red solid: m/2 442.5 (885 calcd
for C.sub.46H.sub.56N.sub.6O.sub.12).
[0453] A solution of compound 32 (0.18 mmol, 0.18 g) in 1:1
methanol/dioxane (8 mL) was treated with a 1 M solution of
potassium hydroxide (KOH, 2.0 mL, 2.0 mmol) at rt. After stirring
overnight the reaction pH was lowered to 8 with aqueous citric
acid, and the volatiles removed in vacuo. The resulting red residue
was purified by column chromatography on Sephadex LH-20 using water
as eluant. Pure product fractions were pooled and lyophilized to
give compound 33 as 0.15 g of a red powder: m/z 828 (828 calcd for
C.sub.42H.sub.47N.sub.6O.sub.12).
[0454] The pH of a solution of compound 33 (30 mg, 0.03 mmol) in 5
mL water was raised to 9.6 with aqueous sodium carbonate. A
solution of 4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl
ester (Molecular Probes product 2522, 14 mg, 0.04 mmol) in 2 mL
dioxane was added. After 2 h the volatiles were removed in vacuo,
and the residue purified by column chromatography on Sephadex LH-20
using water as eluant. Pure product fractions were pooled and
lyophilized to give compound 34 as 34 mg of a red powder: m/z 1041
(1041 calcd for C.sub.49H.sub.42N.sub.9O.sub.13F.sub.4).
##STR00035## ##STR00036##
Example 53
Preparation of Compound 36
[0455] To a solution of compound 32 (0.22 g, 0.20 mmol) in 5 mL DMF
was added DIEA (34 .mu.L, 0.20 mmol) and a solution of
4-benzoylbenzoic acid, succinimidyl ester (Molecular Probes product
1577, 64 mg, 0.20 mmol) in 5 mL DMF. After stirring overnight the
volatiles were removed in vacuo, giving compound 35 as a red
residue that was used immediately in the next step.
[0456] To a red solution of compound 35 (.about.0.20 mmol) in 1:1
methanol/dioxane (6 mL) was added a 1 M solution of KOH (1.0 mL,
1.0 mmol). The resulting light brown solution was stirred at rt for
3 h, and the pH was lowered to 9.5 with aqueous citric acid. The
volatiles were removed in vacuo, and the resulting red residue was
purified by column chromatography on Sephadex LH-20 using water as
eluant. Pure product fractions were pooled and lyophilized to give
compound 36 as 125 mg of a hygroscopic red powder: m/z 1035 (1035
calcd for C.sub.56H.sub.51N.sub.6O.sub.14).
##STR00037##
Example 54
Preparation of Compound 39
[0457] To a solution of compound 37 as its bis-trifluoroacetate
salt (prepared by condensation of 2 eq of ethylenediamine with
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid
(Molecular Probes product 6103)) (0.14 g, 0.22 mmol) in 10 mL water
was added DIEA (35 .mu.L, 0.20 mmol). A solution of
4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester
(Molecular Probes product 2522, 73 mg, 0.22 mmol) in 8 mL dioxane
was added dropwise with stirring over 10 minutes. After 1 h, the pH
was lowered to 2.5 with aqueous HCl, followed by concentration in
vacuo. The residue was purified by column chromatography on
Sephadex LH-20 using water as eluant. Pure product fractions were
pooled and lyophilized to give compound 38 as 53 mg of an orange
powder.
[0458] To a solution of BAPTA isothiocyanate (Molecular Probes
product 14390, 4 mg, 0.008 mmol) in 1 mL dioxane was added a
solution of compound 38 (6 mg, 0.008 mmol) in 1 mL water. The
reaction pH was raised to 9.3 with aqueous sodium carbonate. After
2 h the volatiles were removed in vacuo, and the residue was
purified by column chromatography on Sephadex LH-20 using water as
eluant. Pure product fractions were pooled and lyophilized to give
compound 39 as 33 mg of a pale orange powder. Further purification
was accomplished by dissolving all of compound 39 in 2 mL water.
The pH was lowered to 2 with aqueous HCl. The resulting precipitate
was collected by centrifugation and then suspended in 2 mL water.
Dissolution was affected by addition of one drop of saturated
aqueous sodium bicarbonate. The resulting solution was lyophilized
to give compound 39 as 5 mg of an orange powder: m/z 1171 (1171
calcd for C.sub.49H.sub.45N.sub.12O.sub.13BF.sub.6S).
##STR00038##
Example 55
Preparation of Compound 42
[0459] To a solution of
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid
(Compound 40, Molecular Probes product 6103)) (0.10 g, 0.30 mmol)
in 15 mL anhydrous acetonitrile under argon was added
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC,
57 mg, 0.30 mmol). The resulting solution was stirred for 20
minutes, and 4-aminomethylbenzophenone (74 mg, 0.30 mmol) and DIEA
(52 .mu.L, 0.30 mmol) were added. The resulting orange mixture was
stirred at rt for 5 h, then partitioned between ethyl acetate (50
mL) and 10% citric acid (40 mL). The organic layer was washed with
brine (1.times.20 mL), dried over sodium sulfate, and concentrated
to an amber residue. This residue was purified by flash
chromatography on silica gel using methanol in chloroform as eluant
to give compound 41 as 54 mg of an amber powder: m/z 529 (520 calcd
for C.sub.29H.sub.26N.sub.3O.sub.4BF.sub.2).
[0460] To a solution of compound 41 (53 mg, 0.10 mmol) in 7 mL
anhydrous THF under argon was added oxalyl chloride (18 .mu.L, 0.20
mmol) and 3 drops of DMF. The resulting mixture was stirred at rt
for 20 minutes, then concentrated in vacuo. The residue was
dissolved in 6 mL anhydrous dioxane, and the resulting solution was
added dropwise to a solution of 5-amino-BAPTA free acid (49 mg,
0.10 mmol) in 6 mL aqueous sodium carbonate at pH 9-9.5. After
stirring for 1.5 h, the reaction pH was lowered to 7.2 with aqueous
citric acid, and concentrated in vacuo. The resulting residue was
purified by column chromatography on Sephadex LH-20 using water as
eluant. Pure product fractions were pooled and lyophilized to give
compound 42 as 15 mg of an orange powder: m/z 999 (999 calcd for
C.sub.51H.sub.45N.sub.6O.sub.13BF.sub.2).
##STR00039##
Example 56
Preparation of Compound 44
[0461] The pH of a solution of the Compound 43 (prepared by
condensing cadaverine with 4'-carboxy-fluo-4, tetramethyl ester
followed by saponification) (12 mg, 0.012 mmol) in 3 mL water was
raised to 10.3 with aqueous sodium carbonate. A solution of
4-benzoylbenzoic acid, succinimidyl ester (Molecular Probes product
1577, 10 mg, 0.030 mmol) in 2 mL dioxane was added. After stirring
at rt overnight, the volatiles were removed in vacuo. The residue
was purified by column chromatography on Sephadex LH-20 using water
as eluant. Pure product fractions were pooled and lyophilized to
give compound 44 as 14 mg of an orange powder: m/z 1074 (1074 calcd
for C.sub.56H.sub.46N.sub.4O.sub.16F.sub.2).
##STR00040##
[0462] The reagents employed in the preceding examples are
commercially available or can be prepared using commercially
available instrumentation, methods, or reagents known in the art or
whose preparation is described in the examples. It is evident from
the above description and results that the subject invention is
greatly superior to presently available methods for labeling
phosphorylated target molecules in a biological sample, as an
unprecedented 500-1000 fold concentration range of phosphorylated
target molecules can be detected. The subject invention overcomes
the shortcomings of the currently used methods by allowing labeling
as well as isolation of phosphorylated target molecules in a simple
procedure that has increased sensitivity. It is appreciated that
the methods of the present invention provide labeling of
phosphorylated target molecules in solution or immobilized and that
the phosphate-binding compounds can be either immobilized or in
solution, allowing for identification of enzymes responsible for
phosphorylation of these target molecules. The examples are not
intended to provide an exhaustive description of the many different
embodiments of the invention. Thus, although the foregoing
invention has been described in extensive detail by way of
illustration and example for purposes of clarity for understanding,
those of ordinary skill in the art will readily realize that many
changes and modifications can be made thereto without departing
from the spirit or scope of the appended claims.
[0463] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
* * * * *