U.S. patent application number 11/823775 was filed with the patent office on 2012-09-27 for immunoaffinity isolation of modified peptides from complex mixtures.
This patent application is currently assigned to CELL SIGNALING TECHNOLOGY, INC.. Invention is credited to Michael J. Comb, John Rush, Yi Tan, Xiangming Zha, Hui Zhang.
Application Number | 20120244594 11/823775 |
Document ID | / |
Family ID | 43625490 |
Filed Date | 2012-09-27 |
United States Patent
Application |
20120244594 |
Kind Code |
A9 |
Rush; John ; et al. |
September 27, 2012 |
Immunoaffinity isolation of modified peptides from complex
mixtures
Abstract
The invention provides methods for isolating a modified peptide
from a complex mixture of peptides, the method comprising the steps
of: (a) obtaining a proteinaceous preparation from an organism,
wherein the preparation comprises modified peptides from two or
more different proteins; (b) contacting the preparation with at
least one immobilized modification-specific antibody; and (c)
isolating at least one modified peptide specifically bound by the
immobilized modification-specific antibody in step (b). The method
may further comprise the step of (d) characterizing the modified
peptide isolated in step (c) by mass spectrometry (MS), tandem mass
spectrometry (MS-MS), and/or MS.sup.3 analysis, or the step of (e)
utilizing a search program to substantially match the spectra
obtained for the modified peptide during the characterization of
step (d) with the spectra for a known peptide sequence, thereby
identifying the parent protein(s) of the modified peptide. Also
provided are an immunoaffinity isolation device comprising a
modification-specific antibody, and antibodies against novel UFD1
and PTN6 phosphorylation sites.
Inventors: |
Rush; John; (Brookline,
MA) ; Zhang; Hui; (Seattle, WA) ; Zha;
Xiangming; (Iowa City, IA) ; Comb; Michael J.;
(Manchester, MA) ; Tan; Yi; (Lynnfield,
MA) |
Assignee: |
CELL SIGNALING TECHNOLOGY,
INC.
|
Prior
Publication: |
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Document Identifier |
Publication Date |
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US 20110053242 A1 |
March 3, 2011 |
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Family ID: |
43625490 |
Appl. No.: |
11/823775 |
Filed: |
June 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10777893 |
Feb 12, 2004 |
7300753 |
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11823775 |
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10175486 |
Jun 19, 2002 |
7198896 |
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10777893 |
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09535364 |
Mar 24, 2000 |
6982318 |
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10175486 |
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09148712 |
Sep 4, 1998 |
6441140 |
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09535364 |
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60299893 |
Jun 21, 2001 |
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60337012 |
Nov 8, 2001 |
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Current U.S.
Class: |
435/174 ;
250/282; 422/261; 435/272; 436/501; 530/344; 530/389.8; 530/391.1;
73/61.52 |
Current CPC
Class: |
G01N 2440/14 20130101;
G01N 30/7233 20130101; C07K 16/44 20130101; B01D 15/3828 20130101;
G01N 33/6854 20130101; B01D 15/3809 20130101; C07K 1/22 20130101;
G01N 33/6848 20130101; G01N 2030/8831 20130101; C07K 16/18
20130101; G01N 33/6842 20130101 |
Class at
Publication: |
435/174 ;
435/272; 436/501; 530/344; 530/389.8; 530/391.1; 250/282; 73/61.52;
422/261 |
International
Class: |
C12N 11/00 20060101
C12N011/00; G01N 33/566 20060101 G01N033/566; C07K 1/14 20060101
C07K001/14; C07K 16/18 20060101 C07K016/18; B01D 11/02 20060101
B01D011/02; C07K 16/40 20060101 C07K016/40; C07K 1/22 20060101
C07K001/22; H01J 49/26 20060101 H01J049/26; G01N 30/72 20060101
G01N030/72; C12S 3/14 20060101 C12S003/14; C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 20, 2002 |
US |
PCT/US02/19480 |
Claims
1. A method for isolating a modified peptide from a complex mixture
of peptides, said method comprising the steps of: (a) obtaining a
proteinaceous preparation from an organism, wherein said
proteinaceous preparation comprises modified peptides from two or
more different proteins; (b) contacting said proteinaceous
preparation with at least one immobilized modification-specific
antibody; and (c) isolating at least one modified peptide
specifically bound by said immobilized modification-specific
antibody in step (b).
2. The method of claim 1, further comprising the step of (d)
characterizing said modified peptide isolated in step (c) by mass
spectrometry (MS), tandem mass spectrometry (MS-MS), and/or
MS.sup.3 analysis.
3. The method of claim 2, wherein said mass spectrometry comprises
MALDI-TOF MS, wherein said tandem mass spectrometry comprises
LC-MS/MS, and wherein said MS.sup.3 analysis comprises
LC-MS.sup.3.
4. The method of claim 2 or 3, further comprising the step of (e)
utilizing a search program to substantially match the spectra
obtained for said modified peptide during the characterization of
step (d) with the spectra for a known peptide sequence, thereby
identifying the parent protein(s) of said modified peptide.
5. The method of claim 1, wherein said proteinaceous preparation
comprises a digested biological sample selected from the group
consisting of a digested crude cell extract, a digested tissue
sample, a digested serum sample, a digested urine sample, a
digested synovial fluid sample, and a digested spinal fluid
sample.
6. The method of claim 5, wherein said digested preparation is
obtained using at least one proteolytic enzyme or chemical
cleavage.
7. The method of claim 6, wherein said proteolytic enzyme is
immobilized.
8. The method of claim 6, wherein said proteolytic enzyme is
soluble, and wherein said digested preparation is treated with a
proteolysis inhibitor prior to said contacting step (b).
9. The method of claim 1, wherein step (a) further comprises
pre-purifying said proteinaceous preparation by immobilized metal
affinity chromatography (IMAC).
10. The method of claim 1, wherein said immobilized antibody of
step (b) is covalently-linked to a chromatography resin or
noncovalently-linked to protein-A- or protein-G-agarose.
11. The method of claim 10, wherein said resin is contained within
a column or micropipette tip.
12. The method of claim 2, wherein said immobilized antibody of
step (b) is immobilized in chromatography resin within a column,
said column being coupled to a mass spectrometer for said
characterization of step (d).
13. The method of claim 1, wherein said modification comprises
phosphorylation.
14. The method of claim 1, wherein said modified peptide(s)
comprise(s) a phosphopeptide.
15. The method of claim 1, wherein said modification-specific
antibody comprises a motif-specific, context-independent antibody
that recognizes a motif comprising at least one phosphorylated
amino acid.
16. The method of claim 15, wherein said motif consists of a single
phosphorylated amino acid.
17. The method of claim 15, wherein said motif comprises all or
part of a kinase consensus substrate motif or a protein-protein
binding motif.
18. The method of claim 17, wherein said kinase consensus substrate
motif is selected from the group consisting of MAPK consensus
substrate motifs, CDK consensus substrate motifs, PKA consensus
substrate motifs, AKT consensus substrate motifs, PKC consensus
substrate motifs, phosphothreonine-X-arginine, and ATM consensus
substrate motifs, and wherein said protein-protein binding is a
14-3-3 binding motif or a PDK1 docking motif.
19. The method of claim 1, wherein said modification-specific
antibody is a monoclonal antibody or a polyclonal antibody.
20. The method of claim 1, wherein said modified peptide isolated
in step (c) corresponds to a known marker of disease.
21. The method of claim 4, wherein said modified peptide
characterized in step (d) comprises an unknown modification site of
said parent protein.
22. The method of claim 2 or 3, further comprising the step of (e)
comparing the modification state of said modified peptide
characterized in step (d) with the modification state of a
corresponding peptide in a reference sample, thereby to compare
protein activation in said proteinaceous preparation with protein
activation in said reference sample.
23. The method of claim 22, wherein said proteinaceous preparation
corresponds to a diseased organism and said reference sample
corresponds to a normal organism, whereby comparison of protein
activation provides information on activation changes resulting
from said disease.
24. The method of claim 22, wherein said proteinaceous preparation
is obtained from a tissue biopsy cell or a clinical fluid sample
and said reference sample corresponds to a diseased organism,
whereby the comparison of protein activation provides information
useful for diagnosis of said disease.
25. The method of claim 22, wherein said protein preparation
corresponds with an organism or preparation treated with at least
one test compound and said reference sample corresponds with an
untreated organism or preparation, whereby the comparison of
protein activation provides information on activation changes
resulting from treatment with said test compound.
26. The method of claim 23, wherein the comparison of protein
activation identifies the modified peptide characterized in step
(d) as corresponding to a parent protein not previously reported as
so modified in said disease.
27. The method of claim 24 or 25, wherein said disease is
cancer.
28. The method of claim 25, wherein said test compound comprises a
cancer therapeutic.
29. The method of claim 25, wherein said test compound comprises a
kinase inhibitor.
30. A method for isolating a phosphopeptide from a complex mixture
of peptides, said method comprising the steps of: (a) obtaining a
proteinaceous preparation from an organism, wherein said
proteinaceous preparation comprises phosphopeptides from two or
more different proteins; (b) fractionating phosphopeptides in said
proteinaceous preparation by reversed-phased chromatography to
produce a fractionated proteinaceous preparation; (c) contacting
said fractionated proteinaceous preparation with at least one
immobilized motif-specific, context-independent antibody that binds
a motif comprising at least one phosphorylated amino acid; (d)
isolating at least one phosphopeptide specifically bound by said
immobilized antibody in step (c); and (e) characterizing said
phosphopeptide isolated in step (d) by mass spectrometry (MS),
tandem mass spectrometry (MS-MS), and/or MS.sup.3 analysis.
31. The method of claim 30, further comprising the step of (f)
utilizing a search program to substantially match the mass spectra
obtained for said phosphopeptide during the characterization of
step (e) with the mass spectra for a peptide of one or more known
protein(s), thereby identifying the parent protein(s) of said
modified peptide.
32. The method of claim 32, wherein said mass spectrometry
comprises MALDI-TOF MS, wherein said tandem mass spectrometry
comprises LC-MS/MS, and wherein said MS.sup.3 analysis comprises
LC-MS.sup.3.
33. The method of claim 32, wherein step (a) further comprises
digesting said proteinaceous preparation to produce a complex
mixture of peptides.
34. The method of claim 30, wherein said motif-specific,
context-independent antibody of step (c) comprises a general
phosphotyrosine-specific antibody, a general
phosphothreonine-specific antibody, or a general
phosphoserine-specific antibody.
35. The method of claim 30, wherein said motif-specific,
context-independent antibody of step (c) is specific for a
phosphorylated kinase consensus substrate motif or protein-protein
binding motif.
36. The method of claim 35, wherein said kinase consensus substrate
motif is selected from the group consisting of MAPK consensus
substrate motifs, CDK consensus substrate motifs, PKA consensus
substrate motifs, AKT consensus substrate motifs, PKC consensus
substrate motifs, phosphothreonine-X-arginine, ATM/ATR consensus
substrate motifs, p85 PI3K binding motif, phosphothreonine-proline
motif, Arg-X-Tyr/Phe-X-phosphoserine motif,
phosphoserine/phosphothreonine-Phe motif, PLK consensus substrate
motifs, and DNA damage-induced substrate motifs, and wherein said
protein-protein binding is a 14-3-3 binding motif or a PDK1 docking
motif.
37. The method of claim 30, wherein said reversed-phased
chromatography of step (b) comprises a C18 column.
38. The method of claim 30, further comprising the step of (f)
quantifying said isolated phosphopeptides of step (e).
39. The method of claim 38, wherein step (f) comprises quantifying
said isolated phosphopeptides using stable isotope labeling by
amino acids in cell culture (SILAC) and/or absolute quantification
of peptides (AQUA) techniques.
40. An immunoaffinity isolation device for the isolation of
modified peptides a complex mixture, said device comprising a
support comprising at least one modification-specific antibody
immobilized to a rigid, non-porous or macroporous resin.
41. The device of claim 40, wherein said support is selected from
the group consisting of a thin capillary column having an internal
diameter of about 50 to 300 micrometers and a micropipette tip.
42. The device of claim 41, wherein said modification-specific
antibody comprises a motif-specific, context-independent
antibody.
43. The device of claim 41, wherein said column is adapted to be
coupled to an electrospray source on a mass spectrometer.
44. An antibody that binds ubiquitin fusion degradation protein 1
(UFD1) only when phosphorylated at serine 335, but does not
substantially bind to UFD1 when not phosphorylated at this
residue.
45. An antibody that binds protein-tyrosine phosphatase 1c (PTN6)
only when phosphorylated at serine 588, but does not substantially
bind to PTN6 when not phosphorylated at this residue.
46. An antibody that binds a protein phosphorylation site listed in
Column 5 of Table 5 only when phosphorylated at the
phosphorylatable residue indicated in Column 5, but does not
substantially bind to the phosphorylation site when not
phosphorylated at the indicated residue.
47. An antibody that binds a protein phosphorylation site listed in
Column 5 of Table 6 only when not phosphorylated at the
phosphorylatable residue indicated in Column 5, but does not
substantially bind to the phosphorylation site when phosphorylated
at the indicated residue.
48. An antibody that binds a protein phosphorylation site listed in
Column 4 of Table 7 only when not phosphorylated at the
phosphorylatable residue indicated in Column 4, but does not
substantially bind to the phosphorylation site when phosphorylated
at the indicated residue.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
10/777,893, filed Feb. 12, 2004, presently pending, the disclosures
of which are hereby incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to peptides and methods of isolating
and characterizing the same.
BACKGROUND OF THE INVENTION
[0003] The activation of proteins by modification represents an
important cellular mechanism for regulating most aspects of
biological organization and control, including growth, development,
homeostasis, and cellular communication. For example, protein
phosphorylation plays a critical role in the etiology of many
pathological conditions and diseases, including cancer,
developmental disorders, autoimmune diseases, and diabetes. In
spite of the importance of protein modification, it is not yet well
understood at the molecular level. The reasons for this lack of
understanding are, first, that the cellular modification system is
extraordinarily complex, and second, that the technology necessary
to unravel its complexity has not yet been fully developed.
[0004] The complexity of protein modification on a proteome-wide
scale derives from three factors: the large number of modifying
proteins, e.g. kinases, encoded in the genome, the much larger
number of sites on substrate proteins that are modified by these
enzymes, and the dynamic nature of protein expression during
growth, development, disease states, and aging. The human genome
encodes, for example, over 520 different protein kinases, making
them the most abundant class of enzymes known. See Hunter, Nature
411: 355-65 (2001). Each of these kinases phosphorylates specific
serine, threonine, or tyrosine residues located within distinct
amino acid sequences, or motifs, contained within different protein
substrates. Most kinases phosphorylate many different proteins: it
is estimated that one-third of all proteins encoded by the human
genome are phosphorylated, and many are phosphorylated at multiple
sites by different kinases. See Graves et al., Pharmacol. Ther. 82:
111-21 (1999).
[0005] Many of these phosphorylation sites regulate critical
biological processes and may prove to be important diagnostic or
therapeutic targets for molecular medicine. For example, of the
more than 100 dominant oncogenes identified to date, 46 are protein
kinases. See Hunter, supra. Oncogenic kinases such as ErbB2 and
Jak3, widely expressed in breast tumors and various leukemias,
respectively, transform cells to the oncogenic phenotype at least
in part because of their ability to phosphorylate cellular
proteins. Understanding which proteins are modified by these
kinases will greatly expand our understanding of the molecular
mechanisms underlying, e.g., oncogenic transformation. Thus, the
ability to selectively identify modification sites, e.g.
phosphorylation sites, on a wide variety of cellular proteins
represents an important new tool for understanding the key
signaling proteins and pathways implicated in diseases, such as
cancer.
[0006] Although several methods for purifying phosphopeptides have
been described, these methods have significant limitations that
render them unsuitable for the isolation or purification of
modified peptides from complex mixtures of peptides on a genome- or
cell-wide basis. In one method, which employs reversed-phase HPLC,
proteins are labeled in vivo or in vitro with radioactive
phosphate, and the protein of interest is purified to near
homogeneity (so that it represents at least 95% of the protein in
the sample) before analysis. See, e.g. Wettenhall et al. Methods
Enzymol. 201: 186-199 (1991). The highly purified protein is then
digested with a proteolytic enzyme to produce peptides, and the
radioactively labeled peptides containing a phosphorylation site of
the single protein are purified by reversed-phase HPLC.
Phosphorylated peptides are distinguished from nonphosphorylated
peptides by measuring the radioactivity associated with each HPLC
fraction, and then chemically sequenced.
[0007] The reversed-phase HPLC method has several important
limitations that render it unsuitable for the purification of
modified peptides from complex mixtures of peptides, e.g. cellular
digests. The method cannot be applied to biological samples that
cannot be radioactively labeled, such as tissue biopsy samples.
Selective peptide loss during purification by this method can
introduce biases, so that the most prominent modified peptide
before and after the HPLC step is not necessarily the same. This
problem is addressed by first purifying the protein so its level of
radioactivity can be measured and then rigorously accounting for
sample recovery during all subsequent purification and analysis
steps. Accordingly, modified sites cannot be identified from
complex peptide mixtures. The HPLC method is often unsuccessful
when applied to proteins that are modified at low levels, for
example, where only a small percentage (less than 10%) of the
protein is phosphorylated at one site. This problem results from
the difficulty of purifying a phosphopeptide to homogeneity against
a high background of nonphosphorylated peptides, and the need for a
nearly homogenous phosphopeptide during chemical sequencing.
Additional shortcomings of this method exist.
[0008] Several researchers have employed immobilized
phospho-specific antibodies, along with mass spectrometry (MS or
MS/MS), to identify phosphorylation sites in proteins. Immobilized
anti-phosphotyrosine antibodies have been used to purify
phosphopeptides from digests of gelsolin, an actin binding-protein.
See De Corte, et al., Prot. Sci. 8: 234-241 (1999). However the
single protein of interest, gelsolin, was first purified and
phosphorylated in vitro, before digesting to yield
gelsolin-specific phosphopeptides. Immobilized anti-phosphotyrosine
antibodies have similarly been employed to identify EphB
phosphopeptides from purified EphB digests (Kalo et al., Biochem.
38: 14396-408 (1999)) and to purify alpha-enolase phosphopeptides
from a purified digest of human alpha-enolase (Marcus et al.,
Electrophoresis 21: 2622-2636 (2000)). However, in the latter
attempt the method failed, and the authors expressly concluded that
the low binding affinity between the antibody and the
phosphopeptides makes the detection of phosphorylation sites almost
impossible (Id. at p. 2635). The prevailing view (enunciated by
Marcus et al.) that phosphospecific antibodies are not generally
suitable for isolating phosphopeptides has recently been reiterated
in a review on protein phosphorylation analysis authored by
recognized leaders in the field of biological mass spectrometry.
Mann et al., Trends in Biotech. 20: 261-268 (2002).
[0009] The identification of Ty1 Gag protein epitopes in digested
yeast cell extract using an immobilized epitope-specific antibody
has also been reported. See Yu et al., J. Am. Soc. Mass. Spec. 9:
208-215 (1998). However, the immobilized antibody was a Ty1 Gag
epitope-specific antibody (i.e. was not a general
modification-specific antibody), was not phospho-specific, and
recognized only peptides from a single protein, Ty1 Gag. None of
these methodologies are suitable for the selective isolation of
phosphopeptides from complex mixtures of peptides that are derived
from multiple, unpurified proteins, and most require the timely
pre-purification of desired proteins. Reviewed in Mann et al., Ann.
Rev. Biochem. 70: 437-73 (2001).
[0010] Another widely used method for purifying modified peptides
is immobilized metal affinity chromatography (IMAC). This
pseudo-affinity purification method is based on the interaction of
metal ions and negatively charged peptide moieties, such as
phosphate. See, e.g. Posewitz et al., Anal. Chem. 15: 2883-2892
(1999). Pre-purified, phosphorylated proteins are digested to
peptides, and the phosphorylated peptides are then purified by
passing the digest through a miniaturized chromatography column
containing a resin with a covalently attached metal chelator, e.g.
iminodiacetic or nitrilotriacetic acid. A cation is non-covalently
attached to the chelator by treating the resin with one of several
metal salts, such as Fe.sup.3+, Ni.sup.2+, Ga.sup.3+, or Cu.sup.2+.
When the protein digest is applied to the column, peptides with a
sufficiently high negative charge density, such as from a phosphate
group, can bind to the metal cation. Eluted peptides can then be
analyzed by chemical sequencing or by mass spectrometry (MS or
MS/MS) to assign phosphorylation sites.
[0011] As with the reversed-phase HPLC method, IMAC purification of
modified peptides has several limitations that render it unsuitable
for the purification of modified peptides from complex mixtures of
peptides, such as cellular digests. The method must be adjusted for
each desired sample, since, phosphopeptides, for example, are
sensitive to the exact conditions used for IMAC. It is not unusual
to test peptide binding to all 4 commonly used cations in
combination with 3 different pH conditions (12 test conditions
altogether) in order to find the metal-pH combination best suited
for purification of a single, specific phosphopeptide. Isolating a
second, different phosphopeptide from the same, or different,
protein may require a second metal-pH combination that is unique.
The IMAC method is not specific for phosphopeptides, and peptides
with several negatively charged amino acid residues (such as
aspartic acid and glutamic acid) and without phosphate can bind to
IMAC resins and contaminate any purified phosphopeptides. This
drawback is especially problematic when only a small percentage of
the protein sample is modified, e.g. a partially phosphorylated
protein, because the background level of contaminating
nonphosphorylated peptides can overwhelm the level of
phosphopeptides. For this reason, the IMAC method is not suitable
for the isolation of desired modified peptides from complex peptide
mixtures. Further, the method is not specific for the type of
modified residue, e.g. phosphorylated residue, thus peptides with
phosphoserine, phosphothreonine, or phosphotyrosine all bind and
elute from IMAC resins.
[0012] Accordingly, there remains a need in the art for the
development of simple peptide isolation/purification methods that
are suitable for the isolation of modified peptides from complex
mixtures of peptides, e.g. digested cell extracts, which contain a
wide variety of different, modified proteins, and yet do not
require timely or costly pre-purification steps. The development of
suitable peptide isolation methods that are simple and can be
readily automated would, for example, enable the rapid profiling of
activation states on a genome-wide basis and the identification of
new diagnostic or therapeutic targets within cell signaling
pathways that are at the forefront of the proteomics era currently
underway. The unresolved need for such high-throughput methods has
recently been recognized. See, e.g. Mann, Nat. Biotech. 17: 954-55
(1999).
SUMMARY OF THE INVENTION
[0013] The present invention provides methods for isolating a
modified peptide from a complex mixture of peptides (such as exists
in a cell extract digest) by the steps of: (a) obtaining a
proteinaceous preparation from an organism, in which modified
peptides from two or more different proteins are present; (b)
contacting the proteinaceous preparation with at least one
immobilized modification-specific antibody; and (c) isolating at
least one modified peptide specifically bound by the immobilized
modification-specific antibody. The method may further include the
step of (d) characterizing the modified peptide(s) isolated in step
(c) by mass spectrometry (MS), tandem mass spectrometry (MS-MS),
and/or MS.sup.3 analysis. The method may also further include the
step of (e) utilizing a search program (such as Sequest) to
substantially match the spectra obtained for the modified
peptide(s) during the characterization of step (d) with the spectra
for a known peptide sequence, thereby identifying the parent
protein(s) of the modified peptide(s). The invention encompasses
the isolation of modified peptides containing virtually any type of
modified amino acids, including but not limited to phosphorylated,
acetylated, methylated, nitrosylated, and/or glycosylated residues.
Motif-specific, context-independent antibodies that bind single
modified amino acids or that bind conserved modified motifs
comprising multiple amino acids are advantageously employed in the
disclosed methods.
[0014] Also provided are an immunoaffinity isolation device for the
isolation of modified peptides from a complex mixture according to
the method of the invention, and antibodies to novel UFD1 and PTN6
phosphorylation sites discovered by the practice of the disclosed
methods.
[0015] The method of the invention enables the rapid, efficient,
and direct isolation (and subsequent characterization) of modified
peptides from complex mixtures, such as crude cell extracts,
without the need for costly and timely pre-purification of desired
peptides or proteins. The method enables the single-step
immunoaffinity isolation, and subsequent characterization of
multiple different modified peptides, corresponding to a multitude
of different modified proteins and signaling pathways, with a
single antibody. Alternatively, the method can further employ a
simple pre-fractionation step. The simplicity of the disclosed
method also renders it readily automatable, as only a single
isolation step is required. Further advantages and preferred
embodiments of the invention are described in detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1--is a flow-diagram representation of the method of
the invention.
[0017] FIG. 2--depicts a MALDI-TOF mass spectrum of an unpurified
mixture of 10 different phosphorylated and nonphosphorylated
peptides, using alpha-cyano-4-hydroxycinnamic acid as matrix. Peaks
labeled with stars are phosphorylated peptides, and peaks labeled
with circles correspond to nonphosphorylated peptides. Unmarked
peaks are synthetic peptide byproducts.
[0018] FIG. 3--depicts a MALDI-TOF mass spectrum of the
phosphotyrosine peptide mixture described in FIG. 2, after
isolation of phosphopeptides with monoclonal P-Tyr-100
antibody-resin, according to the method of the invention. Peaks
labeled with stars are phosphorylated peptides, and peaks labeled
with primed stars correspond to a phosphopeptide artifact with a
mass of M-78. These artifacts are also present in the unpurified
peptide mix (FIG. 2) but are obscured by the peaks from
nonphosphorylated peptides (M-80).
[0019] FIG. 4--depicts a MALDI-TOF mass spectrum of the purified
and unpurified phosphotyrosine peptide mix described in FIG. 2,
using alpha-cyano-4-hydroxycinnamic acid as matrix. The top panel
shows the peptide mix before purification (as in FIG. 2), and the
bottom panel shows the peptide mix after purification (FIG. 3).
[0020] FIG. 5--depicts a MALDI-TOF mass spectrum of an unpurified
mixture of 4 different phosphorylated and nonphosphorylated
peptides, using alpha-cyano-4-hydroxycinnamic acid as matrix. Peaks
labeled with stars are phosphorylated peptides, and peaks labeled
with circles correspond to nonphosphorylated peptides. Unmarked
peaks are synthetic peptide byproducts.
[0021] FIG. 6--depicts a MALDI-TOF mass spectrum of the bound and
unbound peptide fractions after immunoaffinity
isolation/purification of the phosphothreonine peptide mix
described in FIG. 5, using alpha-cyano-4-hydroxycinnamic acid as
matrix. The top panel shows the fraction of the peptide mix that
did not bind to a polyclonal P-Thr-antibody-resin, and the bottom
panel shows the fraction of the peptide mix that did bind to and
was eluted from the polyclonal P-Thr-antibody-resin. Peaks labeled
with stars are phosphorylated peptides, and peaks labeled with
circles correspond to nonphosphorylated peptides.
[0022] FIG. 7--depicts a MALDI-TOF mass spectrum of the unpurified
and purified phosphotyrosine peptide mix described in FIG. 2, using
alpha-cyano-4-hydroxycinnamic acid as matrix. This isolation is
similar to the one described in FIGS. 2-4, except that the amount
of phosphotyrosine peptide mix was reduced to a low level. In all
panels, peaks labeled with stars are phosphorylated peptides, peaks
labeled with circles correspond to nonphosphorylated peptides, and
peaks labeled with primed stars correspond to a phosphopeptide
artifact with a mass of M-78. The top panel shows the unpurified,
complex phosphotyrosine peptide mix. The second panel shows the
peptides that did not bind to the monoclonal P-Tyr-100
antibody-resin, and the third panel shows the peptides that did
bind and elute from the antibody-resin. The bottom panel shows the
bound and eluted peptide fraction after treatment with a
phosphatase enzyme, to remove phosphate groups from
phosphopeptides, reducing the observed mass by 80. Lines drawn
between the third panel and the bottom panel show the relationships
between phosphopeptides and dephosphorylated phosphopeptides.
[0023] FIG. 8--depicts a MALDI-TOF mass spectrum of the unpurified
and purified phospho-Akt substrate peptide mix, using
alpha-cyano-4-hydroxycinnamic acid as matrix. Peaks labeled with
stars are phosphorylated peptides, peaks labeled with circles
correspond to nonphosphorylated peptides, and peaks labeled with
squares are metastable-decomposition phosphopeptide products. The
top panel shows the peptide mix before purification and the bottom
panel shows the peptide mix after purification.
[0024] FIG. 9--depicts a MALDI-TOF mass spectrum of the unpurified
and purified 14-3-3 binding motif peptide mix, using
alpha-cyano-4-hydroxycinnamic acid as matrix. Peaks labeled with
stars are phosphorylated peptides, peaks labeled with circles
correspond to nonphosphorylated peptides, and peaks labeled with
squares are metastable-decomposition phosphopeptide products. Peaks
labeled with filled stars are phosphopeptides that are not expected
to bind to the 14-3-3 binding motif antibody because their
sequences do not fit the antibody's known specificity. The top
panel shows the peptide mix before purification and the bottom
panel shows the peptide mix after purification.
[0025] FIG. 10--depicts a MALDI-TOF mass spectrum of the peptides
purified by immobilized P-Tyr-100 antibody from a mixture
containing a digested crude 3T3 cell extract, the phosphotyrosine
peptide mix, and the phospho-Akt substrate peptide mix, using
alpha-cyano-4-hydroxycinnamic acid as matrix (top panel). Peaks
labeled with stars are phosphorylated peptides. The bottom panel
shows the bound and eluted peptide fraction after treatment with a
phosphatase enzyme, to remove phosphate groups from
phosphopeptides, reducing the observed mass by 80. Arrows drawn
between the top panel and the bottom panel show the relationships
between phosphopeptides and dephosphorylated phosphopeptides.
[0026] FIG. 11--depicts a MALDI-TOF mass spectrum of the peptides
purified by immobilized phospho-Akt substrate antibody from a
mixture containing a digested crude 3T3 cell extract, the
phosphotyrosine peptide mix, and the phospho-Akt substrate peptide
mix, using alpha-cyano-4-hydroxycinnamic acid as matrix (top
panel). Peaks labeled with stars are phosphorylated peptides, and
peaks labeled with squares are metastable-decomposition
phosphopeptide products. The bottom panel shows the bound and
eluted peptide fraction after treatment with a phosphatase enzyme,
to remove phosphate groups from phosphopeptides, reducing the
observed mass by 80. Arrows drawn between the top panel and the
bottom panel show the relationships between phosphopeptides and
dephosphorylated phosphopeptides.
[0027] FIG. 12--depicts a MALDI-TOF mass spectrum of the peptides
purified by immobilized 14-3-3 binding motif antibody from a
mixture containing a digested crude 3T3 cell extract and the 14-3-3
binding motif peptide mix, using alpha-cyano-4-hydroxycinnamic acid
as matrix. The top panel shows the peptide mix before purification,
and the middle panel shows the peptide mix after purification.
Peaks labeled with stars are phosphorylated peptides, peaks labeled
with circles correspond to nonphosphorylated peptides, and peaks
labeled with squares are metastable-decomposition phosphopeptide
products. The bottom panel shows the bound and eluted peptide
fraction after treatment with a phosphatase enzyme, to remove
phosphate groups from phosphopeptides, reducing the observed mass
by 80.
[0028] FIG. 13--depicts a Western blot of A431 cells overexpressing
the epidermal growth factor receptor (EGFR) and probed with
P-Tyr-100 antibody. Induction of EGFR expression is shown by the
major band that appears after treating the cells with EGF.
[0029] FIG. 14--depicts a MALDI-TOF mass spectrum of modified
peptides (phosphotyrosine) isolated from an A431 cell extract with
P-Tyr-100 antibody-resin, using alpha-cyano-4-hydroxycinnamic acid
as matrix. This cell line overexpresses the EGF receptor and was
treated with EGF to induce phosphorylation at specific sites in the
EGF receptor, as shown in FIG. 13. Peaks labeled with stars are
phosphopeptides, and peaks labeled with circles correspond to
nonphosphorylated peptides. Phosphopeptides purified from the
digested lysate with P-Tyr-100 antibody-resin corresponded to two
known major phosphorylation sites in the EGF receptor, as expected
(top, panel). The fraction was treated with phosphatase and
reanalyzed (bottom panel) to confirm isolation of phosphopeptides.
Lines drawn between the top and bottom panels indicate the
relationships between phosphopeptides and dephosphorylated
phosphopeptides.
[0030] FIG. 15--depicts a Western blot of 3T3 cells stably
transfected to express active Src protein kinase constituitively
and probed with P-Tyr-100 antibody. Comparison to untransfected
cells shows the effect of Src expression on the number and level of
proteins recognized by the P-Tyr-100 antibody.
[0031] FIG. 16--depicts a MALDI-TOF mass spectrum of modified
peptides isolated from an extract of 3T3 cells transfected with Src
protein kinase (as shown in FIG. 15) with immobilized P-Tyr-100
antibody, using alpha-cyano-4-hydroxycinnamic acid as matrix (top
panel). Peaks labeled with stars are phosphorylated peptides, and
peaks labeled with circles correspond to nonphosphorylated
peptides. This bound-and-eluted peptide fraction was treated with
phosphatase and reanalyzed (bottom panel) to confirm isolation of
phosphopeptides.
[0032] FIG. 17--depicts an LC-MS/MS spectrum of one of the modified
peptides purified from an extract of 3T3 cells transfected with Src
protein kinase (as shown in FIG. 15) with immobilized P-Tyr-100
antibody. Portions of the spectrum were amplified to show
low-intensity product ions. Sequest assigned this particular
spectrum to a phosphotyrosine-peptide from enolase A. The peptide
sequence and pertinent Sequest scores are shown. Peaks labeled "b"
indicate product ions that contain the amino-terminus of the
peptide, and "y" indicates product ions that contain the
carboxyl-terminus. The number following the "b" or "y" label
indicates the number of peptide residues in that ion.
Doubly-protonated ions, i.e., ions with a charge (z) of 2, are
labeled "++".
[0033] FIG. 18--depicts a Western blot of Jurkat cells treated with
TPA and probed with phospho-(Ser) PKC substrate antibody.
Comparison to untreated cells shows the effect of TPA treatment on
the number and level of proteins recognized by the phospho-PKC
substrate antibody.
[0034] FIG. 19--depicts a MALDI-TOF mass spectrum of modified
peptides isolated from a TPA-treated Jurkat cell extract (as shown
in FIG. 18) with immobilized phospho-PKC substrate motif antibody,
using alpha-cyano-4-hydroxycinnamic acid as matrix (top panel).
Peaks labeled with stars are phosphorylated peptides, peaks labeled
with circles correspond to nonphosphorylated peptides, and peaks
labeled with squares are metastable-decomposition phosphopeptide
products. This bound-and-eluted peptide fraction was treated with
phosphatase and reanalyzed (bottom panel) to confirm isolation of
phosphopeptides.
[0035] FIG. 20--depicts various chromatograms obtained by LC-MS/MS
analysis of the modified peptides purified from a TPA-treated
Jurkat cell extract (as shown in FIG. 18) with immobilized
phospho-PKC substrate motif antibody. The top panel shows where
survey MS scans were collected (the y-axis value is the height of
the tallest peak in each individual spectrum), and the second panel
shows where MS/MS spectra were collected (the y-axis value is the
sum of the heights of all peaks in each individual spectrum). The
third, fourth, and fifth panels show where neutral loss of 49,
32.7, and 24.5, respectively, was detected (the y-axis value is the
height of the neutral-loss ion). The peaks in each chromatogram are
labeled with their corresponding spectrum numbers.
[0036] FIG. 21--depicts properties of the peptides that were
observed to undergo neutral-loss during the LC-MS/MS analysis shown
in FIG. 20, such as mass, phosphate content, and correspondence to
peaks in the MALDI-TOF mass spectrum shown in FIG. 19.
[0037] FIG. 22--depicts some of the MS/MS spectra (left panels) and
MS.sup.3 spectra (right panels) acquired during LC-MS.sup.3
analysis of the modified peptides purified from a TPA-treated
Jurkat cell extract (as shown in FIG. 18) with immobilized
phospho-PKC substrate motif antibody. Each MS.sup.3 spectrum is
grouped with its corresponding MS/MS spectrum, which caused the
data-dependent MS.sup.3 spectrum to be acquired. Sequest was able
to assign parent proteins with good confidence to the three
MS.sup.3 spectra shown.
[0038] FIG. 23--depicts the MS/MS spectra (left panels) and
MS.sup.3 spectra (right panels) that confirm an assignment made by
Sequest to one of the spectra in FIG. 22. The top panels show the
spectra collected for a biological peptide and assigned by Sequest
to UFD1_HUMAN residues 333-343 with phosphoserine at residue 335.
The bottom panels are the spectra collected for a peptide that was
synthesized with this sequence and phosphorylation site. The close
correspondence of the biological peptide spectra and the synthetic
peptide spectra confirms the assignment made by Sequest. Portions
of the MS/MS spectra were amplified to show weak-intensity product
ions.
[0039] FIG. 24--depicts a Western blot of 3T3 cells stably
transfected to express active Akt protein kinase constituitively
and treated with PDGF. The extract was analyzed by SDS-PAGE,
blotted, and probed, using untransfected, untreated cells as a
negative control. The top panel is probed with a general Akt
antibody, the second panel with an antibody specific for
phosphorylation at Akt residue Thr308, and the third panel with an
antibody specific for phosphorylation at Akt residue Ser 473. The
bottom panel is probed with phospho-(Ser/Thr) Akt substrate motif
antibody. This shows that activation of Akt protein kinase is
accompanied by an increase in the number and level of proteins
recognized by the phospho-Akt substrate antibody. Other blotting
experiments showed the major protein recognized by the phospho-Akt
substrate antibody is the ribosomal protein S6.
[0040] FIG. 25--depicts a MALDI-TOF mass spectrum of modified
peptides purified from an extract of 3T3 cells transfected with Akt
protein kinase and treated with PDGF (as shown in FIG. 24), using
alpha-cyano-4-hydroxycinnamic acid as matrix (top panel).
Immobilized phospho-(Ser/Thr) Akt substrate motif antibody was used
to purify modified peptides from the digested extract. Peaks
labeled with stars are phosphorylated peptides, peaks labeled with
circles correspond to nonphosphorylated peptides, and peaks labeled
with squares are metastable-decomposition phosphopeptide products.
All four phosphopeptides in the top panel are accompanied by
metastable-decomposition products arising from neutral loss of
phosphate. Two of these fit the expected masses for phosphopeptides
from the ribosomal protein S6 (2,254.5 and 2,334.4). This fraction
was treated with phosphatase and reanalyzed (bottom panel) to
confirm isolation of phosphopeptides. Lines drawn between the top
and bottom panels indicate the relationships between
phosphopeptides and dephosphorylated phosphopeptides.
[0041] FIG. 26--depicts various chromatograms obtained by LC-MS/MS
analysis of the modified peptides purified from a PDGF-treated 3T3
cell extract (as shown in FIG. 24) with immobilized
phospho-(Ser/Thr) Akt substrate motif antibody. The top panel shows
where survey MS scans were collected (the y-axis value is the
height of the tallest peak in each individual spectrum), and the
second panel shows where MS/MS spectra were collected (the y-axis
value is the sum of the heights of all peaks in each individual
spectrum). The third, fourth, and fifth panels show where neutral
loss of 49, 32.7, and 24.5, respectively, was detected (the y-axis
value is the height of the neutral-loss ion). The peaks in each
chromatogram are labeled with their corresponding spectrum
numbers.
[0042] FIG. 27--depicts properties of the peptides that were
observed to undergo neutral-loss during the LC-MS/MS analysis shown
in FIG. 26, such as mass, phosphate content, and correspondence to
peaks in the MALDI-TOF mass spectrum shown in FIG. 25.
[0043] FIG. 28--depicts three MS/MS spectra acquired during the
LC-MS/MS analysis shown in FIG. 26. These three spectra have been
tentatively assigned to the multiply phosphorylated peptide from
the ribosomal protein S6 with one (panel 1), two (panel 2), or
three (panel 3) phosphate groups. Neutral loss of one, two, or
three phosphate groups is readily apparent.
[0044] FIG. 29--depicts a Western blot of COS-1 cells treated with
insulin and an analog of cAMP and probed with phospho-(Ser) 14-3-3
binding motif antibody. Comparison to untreated cells shows the
effect of treatment on the number and level of proteins recognized
by the phospho-(Ser) 14-3-3 binding motif antibody.
[0045] FIG. 30--depicts a MALDI-TOF mass spectrum of modified
peptides isolated from a treated COS-1 cell extract (as shown in
FIG. 29) with immobilized phospho-(Ser) 14-3-3 binding motif
antibody, using alpha-cyano-4-hydroxycinnamic acid as matrix (top
panel). Peaks labeled with stars are phosphorylated peptides, peaks
labeled with circles correspond to nonphosphorylated peptides, and
peaks labeled with squares are metastable-decomposition
phosphopeptide products.
[0046] FIG. 31--depicts various chromatograms obtained by LC-MS/MS
analysis of the modified peptides purified from a treated COS-1
cell extract (as shown in FIG. 29) with immobilized phospho-(Ser)
14-3-3 binding motif antibody. The top panel shows where survey MS
scans were collected (the y-axis value is the height of the tallest
peak in each individual spectrum), and the second panel shows where
MS/MS spectra were collected (the y-axis value is the sum of the
heights of all peaks in each individual spectrum). The third,
fourth, and fifth panels show where neutral loss of 49, 32.7, and
24.5, respectively, was detected (the y-axis value is the height of
the neutral-loss ion). The peaks in each chromatogram are labeled
with their corresponding spectrum numbers.
[0047] FIG. 32--depicts properties of the peptides that were
observed to undergo neutral-loss during the LC-MS/MS analysis shown
in FIG. 31, such as mass, phosphate content, and correspondence to
peaks in the MALDI-TOF mass spectrum shown in FIG. 30.
[0048] FIG. 33--depicts two MS/MS spectra acquired during the
LC-MS/MS analysis of two different samples, one prepared with
phospho-(Ser/Thr) Akt substrate motif antibody (FIG. 26) (left
panels of this figure), the other prepared with phospho-(Ser)
14-3-3 binding motif antibody (FIG. 31) (right panels of this
figure). In addition to prominent neutral-loss ions, the spectra
have another prominent product ion in common. These spectra are
thought to correspond to peptides that are present in both samples,
due to similar induction conditions and to overlapping motifs
recognized by the antibodies used for purification.
[0049] FIG. 34--depicts an LC-MS/MS spectrum of one of the modified
peptides purified from a treated COS-1 cell extract (as shown in
FIG. 29) with immobilized phospho-(Ser) 14-3-3 binding motif
antibody. Portions of the spectrum were amplified to show
low-intensity product ions. Sequest assigned this particular
spectrum to a phosphoserine-peptide from heat shock 27 kDa protein.
The peptide sequence and pertinent Sequest scores are shown. Peaks
labeled "b" indicate product ions that contain the amino-terminus
of the peptide, and "y" indicates product ions that contain the
carboxyl-terminus. The number following the "b" or "y" label
indicates the number of peptide residues in that ion.
Doubly-protonated ions, i.e., ions with a charge (z) of 2, are
labeled "++".
DETAILED DESCRIPTION OF THE INVENTION
[0050] In accordance with the present invention, there is provided
a general method for isolating a modified peptide (derived from a
post-translationally modified protein) from a complex mixture of
peptides, such as a digested cell lysate. In general, the method
comprises the steps of: (a) obtaining a proteinaceous preparation
from an organism, the protein preparation comprising modified
peptides from two or more different proteins; (b) contacting the
proteinaceous preparation with at least one immobilized
modification-specific antibody; and (c) isolating at least one
modified peptide specifically bound by the immobilized antibody in
step (b). In a preferred embodiment, the method further comprises
the step of (d) characterizing modified peptide(s) isolated in step
(c) by mass spectrometry (MS), tandem mass spectrometry (MS-MS),
and/or MS.sup.3 analysis, or other equivalent method.
[0051] In another preferred embodiment, the invention provides a
method for isolating a phosphopeptide from a complex mixture of
peptides, the method comprising the steps of: (a) obtaining a
proteinaceous preparation from an organism, wherein the
proteinaceous preparation comprises phosphopeptides from two or
more different proteins; (b) contacting the proteinaceous
preparation with at least one immobilized motif-specific,
context-independent antibody that binds a motif comprising at least
one phosphorylated amino acid; (c) isolating at least one
phosphopeptide specifically bound by the immobilized antibody in
step (b); and (d) characterizing said modified peptide isolated in
step (c) by mass spectrometry (MS), tandem mass spectrometry
(MS-MS), and/or MS.sup.3 analysis. In a preferred embodiment, step
(a) further comprises digesting said proteinaceous preparation to
produce a complex mixture of peptides. In another preferred
embodiment, the motif of step (b) comprises all or part of a kinase
consensus substrate motif or a protein-protein binding motif, or
consists of a single phosphorylated amino acid.
[0052] In some preferred embodiments, the methods further comprise
the step of (e) utilizing a search program to substantially match
the spectra obtained for the isolated, modified peptide during the
characterization of step (d) with the spectra for a known peptide
sequence, thereby identifying the parent protein(s) of said
modified peptide. In other preferred embodiments, the method
further includes a quantification step employing, e.g. SILAC or
AQUA, to quantify isolated peptides in order to compare peptide
levels to a baseline or control state.
[0053] The method of the invention enables the single-step
isolation (and subsequent characterization) of multiple different
modified peptides, corresponding to a multitude of different
modified proteins and signaling pathways, with a single antibody.
The method is, therefore, suitable for genome-wide (e.g. cell-wide
or organism-wide) profiling of activation states, and is readily
automatable. The method allows, for example, the rapid, cell-wide
profiling of modification states, such as phosphorylation, of many
different proteins in a test cell or fluid (e.g. a diseased cell)
as compared to a reference cell or fluid (e.g. a normal fluid from
a healthy organism).
[0054] Motif-specific, context-independent antibodies may be
advantageously employed in the disclosed methods. These antibodies
bind short, modified motifs comprising one or more amino acids
including at least one modified residue in a manner that is highly
independent of the differing protein context in which the motif
occurs in multiple signaling proteins within a genome.
Motif-specific, context-independent antibodies, their production,
and their applications are described in U.S. Ser. No. 09/148,712,
Comb et al. (WO 00/14536). Genome-wide profiling of proteins using
motif-specific, context-independent antibodies is generally
described.
[0055] The isolation method of the present invention represents a
significant advance over conventional methods for identifying
modification sites in proteins, particularly with respect to the
following:
[0056] (i) the method is useful for biological samples that have
not been, or cannot be, radioactively labeled;
[0057] (ii) complex mixtures of peptides can be resolved in a
single-step and there is no need for timely and costly purification
before analysis;
[0058] (iii) the method utilizes affinity-chromatography and thus
is more specific than existing methods, such as IMAC, since only
modified peptides are purified, and unmodified peptides do not
contaminate the purified, modified peptide fraction, even when the
overall level of protein phosphorylation is very low;
[0059] (iv) the method specifically isolates the type of modified
residue targeted by the affinity purification, thus, from one
complex, unpurified mixture, the method can be used to isolate
predefined, non-overlapping subsets of modified peptides (e.g.
phosphotyrosine-containing peptides can be purified using a general
protein modification antibody for phosphotyrosine, etc.);
[0060] (v) since the method is based on a stable antibody-antigen
interaction, it does not have to be adjusted as different samples
are analyzed;
[0061] (vi) the recognized problem with existing protein isolation
methods of having non-specific peptides or proteins binding to, and
co-eluting with, bound modified proteins is obviated since
peptides, not proteins, are purified; accordingly, the present
method eliminates the background associated with the non-specific
co-isolation of proteins other than the desired modified protein;
and
[0062] (vii) the method is simpler and easier to use than existing
methods, and is, therefore, particularly well-suited to
high-throughput automation and reproduction.
[0063] As used herein, the following terms have the meanings
indicated:
[0064] "peptide" means a fragment of a whole protein, e.g. a
protease cleavage fragment, having a sequence two or more amino
acids long;
[0065] "modified peptide" means a peptide having an amino acid
sequence comprising at least one, but alternatively more than one,
post-translationally-modified amino acid, for example (but not
limited to), a phosphorylated amino acid such as phosphotyrosine,
phosphoserine, or phosphothreonine, or an acetylated amino acid,
such as acetyl-lysine; modified peptides may contain multiple
modified residues of the same type (e.g. two or more phosphorylated
residues) or may contain multiple modified residues of differing
type (e.g. a phosphorylated residue and a glycosylated
residue);
[0066] "complex mixture of peptides" means a substantially
unpurified mixture of a plurality of different peptides
corresponding to two or more different parent proteins, typically
including both modified and unmodified peptides;
[0067] "proteinaceous preparation" means a preparation of proteins
and/or peptides from one or more cells, tissues, or biological
fluids of an organism, whether unpurified or purified (e.g. IMAC
pre-purified), for example a crude cell extract, a proteolytic
digest, serum, and the like;
[0068] "antibody" means a natural or recombinant antibody,
polyclonal or monoclonal, derivative or fragment thereof, including
F.sub.ab, F.sub.ab', F.sub.(ab')2 and F(v) fragments;
[0069] "modification-specific antibody" means an antibody that
binds at least one modified amino acid, either alone or as part of
a modified motif comprising multiple amino acids, including a
general modification-specific antibody or a motif-specific,
context-independent antibody;
[0070] "general modification-specific antibody" means an antibody
that specifically binds a single modified amino acid, for example a
general phosphotyrosine-specific antibody or a general
acetyl-lysine specific antibody; the term includes, but is not
limited to, a motif-specific, context-independent antibody that
binds a motif consisting of a single modified amino acid;
[0071] "motif-specific, context-independent antibody" means an
antibody that specifically recognizes a short amino acid motif
(typically comprising 1 to 6 invariant amino acids) comprising at
least one modified amino acid in a manner that is highly
independent of the amino acid sequence surrounding (flanking) the
motif in the peptide (i.e. it recognizes the modified motif in
many, if not most, peptides in which it occurs), but does not
substantially recognize peptides containing the unmodified form of
the motif; (the production of such antibodies, which recognize a
plurality of peptides or proteins within a genome that contain the
target motif, has been previously described in Comb et al., WO
00/14536, supra.); the antibody may bind a motif consisting of a
single modified amino acid or a motif comprising multiple amino
acids including at least one modified amino acid (e.g. all or part
of a kinase consensus substrate motif);
[0072] "parent protein" means the protein(s) from which a given
peptide is (or potentially is) derived;
[0073] "phosphopeptide" means a peptide comprising at least one,
but alternatively more than one, phosphorylated amino acid; and
[0074] "protein-protein binding motif" means a short, modified
motif that mediates signal transduction protein binding to a target
protein, for example, 14-3-3 binding motifs, PDK1 docking motifs,
SH2 domains, phosphotyrosine binding domains, and the like.
[0075] The teachings of all references cited in this specification
are hereby incorporated herein by reference. Further aspects,
advantages and uses of the invention are described in more detail
below.
Proteinaceous Preparations
[0076] Proteinaceous preparations containing complex mixtures of
peptides for isolation of modified peptides according to the method
of the invention may be obtained from any desired organism. For
example, the preparation may be obtained from bacteria, yeast,
worms, amphibia, fish, plants, parasites, insects, or mammals. In a
preferred embodiment, the organism is a mammal. In another
preferred embodiment, the mammal is a human. The method can be
applied to a proteinaceous preparation from one or more cell types
or fluid samples derived from any organism. Proteinaceous
preparations may be obtained, for example, by growing cells in
tissue culture according to standard methods, harvesting the cells
from culture media by centrifugation, and lysing the cells by
sonication or other standard means of disrupting cells.
[0077] Proteinaceous preparations may also be obtained directly
from tissue samples. In a preferred embodiment, the tissue sample
is a biopsy sample. These small pieces of living tissue, typically
weighing less than 500 milligrams, are taken directly from an
organism and used directly without growth in tissue culture. The
use of such living tissue allows direct analysis of the biological
state of the tissue without introducing artifacts that may arise as
a consequence of growth in culture. Any desired cell type from a
given organism may be utilized. For example, tumor cells (e.g. from
breast, prostate, etc.) may be cultured or obtained by biopsy to
study proteins with roles in cancer. Neural cells lines are
available to characterize proteins involved in neurotransmission.
Fat cells can be cultured or obtained by biopsy to study proteins
involved in the hormonal mechanisms of fat deposition.
Proteinaceous preparations from tissue samples may contain peptides
or proteins from multiple cell lines or types. In addition, cell
lines with specific, desirable features could be engineered
genetically, e.g., to overexpress a protein thought to have an
important regulatory role in a specific pathway, e.g. cell lines
overexpressing Akt protein. In other preferred embodiments,
proteinaceous preparations are obtained from bodily fluids, such as
serum, urine, spinal fluid, or synovial fluid. Preparations from
blood samples may also be employed, whether cells, e.g.
erythrocytes, are first removed or not.
[0078] Proteinaceous preparations are obtained by standard methods,
e.g. for cells and tissues, by sonication, homogenization,
abrasion, enzymatic digestion, or chemical solubilization.
Generally the method used to lyse cells will be the one most
commonly used for that specific cell type, e.g., enzymatic lysis
for bacteria, abrasion for plant cells, and sonication for animal
cells, but other desired methods may be suitably employed.
Proteinaceous preparations for use in the method of the invention
need not be extensively purified prior to the immunoaffinity
isolation step. For example, urine samples or serum samples may be
directly analyzed. This allows less sample processing, which
increases the likelihood of identifying low-level modifications and
makes it less likely that fractionation methods will bias or skew
the profile of experimentally assigned modifications.
[0079] The mixture can be a crude cell lysate (for example, from
tissue culture, a biopsy, or serum), a partially fractionated
lysate (for example, a highly purified membrane or organelle), or a
known and well-defined composition (for example, an in vitro
modification reaction, that is, a protein modification enzyme
allowed to react with one or more substrate proteins). However, if
desired, simple purifications may be carried out to remove
non-protein elements and/or non-signaling, structural proteins by
standard methods, e.g. by centrifugation to remove erythrocytes,
ultracentrifugation to remove cellular debris and cytoskeletal
proteins, or by treatment with class-specific enzymes such as
nucleases to remove DNA and RNA. In a preferred embodiment, the
proteinaceous preparation is a crude cell extract or fluid, which
has not been extensively purified.
[0080] Preferably; proteinaceous preparations are obtained so as to
reflect the baseline, in vivo activation state, e.g.
phosphorylation state, of proteins in a given cell, e.g. a breast
cancer cell. However, proteinaceous preparations may be obtained
from cells or organisms pre-treated with inducers. For example,
cells grown in tissue culture can be exposed to chemicals such as
calyculin or okadaic acid, which broadly elevate cellular
phosphoprotein levels by inhibiting cellular phosphatases.
Alternatively, a considerably narrower and more specific set of
phosphoproteins in pathways can be induced by treatment with
hormones, such as epidermal growth factor, that activate certain
signaling pathways. Organisms can also be treated with drugs or
infectious agents, and the effects of these treatments can be
evaluated by isolating and analyzing specific tissues or fluids
from the organism.
[0081] To obtain a complex mixture of peptides, the proteinaceous
preparation, which contains a great variety of different proteins,
is digested with a suitable proteolytic enzyme, e.g. trypsin or
chemical cleavage reagent. Any suitable enzyme that yields a
significantly digested proteinaceous preparation (i.e. mostly
peptides as opposed to proteins) may be employed, for example
endoproteinases Lys-C, Glu-C, Asp-N, chymotrypsin, and thermolysin.
In a preferred embodiment, the enzyme is trypsin. If desired,
digestion with two or more different proteolytic enzymes may be
carried out to yield smaller peptides suitable for mass
spectrometry analysis (e.g., peptides of about 30 amino acids in
length or less, for current MS methods). Digestion of proteins may
be carried out in an enzymatic solution or with immobilized
proteolytic enzymes (e.g. trypsin-POROS resin, available from
Applied Biosystems, Inc., Framingham, Mass.; trypsin-Matrix F7m,
available from MoBiTec, Marco Island, Fla.), which can easily be
removed from the digest by centrifugation or filtration before the
preparation is contacted with the immobilized antibody. If soluble
proteolytic enzymes are used, the digests are preferably treated
with inhibitors such as PMSF or alpha-2-macroglobulin before the
proteinaceous preparation is contacted with the immunoaffinity
purification device, so that the proteolytic enzyme will not
degrade the immobilized antibody molecules. Alternatively, soluble
proteolytic enzymes are removed by prefractionating peptides by
solid phase extraction prior to contact with the immobilized
antibody. Digestions are conducted for a period of time sufficient
for complete digestion, that is, for cleavage of all peptide bonds
that can be cleaved by the protease. However, if the digestion is
incomplete, intentionally or not, the overlap between partially
digested peptides can provide further evidence that the
phosphorylation site has been assigned correctly, i.e., when the
same phosphorylation site is found in more than one peptide because
the peptides overlap due to incomplete digestion, the site
assignment is more convincing. In a preferred embodiment, a
protease that can cleave at a large variety of residues, e.g.,
chymotrypsin, thermolysin, or elastase, is used for digestion in a
manner that deliberately generates peptides with overlapping
sequences.
[0082] Preferably, proteinaceous preparations for use in the method
of the invention contain modified peptides, e.g. phosphopeptides,
from two or more different proteins, and in most cases contain
modified peptides from a multitude of different proteins. The
proteinaceous preparation typically contains a complex mixture of
many different types of modified, as well as unmodified, peptides.
For example, such'mixtures may contain peptides modified by
phosphorylation, acetylation, methylation, sulfation,
nitrosylation, or glycosylation, among others. See, e.g. Krishna et
al., Adv. Enzymol. Relat Areas Mol. Biol. 67: 265-98 (1993); Parekh
et al., Curr. Opin. Biotechnol. 8: 718-23 (1997).
[0083] In a preferred embodiment, the proteinaceous preparation
contains phosphopeptides from two or more different proteins.
Accordingly, these complex mixtures of modified peptides reflect
the activation state, e.g. phosphorylation state, of signaling
pathways in a given organism or cell type on a genome-wide or
cell-wide basis, thus providing a snap-shot of activation states in
that organism. The complex mixture of modified peptides in the
proteinaceous preparation reflects the baseline, in vivo activation
status in the given organism or cell line, but may, as discussed
above, reflect activation status in a treated cell, so as to
reflect the effect of treatment upon activation status.
[0084] In certain preferred embodiments, the proteinaceous
preparation comprises a digested biological sample selected from
the group consisting of a digested crude cell extract, a digested
tissue sample, a digested serum sample, a digested blood sample, a
digested urine sample, a digested synovial fluid sample, and a
digested spinal fluid sample. The digested preparation may be
obtained using at least one proteolytic enzyme, such as trypsin. In
a preferred embodiment, the proteolytic enzyme is immobilized. In
another preferred embodiment, the proteolytic enzyme is soluble,
and the said digested preparation is treated with a proteolysis
inhibitor prior to the contacting step (b).
[0085] In one preferred embodiment the method is coupled with
quantitative mass spectrometry approaches, so that modified
peptides are identified and at the same time their levels in the
sample are measured. The absence of a modified peptide is more
meaningful when quantitative mass spectrometry approaches are used,
because that peptide has been sought and not found, as opposed to
overlooked. In addition, modified peptides that are present in two
samples may be present at very different levels, but this
difference is not easily recognized in qualitative identification
studies, even though it may be a key feature of the system being
studied. SILAC (stable isotope labeling by amino acids in cell
culture) can be used for relative quantification of two cell
culture samples, e.g., a cell line treated with a drug can be
compared to the same cell line left untreated. See, e.g. Ong et
al., Mol. Cell. Proteomics 5: 376-86 (2002). Another quantitative
mass spectrometry strategy is termed Aqua for absolute
quantification. See Gerber, Rush et al. Proc. Natl. Acad. Sci.
U.S.A. 100: 6940-5 (2003). In Aqua a sample is compared to a panel
of synthetic peptide standards, which serve as a reference control.
Unlike SILAC, Aqua is better suited for the development of assays
based on mass spectrometry, and it can be applied to tissue samples
as well as cell cultures.
Immunoaffinity Isolation
[0086] The proteinaceous preparation, which contains a complex
mixture of modified and unmodified peptides from a plurality of
different proteins, is contacted with an immobilized,
modification-specific antibody (e.g. anti-phosphothreonine) in
order to isolate many, if not most, peptides containing the
modification for which the immobilized antibody is specific.
Peptides with the appropriate modification bind to the immobilized
antibody, while unmodified peptides and/or peptides with other
modifications do not. Thus, immunoaffinity purification according
to the disclosed method allows the one-step isolation of a broad
range of desired peptides (originating from different proteins)
from substantially unpurified, complex mixtures of peptides.
[0087] In a preferred embodiment, the antibodies are
covalently-linked to an inert chromatography resin, such as
agarose, polystyrene, or silica, by standard techniques. Briefly,
the carbohydrate groups of the antibody molecules are oxidized to
reactive aldehyde groups, which are then covalently bonded to the
hydrazide groups of derivatized chromatography resins. See, e.g.
Hoffman et al., J. Immunol. Methods 9: 113-120 (1988). The
carbohydrate groups of the antibody are not required for antigen
recognition, so the chemical modification does not interfere with
their ability to bind peptides. Using this standard method or
others, antibodies are attached to chromatography supports at high
concentrations, and because the antibodies are attached covalently
to the resin, they do not leach off the support and contaminate
purified samples. Alternatively antibodies may be immobilized by
non-covalent attachment to protein A or protein G, which have been
previously covalently linked to agarose resin, as in another
preferred embodiment. It is simpler to immobilize antibodies to
protein A- or protein G-agarose than it is to covalently immobilize
antibodies to agarose. However antibodies immobilized to protein A
or protein G supports have the disadvantage that they can be used
only once, because the interaction of the antibody with protein A
or protein G is disrupted by the conditions used to elute peptides
from the antibody. When the complex mixture of peptides in the
proteinaceous preparation is contacted with the antibody-resin, in
either batch or column format, the antibody-resin selectively binds
the modified peptides, even when they are present at low levels
(i.e. picomole amounts).
[0088] For example, in batch format, the proteinaceous preparation
is contacted with the antibody-resin by mixing as a slurry, and the
antibody-resin with bound peptides is then removed by
centrifugation, filtration, etc. Alternatively, in column format,
the covalently-linked antibody-resin is contained within/packed in
a chromatography column, and the proteinaceous preparation is
passed through the column, so peptides that are recognized by the
immobilized antibody are retained on the column and unrecognized
peptides pass through the column. The antibody-resin may, in
another preferred embodiment, be contained within a micropipette
tip.
[0089] Column size, flow rates, and conditions (e.g. pH, choice of
buffer) are selected in accordance with standard techniques. For
low-level samples, a substance such as BSA, detergent, or polymer
may be added to the proteinaceous preparation prior to contact with
the immobilized antibody in order to prevent non-specific peptide
loss through adsorption. The immunoaffinity purification step may
be optimized, if desired, to ensure that all modified peptides in
the sample are quantitatively bound to and eluted from the
antibody-resin (i.e. little, if any, desired modified peptide is
unbound). For example, the molar ratio of antibody to modified
peptides, the amount of antibody per unit mass of antibody-resin,
the length of time the sample contacts the antibody-resin
(including recirculating the sample through an antibody-resin
column), the temperature at which contact occurs, the inclusion of
additives (e.g., salts, detergents, organic solvents, or polymers)
that may enhance interaction of modified peptides with the
antibody-resin, etc., may, if desired, each be optimized by the
skilled artisan in practicing the method of the invention.
Generally the metric for evaluating optimization is maximizing the
number of modified peptides observed during analysis, with some
emphasis given to minimizing the number of unmodified peptides that
are isolated, which is a measure of the specificity of the method
for modified peptides. This can be evaluated by MALDI-TOF mass
spectrometry, before and after treatment with phosphatase, an
enzyme that removes phosphate from peptides and lowers the measured
masses of phosphopeptides. It can also be evaluated by the actual
peptide sequences determined by tandem mass spectrometry. Other
optimizations are possible, e.g., reducing the amount of time
needed to analyze a sample without reducing the number of modified
peptides identified and increasing the number of modified peptides
identified by using different types of separation systems or mass
spectrometers for analysis, e.g., capillary electrophoresis for
separating peptides instead of reversed-phase HPLC, or
quadrupole-time-of-flight hybrid mass spectrometry instead of ion
trap mass spectrometry for analysis.
[0090] In a preferred embodiment, immunoaffinity isolation is
carried out by utilizing a device consisting of one or more
modification-specific antibodies immobilized to a rigid, non-porous
or macroporous resin particle, packed into a thin capillary column,
with an internal diameter of about 50 to 300 micrometers. While
capillary columns of this type containing reversed-phase or ion
exchange supports are already widely used, prior to the instant
invention, capillary columns packed with immunoaffinity supports,
as disclosed herein, have not been described. Immunoaffinity
isolation devices of the invention may be constructed of any
suitable material, for example, fused silica capillaries. The ends
of the capillaries are drawn to fine tips, so the internal diameter
at the tip is 3 micrometers or less, using an electronic
microcapillary puller. The capillaries are then packed with
chromatography resin using a Jorgensson and Kennedy pressure bomb,
to force the resin slurry into the column through the back end. See
Gatlin et al. Anal. Biochem. 263: 93-101 (1998). Resin particles
are larger than the diameter of the capillary tip, so the resin
accumulates in the column and is packed by pressure applied through
the bomb. When the packed column has reached the desired length,
the pressure is relieved, the empty back of the capillary is
trimmed away, and the column is stored or used.
[0091] A preferred resin is POROS, a rigid macroporous resin
developed at Perseptive Biosystems for use in perfusion
chromatography. Resin particles are about 20 micrometers in
diameter and are of uniform size. The resin is sold commercially
through Applied Biosystems (Framingham, Mass.), including
chemically derivatized resins for covalently attaching proteins
such as antibodies. Other suitable types of resins known to those
of skill in the art may be employed, for exampled, magnetic
DynaBeads from Dynal.
[0092] This immunoaffinity isolation column can be adapted to be
used as (i.e. coupled to) part of an electrospray source on a mass
spectrometer, so that peptides can be readily analyzed after
isolation with minimal sample loss. The capillary column itself is
fitted directly to the mass spectrometer and acts as a fritless
electrospray interface. For example, using standard low-volume HPLC
fittings, the column is inserted into a plastic (PEEK) micro-tee
fitting (shaped like the letter T). A capillary line from the HPLC
solvent delivery system is attached to the opposite side of the
micro-tee fitting, in line with the column, so different solvents
or a gradient of solvents can be delivered at low flow rates,
typical less than 1 microliter/minute, through the column to elute
samples bound to the column. A gold rod is inserted into the third
stem of the micro-tee, perpendicular to the solvent delivery lines
and column, to supply the electrical connection from the mass
spectrometer through a liquid-metal junction. All three devices are
secured in the fitting with standard PEEK micro-fingertight
fittings and tubing sleeves. The source normally used with the mass
spectrometer is removed and replaced by a metal platform that holds
this micro-tee assembly. The position of the capillary column tip
can be precisely controlled by making adjustments with an XYZ
micromanipulator on the platform, so the position of the spraying
column tip relative to the mass spectrometer orifice is optimized
for maximum ion current signal. In this way microcolumn liquid
chromatography and micro-electrospray ionization may be combined
into one device.
[0093] The solutions used to elute bound samples from
immunoaffinity columns, e.g., 30% acetic acid or 0.1 M glycine, pH
2.3, typically are not compatible with direct analysis by
electrospray mass spectrometry. However, the immunoaffinity
purification device can be used as the first component of a
two-dimensional HPLC system, where an immunoaffinity purification
column and a reversed-phase column are directly connected. A
two-dimensional HPLC system using a strong cation exchange column
upstream of a reversed phase column has been described. See, e.g.
Washburn et al. Nat. Biotech. 19: 242-247 (2001).
[0094] The liquid stream from the HPLC system is diverted to waste
during the immunoaffinity purification step. As samples elute from
the immunoaffinity purification column, they bind to the downstream
reversed-phase capillary column, but the solution components used
for elution do not bind and are diverted to waste. The bound
samples can then be eluted from the reversed-phase column using
solvents that are compatible with direct analysis by electrospray
mass spectrometry. Alternatively, the immunoaffinity step can be
done off-line, using a solid-phase extraction cartridge in a
micropipette tip, as described below, and then applied to a
reversed-phase capillary column in an LC-MS/MS system. In both
cases, the capillary columns are mounted in the mass spectrometer
and samples are ionized as they elute from the column as described
in Gatlin, supra.
[0095] Immunoaffinity isolation devices comprising capillary
columns as described herein are useful not only for peptides that
bind and elute from the column, but also for peptides that bind to
the antibodies with lower affinity and whose passage through the
column is retarded, extending the usefulness of the method. These
columns would be reusable and have lifetimes comparable to other
types of capillary HPLC columns. See, e.g. Gatlin, supra.
[0096] In another preferred embodiment, the immunoaffinity
isolation device is a solid-phase extraction cartridge in a
micropipette tip. Devices that have been constructed with
reversed-phase and other types of HPLC supports (e.g., ZipTips from
Millipore) have been described. See e.g., Erdjument-Bromage et al.,
J. Chromatogr. A 826: 167-181 (1998). These devices are attached to
standard laboratory pipetting devices and are used in the same
manner as pipette tips: as the sample is aspirated into the tip, it
becomes bound to the chromatography support, which is then washed
before eluting the sample in a small volume for analysis. The tip,
for example, may be fabricated by embedding immobilized
antibody-resin in a gel matrix in the dispensing end of a standard
micropipette tip. See, e.g. Chirica et al., Anal. Chem. 72:
3605-3610 (2000). Taking advantage of the general stability of
antibody molecules, these devices may be supplied dry; the end user
would then rehydrate and condition the gel containing immobilized
antibody immediately before use. These high-capacity, small-volume
tips would be used to fractionate (i.e. isolating desired peptide)
one sample and then discarded. Immunoaffinity separation may also
be performed with other types of solid supports, such as porous
filtration membranes or sample supports for MALDI-TOF mass
spectrometry. See, e.g. Weller, Fresenius J. Anal. Chem. 366:
635-645 (2000); Liang et al. Anal. Chem. 70: 498-503 (1998).
[0097] Immunoaffinity isolation according to the method of the
invention may be carried out without additional chromatography
steps (e.g., reversed-phase or ion exchange chromatography).
However, in some preferred embodiments, additional chromatography
methods may be employed in conjunction with, and prior to, the
single-step immunoaffinity isolation of the present method. For
example, a digested cell lysate can be applied to a reversed-phase
solid-phase extraction cartridge and fractionated by increasing the
organic solvent concentration as the cartridge is washed in steps.
Each fraction would thus be enriched for certain peptides, with
minimal overlap between fractions, and the fractionated peptides
could be more concentrated than the peptides in the unfractionated
digested cell lysate. In the same manner, the digested cell lysate
could be prefractionated with an ion-exchange solid-phase
extraction cartridge, which would be developed by washing the
cartridge in steps with increasing concentrations of salt.
[0098] In one preferred embodiment of the method, immobilized metal
affinity chromatography (IMAC) is employed as an upstream
pre-purification/fractionation step prior to immunoaffinity
isolation as disclosed herein. As discussed earlier, although IMAC
can enrich phosphopeptides from peptide mixtures, it has several
important limitations (such as purification of phosphopeptides
without specificity for the particular phosphorylated residue,
purification of acidic peptides that are not phosphorylated,
incomplete purification of phosphopeptides (i.e., some peptides do
not bind or elute from the IMAC support), and poor reproducibility
(which makes it difficult to compare samples)) which render it
unsuitable for the selective and facile isolation of
phosphopeptides from complex mixtures. Despite its limitations as a
stand-alone technology, however, IMAC may be desirably employed as
a bulk phosphopeptide enrichment/pre-purification step upstream of
the method of the present invention.
[0099] For example, IMAC may be performed at very low stringency,
in order to bind as many phosphopeptides as possible without regard
for the much larger number of acidic non-phosphopeptides that would
also bind to the IMAC support under these conditions. After elution
from the IMAC column, the peptides would be fractionated further by
the method of the invention, which would separate acidic
non-phosphopeptides from phosphopeptides and which would further
separate phosphopeptides into discrete subsets based on the
particular residue that is phosphorylated. For example, peptides
that contain phosphotyrosine and peptides that contain the Akt
substrate binding motif would be separated from each other and
could be isolated from the same IMAC-prepurified digested cell
lysate. Thus the method of the invention may be desirably practiced
in conjunction with other methods of phosphopeptide
purification.
[0100] In another preferred embodiment, the method of the invention
is used with several non-overlapping modification-specific
antibodies in series to extract well-defined, distinct modified
peptide populations from a single sample. For example, peptides
containing phosphotyrosine are first extracted from a digested cell
extract using immobilized phosphotyrosine antibody; the digested
extract is then separated from the phosphotyrosine antibody-resin
and treated with a second immobilized antibody, e.g.,
phosphothreonine-proline antibody, to extract phosphopeptides
containing phosphothreonine-proline motifs. This process, which we
have termed "rational fractionation" or "modified peptide sorting",
can be repeated for as many steps as there are non-overlapping
modification-specific antibodies. When each group of extracted,
modified peptides is analyzed by mass spectrometry, identification
of modified sites is simpler than for modified peptides that have
been purified in bulk because the majority of peptides in each
group are highly likely to contain a predetermined modification,
e.g., phosphotyrosine or phosphothreonine-proline. With this
strategy, sample complexity is addressed before analysis rather
than after analysis, resulting in a larger number of
high-confidence identifications.
Modification-Specific Antibodies
[0101] In accordance with the invention, immunoaffinity isolation
is carried out by using at least one modification-specific antibody
that specifically recognizes a given type of post-translational
modification, e.g. phosphorylation, acetylation, methylation,
nitrosylation, glycosylation, etc. Preferably, the
modification-specific antibody is: (i) a general
modification-specific antibody, that is, an antibody that binds a
single modified amino acid residue, e.g. phosphothreonine, but does
not recognize the unmodified amino acid residue, and/or (ii) a
motif-specific, context-independent antibody produced by the method
described in Comb et al., WO 00/14536, supra (also described
below). Motif-specific, context-independent antibodies against many
different phosphorylated kinase consensus substrate motifs and
protein-protein binding motifs are commercially available. (See
CELL SIGNALING TECHNOLOGY, INC. 2003-04 Catalogue & Technical
Reference, pages 12-24).
[0102] The use of such modification-specific antibodies (both
general and/or motif-specific, context-independent) thus allows the
single-step isolation of many, if not most, peptides in a complex
mixture that contain the modification or motif, regardless of the
peptide sequence surrounding the modification or motif (i.e. these
antibodies are not "site-specific" and hence are not limited to
recognition of particular longer peptide sequences presenting a
uniquely-occurring site epitope).
[0103] In a preferred embodiment of the method, the modification
(on the peptides to be isolated) comprises phosphorylation and the
modified peptide(s) isolated comprise(s) a phosphopeptide.
Particularly preferred phosphorylated residues are phosphotyrosine,
phosphoserine, phosphothreonine, or phosphohistidine. Although the
invention is demonstrated in the Examples using phospho-specific
antibodies, it will be recognized by those of skill in the art that
other modification-specific antibodies may be readily employed, for
example, acetylation-specific antibodies. Virtually any desired
modified peptide may be isolated, as described in "Proteinaceous
Preparations" above.
[0104] In certain preferred embodiments, motif-specific,
context-independent antibodies are advantageously employed in the
disclosed method to isolate many, if not most, peptides containing
a desired modified motif. These antibodies and their production
have previously been described. See Comb et al., WO 00/14536,
supra. The antibodies bind to short, modified motifs, which,
because of their small size and degenerate sequences, occur more
than once in a given genome (i.e. occur in two or more different
proteins, as opposed to larger, unique epitopes or "sites" that
statistically occur only once) and thus serve, biologically, as
consensus sequences and conserved binding sites for, e.g. kinases,
in multiple proteins in cellular signaling pathways.
[0105] The invention may utilize antibodies specific for any
desired motif of interest, e.g. signaling pathway motifs,
comprising one or more modified amino acids. In certain preferred
embodiments of the disclosed method, the modification-specific
antibody used to isolate peptides comprises a motif-specific,
context-independent antibody that recognizes a motif comprising at
least one phosphorylated amino acid. In one preferred embodiment,
the motif consists of a single phosphorylated amino acid, such as
phosphotyrosine, phosphothreonine, or phosphoserine. In another
preferred embodiment, the motif comprises all or part of a kinase
consensus substrate motif or a protein-protein binding motif.
[0106] For example, in preferred embodiments, motif antibodies
specific for all or part of any of the following kinase consensus
or protein-protein binding motifs are used for immunoaffinity
isolation: MAPK consensus substrate motifs, CDK consensus substrate
motifs, PKA consensus substrate motifs, AKT consensus substrate
motifs, PKC consensus substrate motifs, PDK1 docking motif (bulky
ring), phosphothreonine-X-arginine, ATM/ATR consensus substrate
motifs, 14-3-3 binding motifs, p85 PI3K binding motif,
phosphothreonine-proline motif, Arg-X-Tyr/Phe-X-phosphoserine
motif, and phosphoserine/phosphothreonine-Phe motif. (See, e.g.
CELL SIGNALING TECHNOLOGY 2003-04 Catalogue at p. 14).
Context-independent antibodies against any desired kinase consensus
substrate motif or protein-protein binding motif may be
advantageously employed in the method of the invention; such motifs
are well described in the literature (see, e.g., Kemp et al.,
Trends in Biochem. Sci. 15: 342-46 (1990); Kemp et al., Methods in
Enzymology 200: 62-81 (1991); al-Obeidi et al., Biopolymers 47:
197-223 (1998); see also L. Cantley, overview in Cell Signaling
Technology, Inc. 2000-2001 Catalogue at p. 198.)
[0107] The preparation of motif-specific, context-independent
antibodies, previously described in Comb et al., WO 00/14536, supra
(the disclosure of which are incorporated by reference in their
entirety) is carried out briefly as follows: [0108] (1)
Motif-specific antibodies that react with any protein or peptide
containing specific target residues independently of the
surrounding amino acids may be obtained by synthesizing a highly
degenerate peptide library. In one preferred embodiment, the
library comprises XXXXXXJ*XXXXXXC where X=all 20 amino acids except
cysteine and J*=a modified (*) amino acid (J). It will be
appreciated that a shorter or longer library may be generated and
less than all of the surrounding amino acids may be varied. For
example, one to four X residues may be selectively biased for 1 or
2 specific amino acids, while the remaining X residues are highly
degenerate. In one preferred embodiment, the peptide library is
about 6 to 14 residues long. While one preferred embodiment
utilizes one fixed amino acid (either modified or unmodified) in a
varied surrounding context, other preferred embodiments may utilize
a motif comprising several fixed amino acids. Likewise, the
surrounding sequence of the library may be varied at more than one
position simultaneously, or, as in the preferred embodiment, varied
at only one surrounding sequence position per degenerate molecule,
such that a library is produced which is completely degenerate at
every position except the fixed residue(s). The peptide library can
be synthesized by standard Fmoc solid phase peptide synthesis using
an ABI peptide synthesizer and using mixtures of each amino acid
during degenerate coupling reactions. [0109] The incorporation of
unmodified amino acids at fixed positions may be selected to mimic
conserved motifs, for example zinc fingers or repeating arginine
residues. [0110] (2) In order to produce as equal a representation
of each amino acid as possible at each degenerate position, several
rounds of altering the amino acid composition, synthesizing, and
peptide sequencing are conducted. Amino acid sequence analysis at
several different positions along the peptide is conducted to
verify a random amino acid representation at each position and that
the random representation is maintained throughout the synthesis.
It will be recognized by one of skill in the art that the number of
rounds may vary in order to achieve an equal distribution of all
amino acids at each position. [0111] (3) The highly diverse peptide
library is used as an antigen, preferably by covalent coupling to a
carrier. In a preferred embodiment, keyhole limpet hemocyanin (KLH)
emulsified in Freund's adjuvant is used as the coupling agent, and
the coupled peptide library injected intradermally into a host,
such as female New Zealand white rabbits. Booster injections may be
given in incomplete Freund's adjuvant until an immune response is
obtained. Antibody titer is measured by a suitable method, such as
ELISA against the motif-specific peptide libraries. Antisera raised
in this manner may be used in either crude or purified
preparations, as outlined below. [0112] (4) Antisera from the most
promising hosts are purified, for example over protein A, and
adsorbed over a J (non-modified) peptide library column. In the
preferred embodiment, the nonadsorbed fraction (flow through) is
then applied to a J* column, eluted at suitable pH, dialyzed and
tested for J* specificity by a suitable method, such as ELISA using
J* and J as antigen. [0113] (5) Antibodies affinity purified in
this fashion recognize the J* peptide library but do not react with
the J library and exhibit a high degree of specificity for J. These
antibodies may be further tested for lack of reactivity against the
unmodified form of the target modified amino acid, J*, or a J*
homologue, utilizing a suitable method, such as ELISA. [0114] (6)
Antibodies may be further tested by western blotting, or another
suitable method, using cell extracts prepared from cells treated
with and without a selected protein modification enzyme inhibitor,
such as protein phosphatase inhibitor okadaic acid. Treatments that
increase protein modification will increase the number of antibody
reactive proteins as well as the intensity of reactivity. The J*
specific antibodies will react with a relatively small number of
proteins from control extracts but will react with a very large
number following treatment. The antibodies will show no reactivity
with the inactive-non-modified versions of these proteins,
demonstrating a high degree of J* specificity and suggesting broad
cross-reactivity to many different modified-target containing
proteins. [0115] (7) The degree of context-independence may be more
carefully examined, for example, by ELISA analysis against
individual J* peptides that are mixed together or tested
individually. Such analysis can indicate if poor reactivity occurs
with certain motifs, such as when J* is followed by proline, for
example. [0116] (8) The context-dependence of J* antibody
recognition may be further examined using an immobilized grid of
modified-peptide libraries. In addition to a fixed target residue,
J*, each different library is synthesized to contain an additional
fixed amino acid at different positions relative to J* but with all
other positions containing all 20 amino acids except cysteine. Each
peptide library is coated, for example, on the bottom of an ELISA
well and exposed to the J* antibodies. Antibodies that do not react
with a particular spot (peptide library) on the grid do not bind
when the specified amino acid is present at the specified position.
This analysis determines whether or not a particular amino acid at
a particular position relative to J* will allow or block binding.
[0117] Alternatively, purified antibodies can be linked to resin,
allowed to bind the modified or unmodified library, unbound
sequences washed away, and bound sequences recovered and subject to
amino acid sequencing to determine the amount of each amino acid
present at each position in the library. This information will
indicate what amino acids are tolerated at each position.
[0118] Antibodies suitable for use in the method of the present
invention may be polyclonal or monoclonal, or may be a fragment
thereof, e.g. an F.sub.ab fragment, or a derivative thereof, e.g. a
humanized antibody. A single antibody, e.g. a general
phosphothreonine antibody, may be used in the immunoaffinity step,
or two or more antibodies may be simultaneously used to isolate
peptides containing different modifications, e.g. acetylated lysine
and phosphothreonine. Alternatively, isolation of peptides with one
modification may first be carried out with one immobilized
antibody, and then peptides with other modifications may
subsequently be purified using other immobilized antibodies and/or
resins. This process, which we have termed "rational fractionation"
or "modified peptide sorting", can be repeated for as many steps as
there are non-overlapping modification-specific antibodies.
[0119] Following contact with the immobilized antibody, the
antibody/resin is thoroughly washed to remove unbound peptides and
then peptides bound to the antibody/resin (i.e. those containing
the desired modification) are isolated from the resin by eluting
with a small volume of an acidic solution, e.g. 30% acetic acid, or
other suitable eluting solution. The eluted peptides are analyzed
directly as described below, or concentrated and desalted with a
micropipette tip containing reversed-phase resin, and then
analyzed. If desired, peptide sequence and/or parent protein
information is obtained by mass spectroscopy.
[0120] In certain circumstances, analysis of purified peptides (as
described below) may indicate that some undesired peptides that
lack the target motif are co-purified along with peptides that
contain the desired target motif. In such cases, the number or
stringency of the resin washes may be increased to eliminate
non-specific peptide binding. Stringency of washes may be increased
according to techniques well known in the art, for example, by
including additives that reduce background binding, such as
detergents, organic solvents, or polymers.
Analysis of Isolated Peptides
[0121] Isolated peptides containing the desired modification may be
analyzed by standard methods to determine peptide sequence,
activation state, and mass. In certain preferred embodiments,
modified peptides isolated according to the method of the invention
are analyzed by mass spectrometry (MS) methods, since MS is
presently the most sensitive method for analyzing peptides. MS
requires less analyte material to provide high-quality information
about peptides than other current methods. It will be recognized by
the skilled artisan that equivalent or subsequently improved
methods of analyzing modified peptides are within the scope of the
invention. For example, at present, peptides of about 30 amino
acids in length or less are most suitable for MS analysis, but
future improvements in methods may allow the analysis of longer
peptides.
[0122] Accordingly, in a preferred embodiment, the general method
of the invention further comprises the step of (d) characterizing
the modified peptide(s) isolated in step (c) by mass spectrometry
(MS), tandem mass spectrometry (MS-MS), and/or MS.sup.3 analysis.
In one preferred embodiment, matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry
is utilized to measure the masses of purified peptides. MALDI-TOF
mass spectrometry is useful for rapidly screening samples before
analyzing them by other, more complex methods such as tandem mass
spectrometry (MS/MS) (see below), and is both sensitive and simple.
For proof-of-principle experiments or diagnostic assays, where the
objective of the isolation is to determine if an expected peptide
is present among the purified modified peptides, the mass of the
purified peptide(s) is calculated from the peptide's known sequence
and searched for in the mass spectrum.
[0123] MALDI-TOF mass analysis of peptides is a rapidly evolving
field, and the preferred methods for preparing isolated modified
peptides for analysis and carrying out such analysis is likely to
change over time. Nonetheless, MALDI-TOF analysis is carried out
according to standard methods (see, e.g. Courchesne et al., Methods
in Mol. Biol. 112: 487-511 (1999)), and improvements in these
methods are within the scope of the present invention. For example,
isolated peptides are prepared for MALDI-TOF analysis using only a
small portion, 1 to 20%, of the isolated (purified) modified
peptide-containing fraction, and analyzed by mixing directly with a
equal volume of saturated matrix solution, e.g.
alpha-cyano-4-hydroxycinnamic acid, and drying the peptide-matrix
solution on the MALDI-TOF sample plate. Other suitable matrix
solutions may be alternatively employed. If necessary, a larger
sample aliquot can be concentrated and desalted with a micropipette
tip containing reversed-phase matrix before mixing it with matrix
solution.
[0124] To confirm that purified peptides contain the desired
modification, a small portion of the sample is preferably analyzed
before and after treatment with an enzyme that removes the modified
group from the peptide. For example, where the modified peptides
being purified are phosphopeptides, phosphate is removed using a
suitable phosphatase, e.g. calf intestinal phosphatase. See, e.g.
Larsen et al. Proteomics 1: 223-238 (2001). This is a simple and
reliable assay to confirm that peptides are modified, and to count
the number of modified groups present in each peptide. For example,
phosphatase treatment will reduce the observed peptide mass by 80
for each phosphate group in the peptide. The mass of a peptide that
is not phosphorylated will not change as a result of phosphatase
treatment. Similarly, any suitable modification-specific enzyme
known in the art may be selected to confirm that peptides isolated
according to the method of the invention contain the desired
modification. See e.g., Krishna, supra.
[0125] If phosphopeptides are being isolated, metastable
decomposition may result in the presence of additional peaks in the
mass spectrum. Metastable decomposition of phosphopeptides has been
noted by others and can be used to recognize and assign
phosphopeptides in a MALDI-TOF mass spectrum (Annan and Carr, Anal.
Chem. 68: 3413-21 (1996)). The peaks for decomposition products are
broader than the peaks for phosphopeptides because the
decomposition products form after ionization and the instrument is
configured to focus ions that are stable during analysis. For
similar reasons, the expected mass shift for loss of phosphate is
-98, but -84 mass shifts are observed because, unlike a stable ion,
the mass of a decomposition product changes during analysis.
Analysis of a large number of synthetic phosphopeptides by
MALDI-TOF mass spectrometry has indicated that some peptides
containing phosphoserine or phosphothreonine--but not
phosphotyrosine--residues undergo metastable decomposition.
Accordingly, metastable decomposition is a reliable indicator of
peptides that contain phosphoserine or phosphothreonine. Metastable
decomposition may be observed in the MALDI-TOF spectra of some
peptides that contain phosphoserine or phosphothreonine, without
additional sample treatment steps and without consuming more
sample.
[0126] In other types of applications, for example in a genome-wide
analysis employing the disclosed method, it may not be possible to
identify the modified peptides isolated from the complex mixture
present in a proteinaceous preparation simply by measuring peptide
masses because many different peptide sequences could produce each
mass observed in the isolated modified peptide fraction.
Accordingly, in another preferred embodiment, modified peptides
isolated from complex mixtures (e.g. crude cell extracts) are
analyzed by tandem mass spectrometry (MS/MS or MS.sup.3), where
peptide ions isolated in one stage of mass spectrometry are
deliberately fragmented by collisions in the mass spectrometer, and
then the fragment masses are measured. See, e.g. Yates, Methods in
Enzymology 271: 351-377 (1996). The fragment masses observed for
each peptide are a property of that peptide's sequence and are a
more specific indicator of the parent protein than the peptide's
mass, i.e. the fragment masses are related to the peptide's
sequence and can be used to identify the protein from which the
peptide originated. If the sequence of the peptide's parent protein
is known, then the peptide can be unambiguously matched to its
parent protein without directly interpreting a sequence from the
fragment mass spectrum.
[0127] A particular peptide's measured mass and partial sequence is
sufficient to unambiguously match it to its parent protein. See
e.g. Eng et al. J. Am. Soc. Mass Spectrom. 5: 976-989 (1994).
Parent protein sequences are increasingly becoming available as the
genomes of common biological model organisms become known. MS/MS
spectra can be collected rapidly (<400 msec per peptide) and in
a data-dependent manner through instrument-control software, so
very complex samples are amenable to analysis. With nanospray
infusion methods, sample volumes of 2 microliters can be analyzed
for an hour or longer. See e.g. Wilm et al., Anal. Chem. 68:1-8
(1996). Accordingly, in a preferred embodiment of the disclosed
method, modified peptides isolated in step (c) are characterized by
tandem MS, for example liquid chromatography (LC)-MS/MS (as
described in Example IV).
[0128] If phosphopeptides are being isolated, it may be observed
that during the fragmentation process of MS/MS, peptides containing
phosphoserine or phosphothreonine often form an ion by simple loss
of phosphate to produce a neutral-loss ion that has a mass 98 lower
than the unfragmented parent ion. If the parent ion has a charge of
+1, the neutral-loss ion has a mass-to-charge value (m/z) of 98/1
or 98 lower than the parent ion mass-to charge value. Likewise,
phosphopeptide parent ions with charges of +2, +3, or +4 will give
neutral-loss ions with m/z values that are 49, 32.7, and 24.5 lower
than the parent ion.
[0129] Neutral loss during MS/MS is the same process as metastable
decomposition during MALDI-TOF mass spectrometry. Therefore many of
the phosphopeptides showing neutral loss during LC-MS/MS are
expected to be the same phosphopeptides that give metastable
decomposition during MALDI-TOF mass spectrometry. For each
neutral-loss MS/MS spectrum, the parent ion mass (m) can be
calculated from the parent ion mass-to-charge value (m/z) and the
charge (z) inferred from the neutral loss value (+2 for neutral
loss of 49, +3 for 32.7, and +4 for 24.5). Some individual peptides
may be observed to undergo neutral loss as +2, +3, and +4 ions. A
comparison of datasets can confirm that the same peptides are
detected by both mass analysis methods.
[0130] Additional steps can be incorporated into the method of the
invention as needed to identify modified peptides that give high
levels of neutral loss during MS/MS and correspondingly low levels
of peptide background fragmentation. One approach is to analyze
these peptides with a mass spectrometer that uses a different
fragmentation mechanism, expecting this will change the
distribution of unproductive neutral-loss fragmentation to
productive peptide backbone fragmentation. For example, analysis
with ion trap mass spectrometers may exacerbate neutral loss
because fragmentation is induced by gradually increasing the energy
of the trapped peptide ion over a relatively long period of time,
whereas analysis with a triple quadrupole mass spectrometer may
allow more backbone fragmentation because the peptide ions become
energized for fragmentation more suddenly. A second approach is to
chemically treat modified peptides that have been purified by the
method of the invention in a manner that removes the group that
undergoes neutral loss during MS/MS but leaves a "remnant"
indicating where the group was. For example, phosphate groups can
be removed chemically from phosphopeptides by beta-elimination.
See, e.g., Byford, Biochem J. 280: 261-5 (1991). Exposing the
peptides briefly to a base, such as NaOH, LiOH, or Ba(OH).sub.2,
results in the removal of phosphoric acid from phosphoserine and
phosphothreonine, leaving dehydroalanine and dehydroaminobutyric
acid, respectively, which can be distinguished from serine and
threonine residues in the peptide by mass: residue masses are 69
for dehydroalanine, 87 for serine, 167 for phosphoserine, and 83
for dehydroaminobutyric acid, 101 for threonine, 181 for
phosphothreonine. Beta-eliminated peptides can be analyzed by
tandem mass spectrometry directly or after further reaction with a
Michael addition reagent. See, e.g., Molloy and Andrews, Anal Chem.
73: 5387-94 (2001).
[0131] Following MS/MS characterization, modified peptides may be
unambiguously identified by analyzing the product ion spectra with
a search program in an attempt to match the spectra obtained for
the modified peptide with the spectra for a known peptide sequence,
thereby identifying the parent protein(s) of the modified peptide.
For example, Sequest, a program that correlates an experimental
spectrum to a library of theoretical spectra derived from protein
sequence databases to find a best-fit match, may advantageously be
used for such a search. It will be recognized that equivalent
search programs may be employed in the practice of disclosed
method. Accordingly, in a preferred embodiment, the method of the
invention further comprises the step of (e) utilizing a search
program to substantially match the spectra obtained for the
modified peptide during the characterization of step (d) with the
spectra for a known peptide sequence, thereby identifying the
parent protein(s) of the modified peptide.
[0132] In certain cases, if phosphopeptides are being isolated, it
may be observed that, during MS/MS, some phosphopeptides undergo
neutral loss to a very high degree, with very little residual
fragmentation along the peptide backbone (which is needed to
produce spectra of a quality high enough for unambiguous
assignments). In such cases, analysis of MS/MS product ion spectra
using a search program (such as Sequest) in an attempt to assign a
phosphorylation site and parent protein to each peptide may not
result in unambiguous assignments. This is a common limitation
encountered during MS/MS analysis of peptides containing
phosphoserine and phosphothreonine. See e.g., DeGnore et al., J.
Am. Soc. Mass Spectrom. 9: 1175-1188 (1998). Even when
phosphopeptides lose phosphate by neutral loss, the position of the
phosphorylation site can be determined, as long as there is
sufficient residual backbone fragmentation, because neutral loss
leaves an unusual residue at the phosphorylation site:
phosphoserine becomes dehydroalanine, and phosphothreonine becomes
dehydroaminobutyric acid.
[0133] Accordingly, in a preferred embodiment of the disclosed
method, isolated modified peptides may be further characterized by
MS.sup.3 (for example LC-MS.sup.3, as in a preferred embodiment)
analysis; that is, the neutral-loss ions may be subjected to an
additional level of MS to give sufficient backbone fragmentation
for identification. This process is simpler to implement on ion
trap mass spectrometers than on other types of mass spectrometers.
As peptides elute from the LC system, a survey MS scan is
performed, and MS/MS spectra are collected for the three most
abundant ions, if they are above a pre-set intensity threshold and
if they have not been recently analyzed by MS/MS already. However,
if neutral loss of 49, 32.7, or 24.5 is detected during MS/MS, then
before collecting another MS/MS spectrum or another survey MS scan,
the instrument first isolates the neutral loss ion, fragments it,
and measures the product ion masses. In the case of
phosphopeptides, if the neutral-loss ion no longer contains
phosphate, it is more likely to fragment like a non-phosphorylated
peptide and give a useful product ion spectrum. With certain
modifications to the instrument control software, MS.sup.3 spectra
can be collected in the same data-dependent manner as MS/MS
spectra, and the MS.sup.3 spectra can be analyzed further with
Sequest. See Tomaino and Rush et al. Abstract ThOE 3:00, presented
at the 50.sup.th ASMS Conference on Mass Spectrometry and Allied
Topics, Jun. 6, 2002.
[0134] Following MS.sup.3 analysis, peptides may again be
identified using a search program such as Sequest. In the event
that a given peptide is unambiguously identified but the program is
unable to distinguish between multiple possible phosphorylation
sites, the most likely phosphorylation site may be chosen by
comparing the sites to the known specificity of the
modification-specific antibody used in the isolation. For example,
two possible phosphorylation sites (encompassing Ser 585 and Ser
588 of PTN6_HUMAN) were distinguished by noting that the sequence
context of one possible site but not the other fits the known
specificity of the phospho-(Ser) PKC substrate motif antibody used
to isolate the peptide (see Example V).
[0135] In cases where peptides comprising multiple modification
sites are isolated, it may be difficult to obtain unambiguous
assignments because of the high level of neutral loss with very
little residual fragmentation along the peptide backbone. At
present, for example, multiply-phosphorylated peptides cannot be
analyzed effectively by LC-MS.sup.3 using the currently available
version of Sequest software. The current data-dependent acquisition
software isolates and fragments the most abundant neutral-loss ion;
for multiply phosphorylated peptides this corresponds to the
peptide with one phosphate removed by neutral loss, leaving one or
more phosphate groups to undergo neutral loss during MS.sup.3.
However, the acquisition software is being revised (per personal
communication) to recognize multiples of neutral loss and to
isolate and fragment the ion with the highest level of neutral
loss, even if it is not the most intense product ion. It is
expected, therefore, that further analysis of multiply-modified
peptides with revised acquisition software will allow the parent
proteins and modification sites of some of these peptides to be
assigned. Accordingly, the scope of the present invention includes
such future revisions and versions of acquisition software, such as
Sequest.
[0136] Following assignment of a peptide sequence and
phosphorylation site to a spectrum, the assignment may be confirmed
by establishing that a synthetic peptide with that sequence and
phosphorylation site gives the same spectrum. This establishes a
formal link between a specific phosphopeptide and its spectrum.
This is a simple and convincing way to further evaluate marginal
Sequest assignments, for example, or to confirm assignments that
are considered especially important.
[0137] A simple confirmation method is essential to strategies that
attempt to assign phosphorylation sites globally, such as the
method of the invention. Neutral loss of phosphate from
phosphoserine or phosphothreonine can make it difficult to assign a
peptide sequence to an MS/MS spectrum and occasionally assignments
will be ambiguous. In contrast to global methods, when analyzing a
single phosphorylated protein, e.g., isolated as a stained band by
SDS-PAGE, the non-phosphorylated peptides from the protein will be
available for analysis and will help to identify the protein,
making assignment of phosphopeptides simpler, because the set of
possibilities can be restricted to peptides that originate from
that identified protein instead of a much larger database of
proteins. However, in a global proteomic method, such as the method
disclosed herein, where, for example, phosphopeptides are isolated
and analyzed separately from non-phosphorylated peptides, often the
only peptide from a particular protein will be the isolated
phosphopeptide, and unambiguous assignments are likely to be more
difficult to achieve. Assignments that are not unambiguous can be
confirmed by synthesizing a peptide with the assigned sequence and
phosphorylation site and analyzing it by LC-MS/MS or LC-MS.sup.3 to
determine whether it produces the same spectrum as the biological
peptide.
[0138] As described above, the tendency of some phosphopeptides to
undergo moderate to excessive neutral loss of phosphate can make it
difficult to assign a sequence to the spectrum of a particular
phosphopeptide. Programs such as Sequest provide a ranked list of
assignments for each spectrum. For non-phosphorylated peptides the
top-ranked assignment made by Sequest is often correct, but for
phosphopeptides the correct assignment may not have the highest
rank because of the additional complexities in the spectrum due to
neutral loss and the inability of Sequest to recognize and take
into account these neutral loss peaks. However a unique feature and
advantage to antibody-based isolation methods, such as the present
invention, is that the known specificity of the antibody can be
used to screen marginal assignments, i.e., assignments that are not
top-ranked, to find ones worth pursuing further. That is,
antibody-based isolation methods have an inherent advantage over
other isolation methods because the antibody's specificity can be
used to partially compensate for some of the limitations associated
with MS/MS analysis of phosphopeptides.
[0139] As discussed above, in practicing the immunoaffinity
isolation methods of the invention, a device for isolating modified
peptides from the proteinaceous preparation may be coupled directly
to a mass spectrometer so that peptides are analyzed as they elute
from the immunoaffinity-isolation device, enabling the mass
spectrometer to analyze even more complex mixtures of peptides. For
example, a liquid chromatography system fractionates complex
peptide mixtures into simpler mixtures, which are then analyzed
immediately by the mass spectrometer without intervening
sample-handling steps. In this manner, the method of the invention
may be readily automated, so as to allow the efficient,
high-throughput isolation of modified peptides from complex
mixtures.
[0140] To increase the tolerance for complex samples even further,
the liquid chromatography system may be multi-modal, i.e. it can
operate in two or more separation modes sequentially. For example,
one set of modified peptides may be eluted from an ion-exchange
support onto a reversed-phase support, followed by a reversed-phase
separation into the mass spectrometer, and then another set of
modified peptides may be analyzed by a more potent elution from the
ion-exchange support onto the reversed-phase support and a second
reversed-phase separation into the mass spectrometer, and so forth,
iteratively. See, e.g. Washburn, supra.
[0141] It is also contemplated that one dimension of multi-modal
liquid chromatography could be immunoaffinity purification, using
general modification-specific antibodies to purify
post-translationally modified peptides, as described herein. In
this sense the immunoaffinity column would resemble a so-called
enzyme reactor column, a column of immobilized protein used
upstream of a mass spectrometer to catalyze a reaction on the
sample to be analyzed. See e.g. Amankwa et al. Protein Sci. 4:
113-125 (1995).
Identification of Novel Sites; Antibodies
[0142] The immunoaffinity isolation methods of the invention allow
the efficient and rapid isolation and identification of peptides
comprising protein modification sites from complex mixtures.
Modified peptides isolated according to the method of the invention
may comprise known modification sites on a particular protein, or
may comprise novel sites of modification previously unreported. For
example, unknown phosphorylation sites of a particular protein may
be identified in accordance with the method of the present
invention. Similarly, the methods of the invention may isolate and
identify sites whose modification (e.g. phosphorylation) is known,
per se, but whose modification in a particular cell, tissue,
disease state, etc. is not known. Thus, the disclosed methods
enable, in part, the identification of modification sites of
particular proteins that are relevant (i.e. the proteins are
activated or de-activated) to a particular disease state.
[0143] The identification of novel protein modification sites
enables the generation of new antibody reagents which are specific
for the novel protein site in its phosphorylated form. For example,
the identification of a novel phosphorylation site (e.g. a
particular phosphoserine site) according to the method of the
invention enables the generation of phospho-specific antibodies
which bind to that protein only when phosphorylated at the novel
site. If a motif-specific, context-independent antibody is employed
for the immunoaffinity isolation, novel sites identified will match
the specificity of the motif-antibody employed. These
modification-specific antibodies against novel sites will be highly
useful reagents for the detection of protein modification, as well
as for diagnostic or therapeutic uses.
[0144] Once a novel modification site is identified,
modification-specific antibodies to that site may be generated by
standard techniques familiar to those of skill in the art. The
antibodies may be polyclonal or monoclonal. Anti-peptide antibodies
may be prepared by immunizing an appropriate host with a synthetic
phospho-peptide antigen comprising the novel modification site,
according to standard methods. See, e.g., ANTIBODIES: A LABORATORY
MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring
Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201:
264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962)).
Monoclonal antibodies may be produced in a hybridoma cell line
according to the well-known technique of Kohler and Milstein.
Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6:
511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
Ausubel et al. Eds. (1989). Motif-specific, context-independent
antibodies may also be produced against the novel site identified
if the site is a motif conserved among a plurality of different
signaling proteins. See Comb et al., WO 00/14536, supra.
[0145] Modification-specific antibodies generated against novel
sites and/or motifs identified by the immunoaffinity methods of the
invention may be screened for epitope and modification-specificity
according to standard techniques. See, e.g. Czernik et al., Methods
in Enzymology, 201: 264-283 (1991). For example, in the case of a
novel phosphorylation site, the antibodies (whether polyclonal or
monoclonal) may be screened against a phospho and non-phospho
peptide library by ELISA to ensure specificity for both the desired
antigen (i.e. that epitope including the novel phosphorylation
site/residue) and for reactivity only with the phosphorylated form
of the antigen. Peptide competition assays may be carried out to
confirm lack of reactivity with other non-target protein
phosphoepitopes. The antibodies may also be tested by Western
blotting against cell preparations containing the parent protein,
e.g. cell lines over-expressing that protein, to confirm reactivity
with the desired phosphorylated target. Specificity against the
desired phosphorylated epitopes may also be examined by
construction of parent/target protein mutants lacking
phosphorylatable residues at positions outside the desired epitope
known to be phosphorylated, or by mutating the desired
phospho-epitope and confirming lack of reactivity.
[0146] In accordance with the present invention, two novel protein
phosphorylation sites were identified by the practice of the
disclosed immunoaffinity isolation methods: (i) a novel ubiquitin
fusion degradation protein 1 (UFD1) phosphorylation site (Ser335,
comprising the sequence GQS*LR) was identified using phospho-(Ser)
PKC substrate motif antibody for immunoaffinity isolation of
modified peptides from a Jurkat cell extract, and (ii) a novel
protein-tyrosine phosphatase 1c (PTN6) phosphorylation site
(Ser588, comprising the sequence KGS*LK) was identified using the
same PKC substrate motif antibody for immunoaffinity isolation of
modified peptides from Jurkat cell extracts (see Example V).
[0147] Phospho-specific antibodies that bind either UFD1 or PTN6,
respectively, only when phosphorylated at these novel sites can now
readily be prepared, according to standard techniques. Synthetic
phospho-peptide antigens comprising the UFD1 or PTN6 sequence
surrounding and including phospho-Ser335 or Ser588, respectively,
may be selected and constructed in accordance with well known
techniques, and used as immunogens to produce poly- or mono-clonal
antibodies. See, e.g., ANTIBODIES: A LABORATORY MANUAL, supra,
Czernik, Methods In Enzymology, supra. The phospho- and
epitope-specificity of these antibodies may be confirmed as
described above.
[0148] Accordingly, in a preferred embodiment, the invention also
provides an antibody that binds ubiquitin fusion degradation
protein 1 (UFD1) only when phosphorylated at serine 335, but does
not substantially bind to UFD1 when not phosphorylated at this
residue. The UFD1 (pSer335) antibody of the invention also does not
substantially bind to proteins other than UFD1, although some
limited cross-reactivity may be observed with proteins containing
sites highly homologous to the UFD1 phospho-Ser335 site.
[0149] In another preferred embodiment, the invention provides an
antibody that binds protein-tyrosine phosphatase 1c (PTN6) only
when phosphorylated at serine 588, but does not substantially bind
to PTN6 when not phosphorylated at this residue. The PTN6 (pSer588)
antibody of the invention also does not substantially bind to
proteins other than PTN6, although some limited cross-reactivity
may be observed with proteins containing sites highly homologous to
the PTN6 phospho-Ser588 site.
Profiling and Diagnostic Applications
[0150] As noted above, the invention enables the rapid, efficient,
and direct isolation of modified peptides from complex mixtures,
such as crude cell extracts or biological fluids, without the need
for costly and time-consuming pre-purification of desired peptides
or proteins. The method makes possible the single-step
immunoaffinity isolation of multiple different modified peptides,
corresponding to a multitude of different modified proteins and
signaling pathways, with a single antibody. Accordingly, the
methods disclosed herein are suitable and highly useful for
genome-wide (e.g. cell-wide) profiling of activation states, for
example. The simplicity of the disclosed method also makes it
readily automatable, as only a single immunoaffinity isolation step
is required.
[0151] Facile isolation of modified peptides aids in the
identification and assignment of modification sites in a great
variety of different proteins. These protein modifications occur in
response to significant events in the life of a cell, and in some
cases the modifications provide a potential target for diagnosing
or preventing the event. As the genome sequences of various
organisms continue to become known, the need to find and assign
these modifications in a given organism will become even more
pronounced. In a broad context, the invention is useful not only to
assign modification sites in well-defined in vitro complexes, but
also to generate genome-wide or cell-wide activation/modification
profiles, that is, to determine how global protein modification
changes within a given cell or tissue in response to environmental
changes, such as stress, inflammation, disease, drug treatment,
etc.
[0152] In contrast to conventional proteomics methods, which focus
on how global protein levels change in response to a particular
treatment, the present invention focuses on cellular changes in
protein modification resulting from a given event, such as disease
or treatment. Protein modification, such as phosphorylation and
dephosphorylation, serves as a molecular switch for modulating many
important biological processes, including cellular transformation
and cancer, programmed cell death, cell cycle control, and
metabolism. Thus, one advantage offered by the present invention is
that it provides a means of focusing on these molecular switching
events, which can occur without an accompanying change in the
amount of a specific protein in a cell, i.e. a cellular response
may be triggered by a change in the modification state of a
specific signaling protein, and not by a change in the amount of
that protein in the cell.
[0153] The immunoaffinity isolation methods of the invention will
be useful for the diagnosis of a condition known to be associated
with the activation (or de-activation) of a given modification site
on a protein. For example, a phosphorylation site on a certain cell
signaling protein which is a known marker of a given disease may be
isolated (from a clinical tissue or fluid sample) in accordance
with the invention to identify the phosphorylation status (i.e.
activation status) of the marker in a patient. This marker
activation information will assist in the diagnosis of disease
and/or identify subjects at risk of disease. Accordingly, in a
preferred embodiment of the disclosed method, the modified peptide
isolated in step (c) corresponds to a known marker of disease.
[0154] The methods of the invention will be useful for profiling
protein activation (i.e. modification) states in a target cell or
fluid, on a genome-wide, or pathway-wide basis, in response to
environmental changes such as disease or drug treatment. For
example, biopsy samples may be obtained from cancer patients and
analyzed against normal, reference tissue or cells from the same
patient. Alternatively, the method will be useful both for
discovering modified protein markers for specific types of cancer,
and as a diagnostic assay for those cancers, perhaps helping to
mark their stage of progression. Accordingly, in one preferred
embodiment of the method, the modified peptide(s) characterized in
step (d) comprise(s) an unknown modification site of a parent
protein. For example, the method may be advantageously employed to
identify phosphorylation sites on particular cell signaling
proteins that are elevated or reduced in cancerous tissue, as
opposed to normal tissue. In a similar manner, the method will be
useful to evaluate the cellular effects of a therapeutic drug (i.e.
changes in protein modification) to gauge if it is having the
desired effect, or to determine when its dosage may induce
toxicity. For example, cells or tissue treated with a test drug
intended to reduce phosphorylation of a particular protein known to
be associated with a certain disease state may be monitored to
determine the phosphorylation state of that protein and/or others.
The method could also be used to monitor the stages and severity of
an infectious disease by monitoring changes in cell-wide
modification state during the course of the disease.
[0155] Accordingly, in a preferred embodiment, the isolation method
of the invention further comprises the step of (e) comparing the
modification state of the modified peptide characterized in step
(d) with the modification state of a corresponding peptide in a
reference sample, thereby to compare protein activation in the
proteinaceous preparation with protein activation in the reference
sample. In one preferred embodiment, the proteinaceous preparation
corresponds to a diseased organism and the reference sample
corresponds to a normal organism, whereby comparison of protein
activation provides information on activation changes resulting
from the disease. In a second preferred embodiment, the
proteinaceous preparation is obtained from a tissue biopsy cell or
a clinical fluid sample and the reference sample corresponds to a
diseased organism, whereby the comparison of protein activation
provides information useful for diagnosis of the disease. In a
third preferred embodiment, the protein preparation corresponds
with an organism or preparation treated with at least one test
compound and the reference sample corresponds with an untreated
organism or preparation, whereby the comparison of protein
activation provides information on activation changes resulting
from treatment with the test compound.
[0156] In another preferred embodiment, the comparison of protein
activation described above identifies the modified peptide
characterized in step (d) as corresponding to a parent protein not
previously reported as so modified in the disease.
[0157] The isolation of modified peptides relevant to a given
disease as outlined above may be carried out for virtually any
disease in which aberrant signal transduction (i.e. protein
activation/modification) is involved or suspected of being
involved. In a preferred embodiment of the method, the disease is
cancer. Similarly, the modified peptide isolation may be employed
to monitor the effects of virtually any test compound or drug on
protein modification. In a preferred embodiment, the test compound
comprises a cancer therapeutic. In a particularly preferred
embodiment, the test compound comprises a kinase inhibitor, such as
STI-571 (Gleevac.RTM.), an inhibitor of Abl kinase for the
treatment of leukemia.
[0158] In the profiling and diagnostic applications described here,
the proteinaceous preparation from which modified peptides will be
isolated may correspond, for example, to a diseased cell or fluid,
tissue biopsy cell or clinical fluid sample, or test cell treated
with a test drug or fluid from an organism treated with a test
drug, and the reference sample may correspond to a normal cell or
fluid, diseased cell or fluid, or untreated cell or fluid from an
untreated organism, whereby the profiling provides information
useful in changes in, e.g. modification state, resulting from
disease or drug treatment, or diagnosis of disease. Alternatively,
the reference sample may correspond to a state of aberrant
signaling (i.e. a diseased sample) and the proteinaceous
preparation may correspond to a normal organism, for example, a
patient being tested for the presence of a marker of disease or
susceptibility to disease.
[0159] The isolation and profiling methods of the invention will be
particularly useful in the high-throughput identification of
modification states on known or unknown proteins on a genome-wide
basis, so as to provide a link between genomic and proteomic
information and actual disease states. The method is readily
automatable, and thus, for example, may be advantageously employed
by pharmaceutical companies wishing to efficiently and rapidly
identify markers of disease for diagnostic or therapeutic
applications.
Quantification of Isolated Peptides
[0160] In one preferred embodiment the method may employ additional
procedures to allow quantification of modified peptides prepared
according to the method of the invention. For example, in SILAC
(stable isotope labeling by amino acids in cell culture) cell
cultures are grown in culture media that has been depleted of
specific amino acids, e.g., arginine and lysine, and then
supplemented with these amino acids in "normal isotope" or stable,
"heavy isotope" forms. See, e.g. Ong et al., Mol. Cell. Proteomics
5: 376-86 (2002). Cells grown in heavy-isotope media can be treated
with a drug candidate that is expected to inhibit one specific
protein kinase, while the same cells grown in normal-isotope media
are left untreated. When these samples are prepared for analysis by
the method of the invention, the cells are harvested and mixed at a
one-to-one ratio. In all subsequent steps these two admixed samples
are handled as one sample, so any mass spectrometry intensity
differences between the samples can be attributed only to drug
treatment and not to sample handling or run-to-run analysis
variations. Differences in the samples are traced to the
drug-treated or untreated sample on the basis of the presence or
absence, respectively, of the heavy-isotope in the analyzed
peptides during mass spectrometry. SILAC provides relative
quantification and is considered to be a promising key method for
discovering biomarkers by comparative, quantitative mass
spectrometry. An alternative method for quantification is Aqua
(absolute quantification). See Gerber, Rush et al. Proc. Natl.
Acad. Sci. U.S.A. 100: 6940-5 (2003). In this method, the modified
peptide to be quantified is synthesized as a heavy-isotope peptide,
which can then serve as an internal quantification standard. A
known amount of the heavy-isotope peptide is added to the sample
prior to digestion with a protease. Because the heavy-isotope
peptide standard is chemically identical to the biologically
derived peptide to be quantified, they will co-purify by the method
of the invention and will be mass analyzed at the same time. By
multiplying the known amount of heavy-isotope peptide standard by
the measured ratio of biological peptide to peptide standard, the
absolute amount of biological peptide in the sample is determined.
In contrast to the SILAC method, the Aqua strategy provides
absolute quantification and can be used with a wide variety of
sample types other than cell cultures. The Aqua method is a
preferred embodiment of the method of the invention when it is
desirable to quantify a limited number, e.g., 1 to 60, of modified
peptides such as in the development of quantitative assays based on
mass spectrometry. A key element of the Aqua strategy is
identifying peptides that can be measured quantitatively because it
is known they can be detected when analyzed by mass spectrometry,
in contrast to peptides that are selected by a de novo process and
that expected to be present but cannot be detected. In a preferred
embodiment, the method of the invention is first used to determine
the peptides that are suitable for measurement by the Aqua strategy
and then later used as part of an Aqua-based mass spectrometry
quantification assay. A third method for quantification is based on
chemical modification of peptides with normal-isotope and
heavy-isotope chemical modification reagents, e.g., reagents that
react with peptide amino groups or carboxylic acid groups. See,
e.g., Zhang et al. Nat. Biotechnol. 21: 660-6 (2003). This follows
the same overall strategy as the SILAC method, except samples are
mixed after the chemical modification step and samples other than
cell cultures can be analyzed.
[0161] The following Examples are provided only to further
illustrate the invention, and are not intended to limit its scope,
except as provided in the claims appended hereto. The present
invention encompasses modifications and variations of the methods
taught herein which would be obvious to one of ordinary skill in
the art.
Example I
A. Isolation of Phosphotyrosine-Containing Peptides from a Peptide
Mixture
[0162] To establish that phosphopeptides can specifically be
purified from complex mixtures without contamination from
nonphosphorylated peptides, the method of the invention was used to
isolate phosphotyrosine (p-Tyr)-containing peptides from a mixture
of phosphorylated and nonphosphorylated synthetic peptides. A
phosphotyrosine peptide mix comprising 5 phosphotyrosine-containing
peptides and their 5 nonphosphorylated partner peptides was
prepared (Table 1); note the nonphosphorylated peptides have the
same sequences as the phosphorylated peptides but are not
phosphorylated, that is, they contain tyrosine instead of
phosphotyrosine. Peptides were synthesized by Fmoc chemistry on a
Rainin/Protein Technologies Symphony peptide synthesis instrument
and using Fmoc-Tyr(PO(OBzl)OH)--OH as the phosphotyrosine monomer.
See Perich, Lett. Pept. Sci. 6: 91 (1999). The peptide mixture
covers a broad mass range designed to resemble a protein
digest.
TABLE-US-00001 TABLE 1 Components of the Phosphotyrosine Peptide
Mix Calculated Protonated Sequence Peptide Mass
KIEKIGEGTY*GVVYKGRHK 2,242.174 (SEQ ID NO: 1) KIEKIGEGTYGVVYKGRHK
2,162.208 (SEQ ID NO: 2) RLIEDNEY*TARQGAKC 1,946.879 (SEQ ID NO: 3)
RLIEDNEYTARQGAKC 1,866.912 (SEQ ID NO: 4) LQERRKY*LKHRC 1,709.878
(SEQ ID NO: 5) LQERRKYLKHRC 1,629.911 (SEQ ID NO: 6)
RQGKDY*VGAIPVDC 1,600.719 (SEQ ID NO: 7) RQGKDYVGAIPVDC 1,520.752
(SEQ ID NO: 8) GKDGRGY*VPATC 1,303.550 (SEQ ID NO: 9) GKDGRGYVPATC
1,223.583 (SEQ ID NO: 10) Y*= phosphotyrosine, Y = tyrosine
The MALDI-TOF mass spectrum of the mixture before immunoaffinity
purification is shown in FIG. 2. Peaks labeled with a star
correspond to phosphorylated peptides, and peaks labeled with open
circles correspond to the nonphosphorylated partner peptides.
[0163] P-Tyr-containing peptides were specifically isolated from
the diverse peptide mixture by contacting the phosphotyrosine
peptide mix (46 nmol total) with a phosphotyrosine monoclonal
antibody P-Tyr-100 immobilized to agarose resin (Cell Signaling
Technology, Inc., product number 9419) (100 .mu.l). The antibody
was incubated with the peptides as a slurry, in a batch
purification format. The slurry was left at room temperature for 10
minutes and on ice for 1 hour. The unbound peptides were removed by
centrifugation through a plastic frit, and the retained
antibody-resin was washed extensively (twice with 1 ml of ice-cold
phosphate buffered saline containing 0.5% NP-40, twice with 1 ml of
ice-cold phosphate buffered saline, and once with water). To elute
bound phosphopeptides, the antibody-resin was resuspended in 400
.mu.l 30% acetic acid, left at room temperature for 10 minutes, and
centrifuged.
[0164] The eluted peptide fraction was dried and resuspended in 80
.mu.l water (the volume of the phosphotyrosine peptide mix before
treatment with antibody-resin), and a 1 .mu.l aliquot was diluted
and analyzed by MALDI-TOF mass spectrometry, as described above
(FIG. 3). FIG. 4 shows the mass spectrum of the phosphotyrosine
peptide mix before (top panel) and after (bottom panel)
immunoaffinity purification. Note that the fraction eluted from the
antibody-resin contains all 5 phosphopeptides but none of the
nonphosphorylated peptide partners. Accordingly, the method of the
invention specifically isolates all desired phosphopeptides
containing a phosphotyrosine, regardless of the different sequences
in which the phosphotyrosine occurs, from a complex mixture of
phosphorylated and nonphosphorylated peptides.
B. Isolation of Phosphothreonine-Containing Peptides from a Peptide
Mixture
[0165] The method of the invention was further demonstrated using a
second general protein modification antibody, a phosphothreonine
polyclonal antibody P-Thr-polyclonal to purify peptides containing
phosphothreonine from a mixture of phosphorylated and
nonphosphorylated synthetic peptides. The mixture consists of 4
synthetic peptides: 2 phosphothreonine-containing peptides and
their 2 nonphosphorylated partner peptides (see Table 2). The
MALDI-TOF mass spectrum, obtained as described above, of the
phosphothreonine peptide mix before immunoaffinity purification
according to the invention is shown in FIG. 5.
TABLE-US-00002 TABLE 2 Components of the Phosphothreonine Peptide
Mix Calculated Protonated Sequence Peptide Mass DTQIKRNT*FVGTPFC
1,806.825 (SEQ ID NO: 11) DTQIKRNTFVGTPFC 1,726.859 (SEQ ID NO: 12)
CKEGLGPGDTTST*F 1,491.620 (SEQ ID NO: 13) CKEGLGPGDTTSTF 1,411.653
(SEQ ID NO: 14) T*= phosphothreonine, T = threonine
A P-Thr-polyclonal antibody (Cell Signaling Technology, Inc.,
product number 9381) was linked to agarose resin using a hydrazide
chemistry (the same chemistry used to produce the P-Tyr-100 agarose
resin used in Example 1A above), using a commercially available
crosslinking kit (BioRad Affi-Gel HZ Immunoaffinity Kit, product
number 153-6060) and following the manufacturer's instructions.
Each milliliter of resin was reacted with 1 milligram of antibody.
P-Thr-containing phosphopeptides were specifically isolated from
this mixture by contacting the phosphothreonine peptide mix (20
pmol total) with this antibody-resin (100 .mu.l), and incubating
the resin and peptides at 4.degree. C. overnight. The resin was
recovered and washed, and the bound peptides were eluted,
processed, and analyzed as described above. FIG. 6 shows the
MALDI-TOF mass spectra of the unbound and bound peptide fractions.
The unbound fraction contains all 4 peptides, including the
phosphopeptides; under the particular conditions utilized, which
were not optimized, some of each isolated phosphopeptide has passed
through the column. The bound fraction contains both
phosphopeptides but does not contain the nonphosphorylated peptide
partners. Accordingly, the method of the invention selectively
isolates all desired phosphopeptides containing a phosphothreonine,
regardless of the different sequences in which the phosphothreonine
occurs, from a mixture of phosphorylated and nonphosphorylated
peptides.
C. Isolation of Phosphotyrosine-Containing Peptides from Low-Level
Samples
[0166] The isolation of modified peptides from low-level samples
(i.e. where the amount of each modified peptide is about 1 pmol or
less) according to the method of the invention was demonstrated
using low-picomole amounts of phosphotyrosine peptides from the
10-peptide mix and the immobilized phosphotyrosine antibody
P-Tyr-100, as described above in Part IA. P-Tyr-containing
phosphopeptides were isolated from this complex mixture, by
contacting the 10-peptide mix (24 pmol total) with the P-Tyr-100
antibody-resin (CST product number 9419) (10 .mu.l). The peptide
mixture contained 100 ng BSA to reduce non-specific peptide loss
through adsorption. The resin was recovered and washed extensively
(twice with 0.5 ml PBS containing 0.5% NP40, twice with 0.5 ml PBS,
and five times with 0.5 ml water). Phosphopeptides bound to the
antibody-resin were eluted by washing the resin three times with 5
.mu.l 0.1 M glycine, pH 2.3. The three elutions were combined, and
an aliquot was desalted with a ZipTip device (Millipore Corp., part
number ZTC18S096), since glycine interferes with MALDI-TOF mass
analysis.
[0167] FIG. 7 shows the mass spectrum for the low-level 10-peptide
mix before (top panel) and after (third panel) immunoaffinity
purification according to the method of the invention. Peaks
labeled with a star correspond to phosphorylated peptides, and
peaks labeled with circles correspond to the nonphosphorylated
partner peptides. All 5 phosphopeptides, although present at low
(picomole) levels, were bound and eluted from the antibody-resin
(third panel). Of the 10 peptides in the mix, only 3 were detected
in the unbound fraction (second panel), and they were all
nonphosphorylated peptides. The bound peptide fraction was
neutralized, treated with calf intestinal alkaline phosphatase, an
enzyme that can remove phosphate from phosphopeptides, and
re-analyzed to confirm the phosphopeptide assignments (FIG. 7,
bottom panel). As expected, the phosphopeptides were completely
dephosphorylated to produce ions with masses 80 lower than the
phosphopeptides. This was particularly helpful in assigning the
peaks at 1,869 and 1,523 to phosphopeptide synthesis artifacts and
the peaks at 1,867 and 1,521 to non-phosphopeptides. Accordingly,
the method of the invention selectively isolates all desired
phosphopeptides, even at low levels, that contain a
phosphotyrosine, regardless of the different sequences in which the
phosphotyrosine occurs, from a low-level mixture of phosphorylated
and nonphosphorylated peptides.
D. Isolation of Akt Substrate Phosphopeptides from a Peptide
Mixture
[0168] The method of the invention was further demonstrated using a
motif-specific, context-independent polyclonal antibody,
phospho-(Ser/Thr) Akt substrate antibody, to purify phosphopeptides
containing the phospho-Akt substrate motif from a mixture of
phosphorylated and nonphosphorylated synthetic peptides. The Akt
protein kinase plays a central role in cell growth (Marte and
Downward, Trends Biochem. Sci. 22: 355-358 (1997)), angiogenesis
(Jiang et al., Proc. Natl. Acad. Sci. USA 97: 1749-1753 (2000)),
and transcriptional regulation (Scheid and Woodgett, Curr. Biol.
10: R191-194 (2000)). The Akt protein kinase is able to
phosphorylate protein substrates at threonine or serine residues
when the target residue occurs within the consensus sequence motif
RXRXX(T/S), where R is arginine, X is any amino acid, and T/S
indicates the target threonine or serine.
[0169] Phospho-(Ser/Thr) Akt substrate polyclonal antibody (Cell
Signaling Technology, Inc., product number 9611) recognizes a
plurality of different phosphorylated proteins that contain the
consensus sequence motif when phosphorylated, but does not
recognize the analogous unphosphorylated motif. The specificity of
the phospho-(Ser/Thr) Akt substrate antibody is that it binds
preferentially to proteins and peptides that contain
phosphothreonine or phosphoserine preceded by lysine or arginine at
positions -5 and -3, i.e., (K/R)X(K/R)XX(T*/S*) (SEQ ID NO: 15), in
a manner substantially independent of the surrounding amino acid
sequence (i.e. the context of the motif). It is now demonstrated
here that this antibody can be used to purify peptides that contain
the phosphorylated Akt consensus substrate motif.
[0170] Akt motif-containing phosphopeptides were selectively
isolated from a mixture of phosphorylated and nonphosphorylated
synthetic peptides according to the method of the invention. The
mixture consisted of 8 synthetic peptides: 3
phosphothreonine-containing peptides, 1 phosphoserine-containing
peptide, and their 4 nonphosphorylated partner peptides (Table 3).
The phospho-Akt substrate consensus sequence is present in all 4
phosphopeptides in this mixture, and it is known from ELISA that
these phosphopeptides are recognized by and can bind to the
phospho-Akt substrate antibody.
TABLE-US-00003 TABLE 3 Components of the Phospho-Akt Substrate
Peptide Mix Calculated Protonated Sequence Peptide Mass
CSPRRRAAS*MDNNSKFA 1,989.889 (SEQ ID NO: 16) CSPRRRAASMDNNSKFA
1,909.923 (SEQ ID NO: 17) CLKDRQGT*HKDAEIL 1,805.872 (SEQ ID NO:
18) SRPRSCT*WPLPREI 1,777.856 (SEQ ID NO: 19) CRSLT*GKPKLFIIQA
1,754.938 (SEQ ID NO: 20) CLKDRQGTHKDAEIL 1,725.906 (SEQ ID NO: 21)
SRPRSCTWPLPREI 1,696.906 (SEQ ID NO: 22) CRSLTGKPKLFIIQA 1,674.972
(SEQ ID NO: 23)
[0171] Phosphopeptides were isolated from this peptide mixture by
contacting the phospho-Akt substrate peptide mix (5 pmol each
peptide) with phospho-(Ser/Thr) Akt substrate antibody immobilized
to agarose resin (Cell Signaling Technology, Inc., part number
9619) (20 .mu.l, 2 .mu.g/.mu.l). The antibody was incubated with
the peptides as a slurry at 4.degree. C. for 2 hours. Unbound
peptides were removed by centrifugation, and the antibody-resin was
washed extensively (two times with 0.5 ml ice-cold PBS containing
0.5% NP-40, two times with 0.5 ml ice-cold PBS, and three times
with 0.5 ml ice-cold water). Bound peptides were eluted with three
10 .mu.l aliquots of 0.1 M glycine, pH 2.3. A 5 .mu.l portion of
the fraction containing bound and eluted peptides was desalted and
concentrated with a reversed-phase ZipTip microcolumn before
analysis by MALDI-TOF mass spectrometry, as described above.
[0172] FIG. 8 shows the mass spectra of the phospho-Akt substrate
peptide mix before (top panel) and after (bottom panel)
immunoaffinity purification. Peaks labeled with a star correspond
to phosphopeptides, peaks labeled with an open circle correspond to
nonphosphorylated peptides, and peaks labeled with a square are
phosphopeptides that have undergone metastable decomposition and
neutral-loss of phosphate (discussed in Example V below). The
fraction of peptides that bound to and eluted from the immobilized
antibody (bottom panel) contains all 4 phosphopeptides but does not
contain the nonphosphorylated partner peptides. Accordingly, the
method of the invention selectively isolates all peptides in this
synthetic peptide mixture that contain the phospho-Akt substrate
motif, whether they contain phosphothreonine or phosphoserine
residues. As desired, peptides that contain the nonphosphorylated
consensus motif are not isolated.
E. Isolation of 14-3-3 Binding Motif #1 Phosphopeptides from a
Peptide Mixture
[0173] The method of the invention was further exemplified using a
second motif-specific, context-independent monoclonal antibody,
phospho-(Ser) 14-3-3 binding motif antibody, to purify
phosphopeptides containing the 14-3-3 binding motif from a mixture
of phosphorylated and nonphosphorylated synthetic peptides. The
14-3-3 proteins regulate several biological processes through
phosphorylation-dependent protein-protein interactions (Muslin et
al., Cell 84, 889-897 (1996)). Nearly all binding partners of
14-3-3 proteins contain at least one of two different
phosphoserine-containing consensus sequences (Yaffe et al. Cell 91,
961-971 (1997)). One consensus sequence, motif #1, is (R/K)SXS*XP,
where R/K indicates arginine or lysine, S is serine, X is any amino
acid, S* is phosphoserine, and P is proline.
[0174] Phospho-(Ser) 14-3-3 binding motif monoclonal antibody (4E2)
(Cell Signaling Technology, Inc., product number 9606) is a
motif-specific antibody that recognizes phosphopeptides that
contain this consensus binding motif #1. The 14-3-3 binding motif
antibody is highly specific for peptides and proteins that contain
this motif (phosphoserine surrounded by proline at the +2 position
and arginine or lysine at the -3 position, i.e., (K/R)XXS*XP (SEQ
ID NO: 24). Recognition is specific for the phosphorylated form of
the motif and is substantially independent of the surrounding amino
acid sequence (i.e. the context of the motif). This antibody weakly
cross-reacts with analogous sequences containing phosphothreonine
instead of phosphoserine in this motif.
[0175] To identify other proteins that bind to 14-3-3 proteins or
to profile known binding partners on a genome-wide (cell-wide)
basis, immobilized 14-3-3 binding motif antibody may be employed to
immunoaffinity purify phosphopeptides from a proteinaceous
preparation in accordance with the method of the invention. To
demonstrate the feasibility of this, this antibody was first
employed to selectively isolate phosphopeptides from a mixture of
phosphorylated and nonphosphorylated synthetic peptides when the
phosphopeptides contain motif sequences that match the antibody's
known specificity. The mixture consisted of 13 synthetic peptides
(Table 4). Four peptides in the mixture contained sequences that
match the antibody's known specificity, 3 with phosphoserine (SEQ
ID NOs: 26, 28, 29) and 1 with phosphothreonine (SEQ ID NO: 27). It
is known by ELISA that these three phosphoserine-containing
peptides are recognized by and can bind to the 14-3-3 binding motif
#1 antibody. The peptide mixture contained 9 other peptides that
should not bind to 14-3-3 binding motif antibody: 2
phosphotyrosine-containing peptides, 2 phospho-Akt substrate motif
peptides, and 5 nonphosphorylated partner peptides.
TABLE-US-00004 TABLE 4 Components of the 14-3-3 Binding Motif
Peptide Mix Calculated Protonated Sequence Peptide Mass
CSPRRRAAS*MDNNSKFA 1,989.889 (SEQ ID NO: 25) CSPRRRAASMDNNSKFA
1,909.923 (SEQ ID NO: 26) FRGRSRS*APPNLWAC 1,797.836 (SEQ ID NO:
27) SRPRSCT*WPLPREI 1,777.856 (SEQ ID NO: 28) TRSRHSS*YPAGTEEC
1,760.705 (SEQ ID NO: 29) CAEYLRSIS*LPVPVL 1,738.896 (SEQ ID NO:
30) LQERRKY*LKHRC 1,709.878 (SEQ ID NO: 31) SRPRSCTWPLPREI
1,696.906 (SEQ ID NO: 32) TRSRHSSYPAGTEEC 1,680.739 (SEQ ID NO: 33)
CAEYLRSISLPVPVL 1,658.929 (SEQ ID NO: 34) MSGRPRTTS*FAESC 1,609.649
(SEQ ID NO: 35) RQGKDY*VGAIPVDC 1,600.719 (SEQ ID NO: 36)
RQGKDYVGAIPVDC 1,520.752 (SEQ ID NO: 37)
[0176] A 14-3-3 binding motif #1 antibody (Cell Signaling
Technology, Inc., product number 9606) was linked to agarose resin
using a hydrazide chemistry (the same chemistry used to produce the
P-Tyr-100 agarose resin used in Example 1A above), using a
commercially available crosslinking kit (BioRad Affi-Gel HZ
Immunoaffinity Kit, product number 153-6060) and following the
manufacturer's instructions. Each milliliter of resin was reacted
with 1 milligram of antibody. Phosphopeptides were selectively
isolated from the 14-3-3 binding motif peptide mixture by
contacting the peptide mix (10 pmol each peptide) with this
antibody-resin (10 .mu.l, 1 .mu.g/.mu.l). The antibody was
incubated with the peptides as a slurry at room temperature for 1
hour and 4.degree. C. for 1 hour. Unbound peptides were removed by
centrifugation, and the antibody-resin was washed extensively
(twice with 1 ml ice-cold PBS and once with 1 ml ice-cold water).
Bound peptides were eluted with one 30 .mu.l aliquot of 0.1 M
glycine, pH 2.0. A 9 .mu.l portion of the eluted peptides was
desalted and concentrated with a reversed-phase ZipTip microcolumn
before analysis by MALDI-TOF mass spectrometry, as described
above.
[0177] The mass spectra of the peptide mix before (top panel) and
after (bottom panel) immunoaffinity purification are shown in FIG.
9. Peaks labeled with a star correspond to phosphopeptides, and
peaks labeled with an open circle correspond to nonphosphorylated
peptides. Peaks labeled with filled stars are phosphopeptides that
are not expected to bind to the 14-3-3 binding motif antibody
because their sequences do not fit the antibody's known
specificity. Of the four phosphopeptides in the mixture that
contain the 14-3-3 binding motif, three were isolated by the 14-3-3
binding motif antibody, and they correspond to the major peaks in
the fraction of peptides that bound to and eluted from the
immobilized antibody (FIG. 9, bottom panel). One 14-3-3 binding
motif phosphopeptide was not isolated (SEQ ID NO: 29, calculated
protonated peptide mass of 1,738.9), but it also could not be
detected in the untreated peptide mix, i.e. it may be a poorly
ionizing peptide. The phosphothreonine-containing peptide, which
contains a slightly variant motif (phosphothreonine in place of
phosphoserine), was also isolated (SEQ ID NO: 27, calculated
protonated peptide mass of 1,777.8); it was, in fact, expected to
cross-react weakly with the antibody. Two peptides that do not
contain sequences that match the antibody's specificity were
isolated, one was a phosphopeptide containing the phospho-Akt
substrate motif (SEQ ID NO: 35, calculated protonated peptide mass
of 1,608.6) and the other was unphosphorylated (SEQ ID NO: 25,
calculated protonated peptide mass of 1,910.9). Several peaks in
the bound and eluted fraction (1,941, 1,770, 1,642, 1,526) are also
present in the bound and eluted fraction of a negative control,
antibody-resin treated with buffer instead of peptide mixtures.
These artifactual peaks appear to originate from the antibody-resin
preparation and can probably be avoided by manufacturing a new lot
of antibody-resin from highly purified antibody or by pre-eluting
the antibody-resin before applying peptide mixtures.
[0178] This result further establishes the generality of the method
of the invention by showing that desired phosphopeptides can be
isolated/enriched by immunoaffinity purification, as described
herein. As previously discussed, in certain cases, as here, some
peptides that contain the target sequence motif may not be
isolated, and/or other peptides that do not contain the target
motif may be inadvertently or artifactually purified, for reasons
that are unclear. Nevertheless, the completeness and specificity of
the disclosed method represents a substantial advance over
alternative phosphopeptide purification methods. As previously
described, it is anticipated that, in cases where undesired
peptides lacking the target motif are co-isolated along with
desired peptides, the former may be avoided by increasing the
number or stringency of the resin washes to remove non-specifically
bound peptides.
Example II
A. Isolation of Phosphotyrosine-Containing and Phospho-Akt
Substrate Peptide Subsets from a Digested Crude Cell Extract
[0179] Example I demonstrates that several phosphorylation-specific
antibodies can be employed in the method of the invention to
selectively separate desired phosphopeptides from
non-phosphopeptides. The antibodies may be general
modification-specific antibodies or motif-specific,
context-independent antibodies that recognize a short non-unique
motif comprising several invariant residues, which motif is present
on a plurality of different peptides or proteins within a genome.
As shown in Example I, the antibodies can distinguish
phosphopeptides from non-phosphopeptides even when the only
difference between the peptides is the presence or absence of a
phosphate group. In the present Example, it is shown that desired
phosphopeptides may be selectively isolated by the method of the
invention from a complex mixture containing phosphopeptides of
different types. The method of the invention isolates the
phosphopeptide subset that would be expected on the basis of the
antibody's specificity. It is also shown that the results obtained
by applying the method to crude cell extracts closely resemble the
results obtained by applying the method to well-defined synthetic
peptide mixtures.
[0180] The exemplary preparation for the isolation described herein
was composed of a digested crude cell extract to which the
phosphotyrosine peptide mix and the phospho-Akt substrate peptide
mix have been added. The crude cell extract was made from 3T3 mouse
fibroblast cells that had been stably transfected to express active
Akt protein kinase constituitively and that had been treated with
50 ng/ml platelet-derived growth factor (PDGF) for 15 minutes. The
cells were washed, harvested, and lysed by sonication, proteins in
the lysate were denatured, and the lysate was cleared by
centrifugation. The extract was then digested to peptides with
endoproteinase Glu-C immobilized to F7m, a polyvinyl matrix bead
(MoBiTec, part number P5101), and the immobilized Glu-C was removed
by centrifugation. The digested extract was treated with
phospho-(Ser/Thr) Akt consensus substrate motif antibody (Cell
Signaling Technology, Inc., product number 9611) to remove
endogenous peptides recognized by this antibody.
[0181] This depleted digested extract was mixed with the
phosphotyrosine peptide mix (Table 1) and the phospho-Akt substrate
peptide mix (Table 3), so that each peptide was present at a
concentration of 10 pmol/ml and the background of peptides from the
digested extract was 250 .mu.g/ml. This peptide-extract mixture (1
ml) was treated with either immobilized P-Tyr-100 antibody (Cell
Signaling Technology, Inc., part number 9419) or immobilized
phospho-Akt substrate antibody (20 .mu.l, 2 .mu.g/.mu.l). After 2
hours at 4.degree. C., each antibody-resin was collected by
centrifugation and extensively washed (three times with 1 ml
ice-cold PBS and two times with 1 ml ice-cold water). Bound
peptides were then eluted with two 15 .mu.l aliquots of 0.1 M
glycine, pH 2.3: Before analysis by MALDI-TOF mass spectrometry, as
described above, a 9 .mu.l portion of the fraction containing bound
and eluted peptides was desalted and concentrated with a
reversed-phase ZipTip microcolumn.
[0182] For P-Tyr-100 antibody, a general modification-specific
antibody, the spectrum shows that the antibody isolated 3 of the 5
phosphotyrosine peptides but none of the 4 phospho-Akt substrate
phosphopeptides, which do not contain phosphotyrosine, and none of
the 9 non-phosphopeptides, as expected due to the antibody's
specificity (FIG. 10, top panel). Peaks labeled with a star
correspond to phosphopeptides, and peaks labeled with an open
circle correspond to nonphosphorylated peptides. Comparison to FIG.
4 shows the 2 phosphotyrosine-containing peptides that were not
identified from the peptide-extract mixture gave low signals from a
relatively simple mixture of synthetic peptides. These peptides may
ionize poorly when other peptides are present because they poorly
compete for protons.
[0183] For the phospho-Akt substrate antibody, a motif-specific,
context-independent antibody, the spectrum shows the antibody
isolated 3 of the 4 phosphopeptides from the phospho-Akt substrate
peptide mix but none of the 5 phosphotyrosine peptides (FIG. 11,
top panel). Comparison to FIG. 8 shows that the single phospho-Akt
substrate peptide that was not identified from the peptide-extract
mixture gave low signals from a synthetic peptide mix.
[0184] The phosphopeptide assignments shown in FIGS. 10 and 11 were
confirmed by treating a portion of the bound peptide fraction with
calf-intestinal phosphatase, which can remove phosphate from
phosphopeptides. As expected, most assigned phosphopeptides were
dephosphorylated to produce ions with masses 80 lower than the
phosphopeptides (FIGS. 10 and 11, bottom panels). Accordingly,
desired phosphopeptides may be selectively isolated from a complex
mixture according to the method of the invention.
B. Isolation of Phosphopeptides Containing the 14-3-3 Binding Motif
from a Digested Crude Cell Extract
[0185] As another example confirming that results obtained with
crude cell extracts closely resemble the results obtained with
well-defined synthetic peptide mixtures, the method of the
invention was employed to isolate 14-3-3 binding motif-containing
phosphopeptides from a complex mixture comprising a cell extract
and a mixture of synthetic peptides.
[0186] The exemplary preparation for the isolation described herein
was composed of a digested crude cell extract to which the 14-3-3
binding motif #1 peptide mix (Table 4) has been added. An
endoproteinase Glu-C-digested crude cell extract was prepared from
3T3 mouse fibroblast cells stably transfected to express active Akt
protein kinase constitutively, as described in Example II(A) above.
This digested extract was mixed with the 14-3-3 binding motif
peptide mix (Table 4), so that each peptide was present at a
concentration of 10 pmol/ml and the background of peptides from the
digested extract was 0.5 mg/ml.
[0187] Immobilized 14-3-3 binding motif antibody was prepared by
mixing 1 mg of 14-3-3 binding motif antibody and 0.1 ml of protein
A-agarose resin (Roche, product number 1 134 515) overnight at
4.degree. C. Unbound antibody was removed by washing the resin
three times with cold PBS. The amount of antibody bound to protein
A-agarose was shown to be 4 mg antibody/ml resin by measuring the
absorbance at 280 nm of the antibody solution before and after
immobilization.
[0188] The peptide-extract mixture (1 ml) was treated with
immobilized 14-3-3 binding motif #1 antibody (20 .mu.l, 1
.mu.g/.mu.l). After 2 hours at 4.degree. C., the antibody resin was
collected by centrifugation and extensively washed (twice with 1 ml
ice-cold PBS and once with 1 ml ice-cold water). Bound peptides
were then eluted with one 30 .mu.l aliquot of 0.1% trifluoroacetic
acid. Before analysis by MALDI-TOF mass spectrometry, as described
above, a 9 .mu.l portion of the fraction containing bound and
eluted peptides was desalted and concentrated with a reversed-phase
ZipTip microcolumn.
[0189] FIG. 12 shows the peptides that were bound and eluted from
the 14-3-3 antibody-resin. Peaks labeled with a star correspond to
phosphopeptides, and peaks labeled with an open circle correspond
to nonphosphorylated peptides. Comparison of FIG. 12, and FIG. 9
shows the method isolated the same four 14-3-3 motif
phosphopeptides from the synthetic peptide mix, even when the
mixture was diluted into a large background of potentially
interfering, non-binding peptides from a digested cell extract.
Accordingly, desired phosphopeptides may be selectively isolated
from a complex mixture according to the method of the
invention.
Example III
Isolation of Phosphotyrosine-Containing Peptides from an Extract of
Cells
Overexpressing Epidermal Growth Factor Receptor
[0190] The selective isolation of modified peptides from a complex
mixture according to the method of the invention was further
demonstrated using a digested whole cell extract and a general
phosphotyrosine antibody to isolate known phosphopeptides. A model
system, the A431 epidermoid carcinoma cell line overexpressing the
human epidermal growth factor receptor (EGFR), was selected since
the modification (phosphorylation) of sites on this protein is
well-studied. Activation of EGFR family members is associated with
many tumors. Five sites of in vivo autophosphorylation have been
identified in EGFR: three major sites (Tyr-1068, Tyr-1148, and
Tyr-1173) and two minor sites (Tyr-992 and Tyr-1086) (Downward et
al., J. Biol. Chem. 260: 14538-546 (1985); Hsuan et al., Biochem.
J. 259: 519-27 (1989); Margolis et al., EMBO J. 9: 4375-380 (1990);
Walton et al., J. Biol. Chem. 265: 1750-54 (1990)). EGFR is the
major phosphorylated protein expected to be expressed in this cell
line.
[0191] A cell preparation was obtained as follows: A431 cells were
treated with 20 ng/ml EGF for 5 minutes and then washed and
harvested. The cells were lysed by sonication, proteins in the
lysate were denatured, and the lysate was cleared by
centrifugation. The cell extract was analyzed by SDS-PAGE and
Western blotting to show the level of phosphorylated EGFR (FIG.
13). Compared to untreated cells (FIG. 13, lane 1), the major
protein recognized by P-Tyr-100 antibody in EGF-treated cells is
EGFR (lane 2). Proteins in the extract supernatant were digested to
peptides with trypsin immobilized to POROS resin (Applied
Biosystems, part number 2-3127-00), and the immobilized trypsin was
removed by centrifugation.
[0192] To selectively isolate phosphotyrosine-containing peptides
from the complex mixture of peptides contained in the proteinaceous
preparation, the trypsin-digested crude extract (about 2.5 mg
protein/mL) was contacted with an immobilized general tyrosine
modification antibody, P-Tyr-100 antibody-resin (Cell Signaling
Technology, Inc., product number 9419) (20 .mu.l). The slurry was
incubated and processed as described above, except that the first
wash was with 0.5 ml PBS containing 0.1% Tween 20. FIG. 14 (top
panel) shows the mass spectrum for the bound peptide fraction from
this complex mixture (digest). Peaks labeled with a star correspond
to two known phosphotyrosine sites in EGF receptor: the protonated
tryptic peptide containing pTyr-1148 has an expected mass of
2,316.0, and the peptide containing pTyr-1086 has an expected mass
of 2,479.2. Note that these EGF receptor peptides were expected to
be the major phosphotyrosine peptides in the bound fraction,
because the cell line overexpresses the EGF receptor.
[0193] To confirm these assignments, the isolated (i.e. bound)
peptide fraction was treated with a phosphatase enzyme, as
described above, and the treated fraction re-analyzed by MALDI-TOF
mass spectrometry, as described above (FIG. 14, bottom panel). As
expected, these two phosphopeptides were completely
dephosphorylated to produce new ions with masses 80 lower than the
phosphopeptides, corresponding to the removal of one phosphate
group from each peptide. Accordingly, the method of the invention
selectively isolates modified peptides, e.g. those containing
phosphotyrosine, from a complex mixture that is present in a
proteinaceous preparation (digested crude cell extract). Similar
isolations may be carried out for any desired proteinaceous
preparation using a desired, immobilized modification-specific
antibody.
Example IV
Isolation of Phosphotyrosine-Containing Peptides from an Extract of
Cells
Expressing Activated Src Protein Kinase or Activated Abl Protein
Kinase
[0194] To demonstrate that the set of phosphopeptides isolated by
the general phosphotyrosine antibody is a property of the cell
extract, the method of the invention was applied to a digested
whole cell extract different from the one used in Example III.
Here, the exemplary system is 3T3 mouse fibroblast cells stably
transfected to express active Src protein kinase constituitively.
The Src family of protein kinases is important in the regulation of
cell growth and differentiation (Thomas and Brugge, Annu. Rev.
Cell. Dev. Biol. 13, 513-609 (1997)). Src protein kinase
participates in many different signaling pathways and can affect
diverse biological processes. Src is known to phosphorylate its
target proteins on Tyr residues, i.e., it is a tyrosine-specific
kinase.
[0195] A digested cell extract was prepared by harvesting 3T3 cells
expressing Src protein kinase. The cells were lysed by sonication,
proteins in the lysate were denatured, and the lysate was cleared
by centrifugation. To show that activated Src protein kinase had
phosphorylated many target proteins, the cell extract was analyzed
by SDS-PAGE and Western blotting (FIG. 15). Activation of Src
protein kinase was shown by blotting extracts of untransfected
(lane 1) and Src-transfected (lane 2) 3T3 cells and probing the
blot with P-Tyr-100 antibody (Cell Signaling Technology, Inc.,
product number 9411). The level and extent of tyrosine
phosphorylation was much greater in cells that had been stably
transfected with Src protein kinase than in untransfected
cells.
[0196] Proteins in the extract were digested to peptides with
immobilized trypsin, and the immobilized trypsin was removed by
centrifugation. Immobilized P-Tyr-100 antibody was prepared by
mixing 1 mg of P-Tyr-100 and 0.1 ml of protein G-agarose resin
(Roche, product number 1 243 233) overnight at 4.degree. C. Unbound
antibody was removed by washing the resin three times with cold
PBS. The amount of antibody bound to protein G-agarose was shown to
be 5 mg antibody/ml resin by measuring the absorbance at 280 nm of
the antibody solution before and after immobilization.
[0197] Phosphotyrosine-containing peptides were isolated from the
complex mixture of peptides contained in the proteinaceous
preparation by contacting the trypsin-digested extract (about 12
mg, 1 mg/ml) with phosphotyrosine antibody P-Tyr-100 that was bound
to protein G resin (20 .mu.l, 5 mg antibody/ml resin) in batch
format at 4.degree. C. for 16 hours. Unbound peptides were removed
by centrifugation, and the antibody-resin was extensively washed
(three times with 1 ml ice-cold PBS and twice with 1 ml ice-cold
water). Bound peptides and antibody were then eluted with 100 .mu.l
of 0.1% trifluoroacetic acid, and the eluted peptides were
separated from eluted antibody by centrifugation through a Microcon
YM-10 membrane (Millipore, product number 42407), which retains
molecules with molecular weights above 10,000. Before analysis by
MALDI-TOF mass spectrometry, a 9 portion of the YM-10 flow-through
fraction was desalted and concentrated with a reversed-phase ZipTip
microcolumn.
[0198] The masses of the peptides that bound to and eluted from the
phosphotyrosine antibody were measured by MALDI-TOF mass
spectrometry before (FIG. 16, top panel) and after (bottom panel)
treating the peptide fraction with shrimp alkaline phosphatase,
which can remove phosphate groups from phosphopeptides and produce
ions with masses 80 lower than phosphopeptides for each phosphate
group in the peptide, to confirm the eluted peptides are
phosphorylated (FIG. 16, bottom panel). The masses of eight
peptides bound and eluted from phosphotyrosine antibody-resin gave
new ions with masses 80 lower than the phosphopeptides after
treatment with phosphatase, indicating they are
phosphopeptides.
[0199] The peptides that bound to and eluted from the
phosphotyrosine antibody were further analyzed by LC-MS/MS. A 25
.mu.l portion of the peptide fraction was desalted and concentrated
with a reversed-phase ZipTip microcolumn and eluted with 2 .mu.l
0.1% trifluoroacetic acid, 40% acetonitrile. An 0.4 .mu.l aliquot
of the eluted fraction was mixed with an ACHA matrix solution and
analyzed by MALDI-TOF mass spectrometry, and it gave a spectrum
similar to the one shown in FIG. 16. The remainder of the eluted
fraction was analyzed by LC-MS/MS.
[0200] LC-MS/MS analysis was performed with a ThermoFinnigan
Surveyor HPLC system coupled to a ThermoFinnigan LCQ Deca ion trap
mass spectrometer. To reduce its acetonitrile concentration to a
level that would allow peptides to bind to a reversed-phase
support, the sample was diluted 10-fold with 0.5% acetic acid,
0.005% HFBA (heptafluorobutyric acid, Pierce Endogen, part number
25003), 5% acetonitrile containing 1% formic acid. Using a pressure
cell, the diluted sample was loaded onto a capillary column (75
.mu.m internal diameter, 15 .mu.m tip, fused silica PicoTip, New
Objective, part number FS360-75-15-N) that had been packed with
Magic C18AQ reversed-phase resin (5 .mu.m particles, 100 Angstrom
pores, Michrom Bioresources, part number 9996610000) and
equilibrated with 0.5% acetic acid, 0.005% HFBA, 5% acetonitrile.
Peptides were eluted from the column by a linear gradient of
increasing acetonitrile concentration at a nominal flow rate of 250
nl/min.
[0201] To induce electrospray at the tip of the column, 2,000 V was
applied to a liquid junction upstream of the column at a cross used
to modulate the flow rate from the HPLC pump, as described by
Gatlin et al., supra. ThermoFinnigan Xcalibur software was used for
instrument control and data acquisition. As peptides eluted from
the LC column, MS/MS spectra were collected in a "top-three"
data-dependent manner: the method performed a survey MS scan and
then collected MS/MS spectra for the three most abundant ions, if
they were above a pre-set intensity threshold and if they were not
recently analyzed by MS/MS already (recognized by using the dynamic
exclusion feature of Xcalibur software).
[0202] Peptides were identified by analyzing all the MS/MS product
ion spectra with Sequest, a program that correlates an experimental
spectrum to a library of theoretical spectra derived from protein
sequence databases to find a best-fit match. One unambiguously
identified phosphopeptide is a phosphotyrosine-containing peptide
from enolase A (FIG. 17), an abundant enzyme. The residue
identified as a phosphorylation site by this method is known to be
phosphorylated in cells transfected with Src (see, e.g. Tanaka et
al. J. Biochem (Tokyo) 117: 554-559 (1995) and Cooper et al. J.
Biol. Chem. 259: 7835-7841 (1984)). This phosphopeptide corresponds
to a prominent peak detected during MALDI-TOF mass spectrometry,
labeled "1,885.2" in FIG. 16, demonstrating, the same
phosphopeptides detected during MALDI-MS can be further analyzed by
LC-MS.
[0203] Accordingly, immunoaffinity isolation of modified peptides
by the disclosed method detected a site known to be phosphorylated
under these cell culture conditions and, as expected, the assigned
site fits the antibody's known specificity. This result, isolation
of a known enolase phosphopeptide from a digested extract of mouse
cells, is in stark contrast to the results reported in Marcus et.
al., supra., where, following the failure to isolate a
phosphotyrosine-containing peptide from gel-purified human enolase
(the same protein), it was expressly concluded that immunoaffinity
purification of phosphopeptides is almost impossible.
[0204] In a separate study the method of the invention was used to
extract phosphotyrosine peptides from NIH/3T3 cells expressing one
of two constituitively active tyrosine kinases, v-Abl and c-Src
Y527F. These cell lines were left untreated during cell culturing
so the bulk of the tyrosine phosphorylation in the cell is expected
to originate from the action of the constituitively active kinase
expressed in the cell. The objective was to determine whether the
method of the invention can be used to detect phosphorylation
indicative of Abl or Src activation in the same cellular
background.
[0205] Lysates were prepared from 2.times.10.sup.8 cells for both
cell lines and digested in 2 M urea with trypsin after treatment
with DTT and iodoacetamide to alkylate cysteine residues. Before
the immunoaffinity step, peptides were prefractionated by
reversed-phase solid phase extraction using Sep-Pak C.sub.18
columns (1 ml column volume per 2.times.10.sup.8 cells) to separate
peptides from other cellular components. The solid phase extraction
cartridges were eluted with steps of 5, 15, 25, and 40%
acetonitrile. Each lyophilized peptide fraction was redissolved in
1 ml PBS and treated with phosphotyrosine antibody (P-Tyr-100, Cell
Signaling Technology product number 9411) immobilized on protein
G-Sepharose (Roche) (60 .mu.g antibody, 15 .mu.l resin) overnight
at 4.degree. C. Antibody-resin was thoroughly washed, and
immunoaffinity-purified peptides were eluted with 75 .mu.l of 0.1%
TFA. A portion of this fraction (40 .mu.l) was concentrated with
Stage tips and analyzed by LC-MS/MS, using a ThermoFinnigan LCQ
Deca XP Plus ion trap mass spectrometer. Peptides were eluted from
a 10 cm.times.75 .mu.m reversed-phase column with a 45-min linear
gradient of acetonitrile delivered at 280 nl/min. MS/MS spectra
were evaluated using the program Sequest with the NCBI mouse
protein database.
[0206] More phosphopeptides were found in 3T3-Src (179) than in
3T3-Abl (83), which was consistent with the level of
phosphotyrosine detected by western blotting. Substantial overlap
was observed between the tyrosine phosphorylation sites found in
3T3-Abl and in 3T3-Src: 62 of the 186 phosphotyrosine sites found
in 3T3/Src were also found in 3T3-Abl. The overlap may be
attributed to activation of a Src-like kinase in 3T3-Abl cells,
which were found to contain IIEDNEpYTAR, the activation loop
phosphopeptide from the Src-family members Hck and Lyn, or
LIEDNEpYTAR, which is found in other Src-family members such as
Fyn, Lck, Src, and Yes. It is clear that at least one Src-family
kinase has been activated in 3T3-Abl cells, but we cannot specify
the activated enzyme because Src-family kinases are so closely
related and these two peptides have identical mass. Some of the
phosphotyrosine sites from known cell signaling proteins found in
3T3-Abl and 3T3-Src are shown in Table 5.
TABLE-US-00005 TABLE 5 Selected phosphopeptides found in 3T3-Abl
and 3T3-Src Protein Name.dagger-dbl. Abl Src Sequence Tyrosine
kinase * Eph receptor A2 .cndot. VLEDDPEATpYTTSGGK * Eph receptor
A4 .cndot. .cndot. VLEDDPEAApYTTR Eph receptor B3 .cndot.
VYIDPFTpYEDPNEAVR focal adhesion kinase .cndot. .cndot.
THAVSVSETDDpYAEIIDEEDTYTMPSTR * focal adhesion kinase .cndot.
.cndot. YMEDSTpYpYK focal adhesion kinase .cndot.
GSIDREDGSFQGPTGNQHIpYQPVGKPDPAAPPK * hemopoietic cell kinase
.cndot. .cndot. IIEDNEpYTAR * fer protein kinase .cndot.
QEDGGVpYSSSGLK * Src .cndot. LIEDNEpYTAR Ser/Thr kinase cdc2a
.cndot. .cndot. IGEGTpYGVVYK * DYRK1a .cndot. .cndot. IYQpYIQSR
DYRK3 .cndot. pYEVLKIIGKGSFGQVAR * GSK3 beta .cndot. .cndot.
GEPNVSpYICSR * HIPK 1 .cndot. .cndot. AVCSTpYLQSR * HIPK 3 .cndot.
TVCSTpYLQSR * MAPK1 .cndot. VADPDHDHTGFLTEpYVATR * p38 MAP Kinase
.cndot. HTDDEMTGpYVATR * pre-mRNA protein kinase .cndot. .cndot.
LCDFGSASHVADNDITPpYLVSR Cdc42 BP kinase beta .cndot.
LPDFQDSIFEpYFNTAPLAHDLTFR Adaptor abl-interactor 1 .cndot.
TLEPVKPPTVPNDpYMTSPAR caveolin .cndot. .cndot. YVDSEGHLpYTVPIR
Cbl-b .cndot. .cndot. ASQDpYDQLPSSSDGSQAPARPPKPR DOK1 .cndot.
.cndot. TVPPPVPQDPLGSPPALpYAEPLDSLR DOK1 .cndot. .cndot.
IPPGPSQDSVpYSDPLGSTPAGAGEGVHSK DOK1 .cndot. .cndot.
LTDSKEDPIpYDEPEGLAPAPPR DOK1 .cndot. LKEEGYELPYNPATDDpYAVPPPR DOK1
.cndot. .cndot. GFSSDTALpYSQVQK GAB1 .cndot. DASSQDCpYDIPR p130 Cas
.cndot. .cndot. TQQGLpYQAPGPNPQFQSPPAK p130 Cas .cndot.
VGQGYVYEAAQTEQDEpYDTPR p130 Cas .cndot. .cndot. EETpYDVPPAFAK PI3K
p85 beta subunit .cndot. .cndot. EYDQLpYEEYTR SHB adaptor protein
.cndot. VTIADDpYSDPFDAK Shc1 .cndot. .cndot. ELFDDPSpYVNIQNLDK
similar to SHB adapt. pro. B .cndot. .cndot. LDpYCGGGGGGDPGGGQR Crk
.cndot. RVPCApYDK disabled homolog 2 .cndot. GPLNGDTDpYFGQQFDQLSNR
DOK1 .cndot. KPLpYWDLpYGHVQQQLLK DOK1 .cndot. GLpYDLPQEPR DOK1
.cndot. LKEEGpYELPpYNPATDDpYAVPPPR HGF reg. tyr. kinase subs.
.cndot. VCEPCpYEQLNK intersectin 2 .cndot. GEPEALpYAAVTK p130 Cas
.cndot. VGQGYVpYEAAQTEQDEpYDTPR p130 Cas .cndot.
HPLILAAPPPDSPAAEDVpYDVPPPAPDLpYDVPPGLR p130 Cas .cndot.
VLPPEVADGSVVDDGVpYAVPPPAER PI3K p85 reg. subunit .cndot.
LNEWLGNENTEDQpYSLVEDDEDLPHHDEK PLC gamma 1 .cndot. IGTAEPDpYGALYEGR
Shc1 .cndot. MAGFDGSAWDEEEEEPPDHQpYpYNDFPGK Src-assoc. adaptor
protein .cndot. SVYLQEFQDKGDAEDGDEpYDDPFAGPADTISLASER Src-assoc.
adaptor protein .cndot. IpYQFTAASPK Src-assoc. adaptor protein
.cndot. SQPIDDEIpYEELPEEEEDTASVK Stam2 .cndot. LVNEAPVYSVpYSK
Wiskott-Aldrich syn.-like .cndot. VIpYDFIEK .dagger-dbl.* indicates
activation loop peptides. .cndot. indicates phosphopeptides found
in 3T3-Abl or 3T3-Src.
[0207] In 3T3-Src cells, phosphorylation was detected in the
activation loop of a Src-family kinase, most likely Tyr-424 of Src.
Tyrosine phosphorylation was also found in proteins that are known
to be substrates of Src, e.g., the non-receptor tyrosine kinase
FAK, the adaptor protein p130Cas, the actin-binding protein
cortactin, the phospholipid-binding protein annexin A2, and the
STAM-interacting protein Hrs. FAK was phosphorylated at Tyr-397,
which creates a binding site for Src-family kinases, and at its
activation loop sites Tyr-576 and Tyr-577, which are known to be
phosphorylated by Src-family kinases. Seven tyrosine
phosphorylation sites were found in p130Cas, all in the
Src-substrate region of p130Cas and six containing the YXXP motif,
which when phosphorylated results in the interaction of p130Cas
with several signaling proteins. Some phosphorylation sites map to
putative Src substrates, e.g., SCAP2 is a homolog of the
Src-associated phosphoprotein SKAPP55 and was found to be
phosphorylated at Tyr-260, which in SKAPP55 is thought to be
phosphorylated by the Src-family kinase Fyn.
Example V
Isolation of Peptides Containing the Phospho-(Ser) PKC Substrate
Motif from an Extract of Jurkat Cells Treated with Tetradecanoyl
Phorbol Acetate
[0208] The method of the invention was further demonstrated using a
motif-specific, context-independent polyclonal antibody,
phospho-(Ser) PKC substrate motif antibody, to purify
phosphopeptides containing the phospho-(Ser) PKC substrate motif
from a digested whole cell extract. Protein kinase C(PKC) family
members are involved in a number of cellular processes such as
secretion, gene expression, proliferation and muscle contraction
(see e.g. Nishikawa et al. J. Biol. Chem. 272: 952-960 (1997) and
Pearson and Kemp. Methods Enzymol. 200: 62-81 (1991)). Conventional
PKC isozymes phosphorylate protein substrates at serine or
threonine residues when the target residue occurs within the
consensus sequence motif (R/K)(R/K)X(S/T)(hyb)(R/K) (SEQ ID NO:
38), where R/K indicates arginine or lysine, X is any amino acid,
S/T indicates the target serine or threonine, and hyb is a
hydrophobic amino acid.
[0209] Phospho-(Ser) PKC substrate motif antibody (Cell Signaling
Technology, Inc., product number 2261) recognizes a plurality of
different phosphorylated proteins that contain the consensus
sequence motif when phosphorylated but does not recognize the
analogous unphosphorylated motif. The specificity of the
phospho-(Ser) PKC substrate antibody is that it binds
preferentially to proteins and peptides that contain phosphoserine
preceded by arginine or lysine at positions -2 and +2 and a
hydrophobic residue at the +1 position, i.e., (R/K)XS*(hyb)(R/K)
(SEQ ID NO: 39), in a manner substantially independent of the
surrounding amino acid sequence. The antibody does not recognize
the non-phosphorylated motif or the motif containing
phosphothreonine. It is demonstrated here that this antibody can be
used in accordance with the method of the invention to purify
peptides that contain this phospho-(Ser) PKC substrate motif, to
identify other proteins that may be phosphorylated by conventional
PKC isozymes on a genome-wide (cell-wide) basis.
[0210] For this example, the model system was Jurkat cells, a human
cell line derived from an acute T cell leukemia, that had been
treated for 10 minutes with a potent activator of protein kinase C,
tetradecanoyl phorbol acetate (TPA). The cells were washed,
harvested, and lysed by sonication, proteins in the lysate were
denatured, and the lysate was cleared by centrifugation.
[0211] To show that TPA had activated protein kinase C and caused
an increased level of PKC-specific protein phosphorylation, the
cell extract was analyzed by SDS-PAGE and Western blotting (FIG.
18). Induction of PKC substrate phosphorylation was shown by
probing a blot of TPA-treated cell extract (lane 2) and untreated
cell extract (lane 1) with phospho-(Ser) PKC substrate antibody
(Cell Signaling Technology, Inc., product number 2261). This showed
that TPA treatment altered the phosphorylation state of a large
number of different proteins that contain the phospho-(Ser) PKC
substrate motif.
[0212] Proteins in the extract were digested to peptides with
endoproteinase Glu-C immobilized to F7m, a polyvinyl matrix bead
(MoBiTec, part number P5101), and the immobilized Glu-C was removed
by centrifugation. Immobilized phospho-(Ser) PKC substrate antibody
was prepared as described above for immobilized P-Tyr-100 antibody
(Example IV). The immobilized antibody was evaluated as described
above and found to contain 4 mg antibody per ml of resin.
[0213] Phosphopeptides containing the phospho-(Ser) PKC substrate
motif were purified from the Glu-C-digested crude cell extract with
the antibody immobilized to protein G-agarose resin. The digest
(about 40 mg, 1 mg/ml protein) was contacted with immobilized
antibody-resin (40 .mu.l, 4 mg/ml) in batch format at 4.degree. C.
for 16 hours, and unbound peptides were removed by centrifugation.
The antibody-resin was washed extensively (three times with 1 ml
ice-cold PBS and twice with 1 ml ice-cold water). Bound peptides
were then eluted with 150 .mu.l 10.1% trifluoroacetic acid, and the
eluted peptides were separated from eluted antibody by
centrifugation through a Microcon YM-10 membrane (Millipore,
product number 42407), which retains molecules with molecular
weights above 10,000. Before analysis by MALDI-TOF mass
spectrometry, a 9 .mu.l portion of the YM-10 flow-through fraction
was desalted and concentrated with a reversed-phase ZipTip
microcolumn.
MALDI-TOF Analysis
[0214] The masses of the peptides that bound to and eluted from the
phospho-(Ser) PKC substrate antibody were measured by MALDI-TOF
mass spectrometry (FIG. 19, top panel). Phosphatase treatment and
metastable decomposition (bottom panel) showed that the
antibody-purified peptide fraction contains several candidate
phosphopeptides with phosphoserine or phosphothreonine, as expected
based on the antibody's specificity. In FIG. 19, peaks labeled with
a star correspond to phosphopeptides, peaks labeled with an open
circle correspond to nonphosphorylated peptides, and peaks labeled
with a square are phosphopeptides that have undergone metastable
decomposition and neutral-loss of phosphate.
[0215] Phosphopeptide peaks detected during MALDI-TOF mass
spectrometry (FIG. 19, top panel) are accompanied by companion
peaks that are broader and apparently 84 lower in mass, e.g., the
peak with a mass of 1989 has a partner peak at 1905, etc. These
companion peaks correspond to metastable decomposition products of
phosphopeptides, formed by neutral-loss of phosphate while the
phosphopeptide ions are traveling to the detector of the mass
spectrometer. The peaks for decomposition products are broader than
the peaks for phosphopeptides because the decomposition products
form after ionization and the instrument is configured to focus
ions that are stable during analysis. For similar reasons, the
expected mass shift for loss of phosphate is -98, but -84 mass
shifts are observed because, unlike a stable ion, the mass of a
decomposition product changes during analysis. Metastable
decomposition of phosphopeptides has been noted by others and can
be used to recognize and assign phosphopeptides in a MALDI-TOF mass
spectrum (Annan and Carr, supra.). Analysis of a large number of
synthetic phosphopeptides by MALDI-TOF mass spectrometry indicates
that some peptides containing phosphoserine or phosphothreonine
(but not phosphotyrosine) residues undergo metastable
decomposition. For this reason, metastable decomposition is a
reliable indicator of peptides that contain phosphoserine or
phosphothreonine.
[0216] The phosphorylation state of the peptide fraction was also
evaluated by treating it with shrimp alkaline phosphatase, which
can remove phosphate groups from phosphopeptides to produce ions
with masses 80 lower than phosphopeptides for each phosphate group
in the peptide. All phosphopeptide candidates were affected by
phosphatase treatment, and four phosphopeptides gave
dephosphorylated peptides that were 80 lower in mass than the
peptides before treatment (FIG. 19, bottom panel).
LC-MS/MS Analysis
[0217] The peptides that bound to and eluted from the phospho-(Ser)
PKC substrate motif antibody were further analyzed by LC-MS/MS. A
40 .mu.l portion of the peptide fraction was desalted and
concentrated with a reversed-phase ZipTip microcolumn and eluted
with 2 .mu.l 0.1% trifluoroacetic acid, 40% acetonitrile. An 0.4
.mu.l aliquot of the eluted fraction was mixed with an ACHA matrix
solution and analyzed by MALDI-TOF mass spectrometry, and it gave a
spectrum similar to the one shown in FIG. 19. An 0.8 .mu.l aliquot
of the eluted fraction was analyzed by LC-MS/MS.
[0218] LC-MS/MS analysis was performed as described above (Example
IV). The chromatogram obtained by analyzing this sample is shown in
FIG. 20. The first panel of FIG. 20 shows where survey MS scans
were collected, and the second panel shows where MS/MS spectra were
collected. The third, fourth, and fifth panels show where neutral
loss of 49, 32.7, and 24.5, respectively, was detected. During the
fragmentation process of MS/MS, peptides containing phosphoserine
or phosphothreonine often form an ion by simple loss of phosphate
to produce a neutral-loss ion that has a mass 98 lower than the
unfragmented parent ion. If the parent ion has a charge of +1, the
neutral-loss ion has a mass-to-charge value (m/z) of 98/1 or 98
lower than the parent ion mass-to charge value. Likewise,
phosphopeptide parent ions with charges of +2, +3, or +4 will give
neutral-loss ions with m/z values that are 49, 32.7, and 24.5 lower
than the parent ion. The occurrence and intensities of neutral-loss
ions are plotted in the third, fourth, and fifth panels of FIG. 20
to help locate candidate phosphopeptides. The neutral loss plots
show that phosphopeptide candidates tend to elute early in the
chromatogram, as expected for phosphopeptides due to the
hydrophilicity of phosphate groups, and that neutral loss is
observed in many of the MS/MS spectra, suggesting this sample is
highly enriched with phosphopeptides.
[0219] As discussed, neutral loss during MS/MS is the same process
as metastable decomposition during MALDI-TOF mass spectrometry. As
expected, many of the phosphopeptides showing neutral loss during
LC-MS/MS (FIG. 20, panels 3-5) are the same phosphopeptides that
gave metastable decomposition during MALDI-TOF mass spectrometry
(FIG. 19, top panel), see FIG. 21. For each neutral-loss MS/MS
spectrum, the parent ion mass (m) can be calculated from the parent
ion mass-to-charge value (m/z) and the charge (z) inferred from the
neutral loss value (+2 for neutral loss of 49, +3 for 32.7, and +4
for 24.5). Some individual peptides were observed to undergo
neutral loss as +2, +3, and +4 ions. For example, LC-MS/MS spectra
533, 534, and 535 show neutral loss and correspond to +4, +3, and
+2 ions, respectively, of a candidate phosphopeptide labeled
"2,413.3" in FIG. 19. Both MALDI-TOF mass spectrometry and LC-MS/MS
give this peptide a mass of 2,413, and the neutral loss observed
during both mass analysis methods show the peptide contains one
phosphate. A comparison of datasets shows the same peptides are
detected by both mass analysis methods, and all the neutral-loss
MS/MS spectra show the peptides contain one phosphate group (FIG.
21).
[0220] All the MS/MS product ion spectra were analyzed with Sequest
in an attempt to assign a phosphorylation site and parent protein
to each peptide, but this did not result in unambiguous
assignments. During MS/MS nearly all phosphopeptides underwent
neutral loss to a very high degree with very little residual
fragmentation along the peptide backbone, which is needed to
produce spectra of a quality high enough for unambiguous
assignments. In general backbone fragmentation was at the same
level as chemical noise, obscuring the features needed to identify
the peptides. As noted above, this is a common limitation
encountered during MS/MS analysis of peptides containing
phosphoserine and phosphothreonine. See e.g., DeGnore et al.,
supra. Even when phosphopeptides lose phosphate by neutral loss,
the position of the phosphorylation site can be determined as long
as there is sufficient residual backbone fragmentation, because
neutral loss leaves an unusual residue at the phosphorylation site:
phosphoserine becomes dehydroalanine, and phosphothreonine becomes
dehydroaminobutyric acid.
LC-MS.sup.3 Analysis
[0221] Some phosphopeptides in this sample were identified by
LC-MS.sup.3, that is, the neutral-loss ions were subjected to an
additional level of MS to give sufficient backbone fragmentation
for identification. This process is simpler to implement on ion
trap mass spectrometers than on other types of mass spectrometers.
As peptides elute from the LC system, a survey MS scan is
performed, and MS/MS spectra are collected for the three most
abundant ions, if they are above a pre-set intensity threshold and
if they have not been recently analyzed by MS/MS already. However,
if neutral loss of 49, 32.7, or 24.5 is detected during MS/MS, then
before collecting another MS/MS spectrum or another survey MS scan,
the instrument first isolates the neutral loss ion, fragments it,
and measures the product ion masses. If the neutral-loss ion no
longer contains phosphate, it is more likely to fragment like a
non-phosphorylated peptide and give a useful product ion spectrum.
With certain modifications to the instrument control software,
MS.sup.3 spectra can be collected in the same data-dependent manner
as MS/MS spectra, and the MS.sup.3 spectra can be analyzed further
with Sequest. See Tomaino and Rush et al., supra.
[0222] Data-dependent LC-MS.sup.3 was performed on the remainder of
the eluted fraction, an 0.8 .mu.l aliquot. FIG. 22 compares the
MS/MS spectra (left panels) and the MS.sup.3 spectra (right panels)
for three phosphopeptides that were identified by this method. Each
MS/MS spectrum contains predominantly one product ion, an intense
peak differing from the parent ion mass by 32.7, consistent with
loss of one phosphate from a phosphopeptide ion with a charge of
+3. Nearly all other peaks in the spectrum are at least 20-fold
less intense than the neutral-loss ion. The MS/MS spectra collected
during LC-MS.sup.3 analysis of this sample are very similar to the
MS/MS spectra collected during LC-MS/MS analysis, described above,
and illustrate how neutral loss can dominate MS/MS spectra of
peptides containing phosphoserine or phosphothreonine. Because
neutral loss of 32.7 was detected during MS/MS, the mass
spectrometer automatically subjected the neutral-loss ion to
MS.sup.3 to produce the spectra shown in the right panels. These
show several product ions of varying intensities distributed
throughout the spectra, and as expected they resemble MS/MS spectra
of non-phosphorylated peptides.
[0223] Using Sequest a phosphorylation site and parent protein can
be assigned to each of the three MS.sup.3 spectra shown in FIG. 22.
As noted in FIG. 21, all three of these assigned phosphopeptides
correspond to candidate phosphopeptides identified during MALDI-TOF
mass spectrometry. Panel 1 corresponds to PTN6_HUMAN, residues
576-595, with phosphorylation at Ser-585 or Ser-588. Although
Sequest unambiguously identified the peptide, in this example, it
could not distinguish the two possible phosphorylation sites.
However, the sites can be distinguished based on the known
specificity of the phospho-(Ser) PKC substrate antibody: the
sequence context of Ser-588 (KGS*LK) fits the antibody's
specificity but the sequence context of Ser-585 does not (EKS*KG)
[underlined residues match the specificity motif
(R/K)XS*(hyb)(R/K)].
[0224] PTN6 is protein-tyrosine phosphatase 1c, also known as
hematopoietic cell protein-tyrosine phosphatase, relevant because
the phosphopeptides in this experiment were purified from a human
cell line derived from an acute T cell leukemia (Jurkat cells).
Brumell et al. (J. Biol. Chem. 272: 875-882 (1997)) have suggested
that this specific tyrosine phosphatase is inhibited by
PKC-mediated serine phosphorylation, but the specific
phosphorylation site has not been identified. Presently, the method
of the invention has identified Ser-588 as a possible site of
PKC-mediated serine phosphorylation.
[0225] Panels 2 and 3 correspond to two overlapping peptides from
UFD1_HUMAN that contain the same phosphorylation site. Sequest
assigned residues 322-343 with phosphorylation at Ser-335 to the
spectrum in panel 2 and residues 333-343 with phosphorylation at
Ser-335 to the spectrum in panel 3. The longer peptide is related
to the shorter peptide by incomplete proteolytic cleavage: Glu-C
did not cleave at Glu-332 completely. In both cases the quality of
the Sequest assignments is good, and the position of the
phosphorylated residue is unambiguous. The sequence context of
Ser-335 (GQS*LR) partially fits the antibody's specificity. UFD1 is
ubiquitin fusion degradation protein 1. This protein has not been
previously shown to be phosphorylated. Presently, the method of the
invention has identified Ser-335 as a novel phosphorylation
site.
Confirmation of Sequence
[0226] For demonstrative purposes, one of the novel phosphorylation
sites was confirmed by showing a synthetic peptide with the
assigned sequence and phosphorylation site gives MS/MS and MS.sup.3
spectra that are identical to the MS/MS and MS.sup.3 spectra of the
biological peptide, i.e., the peptide purified by the method of the
invention from Glu-C-digested Jurkat cells. UFD1 333-343
phospho-Ser-335 was synthesized at Cell Signaling Technology using
Fmoc chemistry. The full-length peptide was purified by HPLC and
then analyzed using the same LC-MS.sup.3 method described
above.
[0227] The MS/MS and MS.sup.3 spectra for the biological peptide
(top panels) and the synthetic peptide (bottom panels) are compared
in FIG. 23. Portions of the MS/MS spectra have been amplified by a
factor of 10 to show ions other than the neutral-loss ion more
clearly. The correspondence between the MS.sup.3 spectra
demonstrates that the assigned peptide sequence and phosphorylation
site are correct. Even though the quality of the MS/MS spectra is
compromised by a dominant neutral-loss ion, there is good
correspondence between the minor peaks of the two spectra.
[0228] In this example, Sequest assigned a peptide sequence and
phosphorylation site to a spectrum, and the assignment was
confirmed by showing a synthetic peptide with that sequence and
phosphorylation site gives the same spectrum. This establishes a
formal link between a specific phosphopeptide and its spectrum.
This is a simple and convincing way to further evaluate marginal
Sequest assignments or to confirm assignments that are considered
especially important.
Marginal Assignments
[0229] As discussed above, neutral loss of phosphate from
phosphoserine or phosphothreonine can make it difficult to assign a
peptide sequence to an MS/MS spectrum and occasionally assignments
will be ambiguous. In a global proteomic method, where
phosphopeptides are isolated and analyzed separately from
non-phosphorylated peptides, often the only peptide from a
particular protein will be the purified phosphopeptide, and
unambiguous assignments are likely to be more difficult to achieve.
Accordingly, marginal assignments may be of higher value, and may
be worth pursuing further. Marginal assignments that are worth
further investigation can be identified by using simple computer
programs to screen the bulk results for assignments that fit the
known specificity of the antibody used to isolate the
phosphopeptides.
[0230] As an example of this, the MS/MS spectra of the sample
described here was further screened for marginal assignments, using
antibody specificity and our higher-confidence MS.sup.3 results as
guides. As described above, MS.sup.3 analysis identified two novel
phosphorylation sites in three different peptides: one mapped to
PTN6 and fit the known phospho-(Ser) PKC substrate motif, and the
other two mapped to UFD1 and fit the motif partially. A comparison
of the peptide sequences showed a variation of the motif might be
sufficient for antibody recognition: the PTN6 site contained the
sequence S*LKRK, and the UFD1 site contained the sequence S*LRKK.
Based on this, all the Sequest output files were searched, which
listed the top 20 candidate peptide sequences for each spectrum,
for marginal results that fit the consensus sequence S*L(R/K)X(R/K)
(SEQ ID NO: 40).
[0231] This search found a fourth candidate phosphopeptide in an
MS/MS spectrum: BRB1_HUMAN, residues 206-233 with phosphorylation
at Ser-228. The sequence contains S*LRTR. This peptide has a mass
of 3,297 and corresponds to a peak observed during MALDI-TOF mass
spectrometry (assigned mass 3,294 in FIG. 21). It is a good example
of a marginal phosphopeptide assignment: it is the
fourteenth-ranked peptide after the initial round of Sequest
scoring, and the eighth-ranked peptide after the final scoring
round. Although there are higher-ranked peptides after the final
round, they all received very poor scores in the initial round,
where they were ranked ninety-fourth or worse. Nevertheless, this
result is worth pursuing because the assigned peptide fits the
antibody's known specificity and it fits well the higher-confidence
assignments made on the basis of MS.sup.3 spectra.
[0232] BRB1 is the B1 bradykinin receptor. It is known that the B1
bradykinin receptor activates protein kinase C (see Christopher et
al. Hypertension 38: 602-605 (2001)). There are no known
phosphorylation sites in the B1 bradykinin receptor, but the B2
bradykinin receptor is phosphorylated at Ser residues in response
to activation of protein kinase C (see Blaukat et al. J. Biol.
Chem. 276: 40431-40 (2001)). Furthermore, protein kinase C
phosphorylation of receptors has been postulated as a general
mechanism for receptor desensitization. It is therefore reasonable
to presume that protein kinase C could phosphorylate B1 receptors
as well. In addition, it is known that the expression of the BRB1
receptor is upregulated on T cells derived from peripheral blood of
patients with multiple sclerosis, relevant because this
phosphopeptide was purified from a human cell line derived from an
acute T cell leukemia (see e.g., Prat et al. Neurology 53:2087-2092
(1999)). The site of phosphorylation tentatively assigned here is
in a domain of the protein that is predicted to be cytoplasmic.
This tentative assignment may be further explored by analyzing a
synthetic peptide with the assigned sequence and phosphorylation
site as described above.
[0233] The ability to filter assignments and extract marginal
assignments that are worth investigating further is a unique
advantage of antibody-based purification methods. Without use of an
antibody and knowledge of the antibody's specificity, these
marginal assignments would be overlooked.
Example VI
Isolation of Peptides Containing the Akt Substrate Motif from an
Extract of Cells Expressing Activated Akt Protein Kinase
[0234] Peptides containing the Akt substrate motif (RXRXXT*/S*,
T*=phosphothreonine, S*=phosphoserine) can be selectively isolated
from a complex mixture of peptides, such as a digested cell lysate.
The Akt protein kinase is an important regulator of cell survival
and insulin signaling, but very few of its in vivo targets have
been identified. Studies with synthetic peptide substrates of Akt
(Alessi et al., FEBS Lett. 399: 333-338 (1996)) as well as the
analysis of known Akt phosphorylation sites on GSK-3 (Franke et al.
Cell 88: 435-437 (1997)), Bad (Pap et al., J. Biol. Chem. 273:
19929-19932 (1998), and Caspase-9 (Cardone et al., Science 282:
1318-1321 (1998)) indicate that Akt phosphorylates its substrates
only at a serine or threonine in a conserved motif characterized by
arginine at positions -5 and -3.
[0235] Phospho-(Ser/Thr) Akt substrate polyclonal antibody (Cell
Signaling Technology, Inc., product number 9611) is a
motif-specific, context-independent antibody that recognizes
phosphopeptides with the consensus substrate motif RXRXX(T*/S*),
where R is arginine, X is any amino acid, and T*/S* indicates
phosphothreonine or phosphoserine. The specificity of the
phospho-(Ser/Thr) Akt substrate antibody is that it binds
preferentially to proteins and peptides that contain
phosphothreonine or phosphoserine preceded by lysine or arginine at
positions -5 and -3, i.e., (K/R)X(K/R)XX(T*/S*) (SEQ ID NO: 15), in
a manner substantially independent of the surrounding amino acid
sequence. To identify potential substrates on a genome-wide
(cell-wide) basis, immobilized phospho-Akt substrate antibody was
used to immunoaffinity purify phosphopeptides from a proteinaceous
preparation in accordance with the method of the invention, as
described below.
[0236] For this example, the model system was 3T3 mouse fibroblast
cells that had been stably transfected to express active Akt
protein kinase constituitively and that had been treated with 50
ng/ml platelet-derived growth factor (PDGF) for 15 minutes. The
cells were washed, harvested, and lysed by sonication, proteins in
the lysate were denatured, and the lysate was cleared by
centrifugation.
[0237] To show that activated Akt protein kinase had phosphorylated
many target proteins, the cell extract was analyzed by SDS-PAGE and
Western blotting (FIG. 24). Activation of Akt protein kinase was
shown by probing a blot of PDGF-treated, transfected cell extract
(lane 2) and untreated, untransfected cell extract (lane 1) with
Akt antibody (Cell Signaling Technology, Inc., product number
9272), phospho-Akt (Thr308) antibody (Cell Signaling Technology,
Inc., product number 9275), and phospho-Akt (Ser473) antibody (Cell
Signaling Technology, Inc., product number 9271). This showed that
PDGF treatment altered the phosphorylation state of Akt protein
kinase (panels 2 and 3) but not its overall cellular expression
level (panel 1). PGDF treatment also altered the phosphorylation
state of a large number of different proteins that contain the
phospho-Akt substrate motif, shown by probing the blot with
phospho-Akt substrate antibody (panel 4). In a separate experiment,
it was shown that the major protein recognized by phospho-Akt
substrate antibody after PDGF treatment (the dark band near the
bottom of panel 4, lane 2) is the ribosomal protein S6, which is
known to be phosphorylated in response to growth factor treatment
(Ferrari and Thomas, Crit. Rev. Biochem. Mol. Biol. 29: 385-413
(1994)).
[0238] Proteins in the extract were digested to peptides with
endoproteinase Glu-C immobilized to F7m, a polyvinyl matrix bead
(MoBiTec, part number P5101), and the immobilized Glu-C was removed
by centrifugation. Phosphopeptides containing the phospho-Akt
substrate motif were purified from the digest with phospho-Akt
substrate antibody immobilized to agarose by hydrazide chemistry,
as described above for the P-Tyr-100 monoclonal antibody; each
milliliter of resin was reacted with 2 milligrams of antibody. The
Glu-C-digested crude cell extract (about 3.5 mg, 0.25 mg/ml
protein) was contacted with immobilized phospho-Akt substrate
antibody-resin (40 .mu.l, 2 .mu.g/.mu.l) in batch format at
4.degree. C. for 16 hours, and unbound peptides were removed by
centrifugation. The antibody-resin was washed extensively (four
times with 0.5 ml ice-cold PBS and three times with 0.5 ml ice-cold
water). Bound peptides were then eluted with 120 .mu.l 10.1 M
glycine, pH 2.3. Before analysis by MALDI-TOF mass spectrometry as
described above, a 9 .mu.l portion of the eluted fraction was
desalted and concentrated with a reversed-phase ZipTip
microcolumn.
MALDI-TOF Analysis
[0239] The masses of the peptides that bound to and eluted from the
phospho-Akt substrate antibody were measured by MALDI-TOF mass
spectrometry before (FIG. 25, top panel) and after (bottom panel)
treating the peptide fraction with calf intestinal phosphatase,
which can remove phosphate groups from phosphopeptides and produce
ions with masses 80 lower than phosphopeptides for each phosphate
group in the peptide, to confirm the eluted peptides are
phosphorylated. In FIG. 25, peaks labeled with a star correspond to
phosphopeptides, peaks labeled with an open circle correspond to
nonphosphorylated peptides, and peaks labeled with a square are
phosphopeptides that have undergone metastable decomposition and
neutral-loss of phosphate.
[0240] Four candidate phosphopeptide peaks (FIG. 25, top panel) are
each accompanied by companion peaks that are broader and apparently
84 lower in mass, e.g., the peak with a mass of 2,404 has a partner
peak at 2,320, and likewise 2,334, 2,324, and 2,254 have partner
peaks at 2,250, 2,241, and 2,171, respectively. These companion
peaks correspond to metastable decomposition products of
phosphopeptides, formed by neutral-loss of phosphate while the
phosphopeptide ions are traveling to the detector of the mass
spectrometer. Metastable decomposition of phosphopeptides has been
noted by others and can be used to recognize and assign
phosphopeptides in a MALDI-TOF mass spectrum (Annan and Carr, Anal.
Chem. 68: 3413-21 (1996)). As described in the previous example, it
has been observed that some synthetic peptides containing
phosphoserine or phosphothreonine but not phosphotyrosine residues
undergo metastable decomposition. The MALDI-TOF mass spectrum in
FIG. 25 indicates these candidate phosphopeptides probably contain
phosphoserine or phosphothreonine, in accordance with the
antibody's known specificity.
[0241] When the bound peptide fraction was treated with calf
intestinal phosphatase, 3 of the 4 candidate phosphopeptides gave
dephosphorylated peptides that were 80 (for one phosphate group) or
160 (for two phosphate groups) lower in mass than the peptides
before treatment (FIG. 25, bottom panel): 2,404 and 2,324 differ
from 2,244 by two or one phosphate groups, respectively, and 2,334
differs from 2,254 by one phosphate group. The presence of
metastable decomposition peaks after phosphatase treatment
indicates these peptides are still phosphorylated, i.e., the
phosphopeptide with a mass of 2,404 probably contains at least
three phosphate groups, and the peptide with a mass of 2,334
probably contains at least two phosphate groups. This is supported
by the LC-MS/MS analysis described below, which defined the
phosphate content of these peptides more precisely.
[0242] It is believed that two of the four immunoaffinity-purified
peptides (FIG. 25, top panel) correspond to a known phosphopeptide
from the ribosomal protein S6 (accession number P10660), which is
the major protein in the PDGF-treated cell extract recognized by
Western blotting with phospho-Akt substrate antibody (FIG. 24): the
phosphopeptides with an observed mass of 2,254.5 and 2,334.4 fit
the expected mass for the Glu-C-peptide from S6 protein QIAKR RRLSS
LRAST SKSE (SEQ ID NO: 41) with 1 and 2 phosphate groups,
respectively (calculated protonated peptide masses of 2,254.2 and
2,334.2). Based on the duration of PDGF treatment in this
experiment and published reports on the order of phosphorylation
(Ferrari et al. J. Biol. Chem. 266: 22770-5 (1991)), it is expected
that only 2 of the 5 phosphorylation sites in this peptide are
phosphorylated, Ser235 and Ser236, underlined in the peptide
sequence shown above. Furthermore these two sites in the peptide
fit the known specificity of the phospho-Akt substrate antibody:
Ser235 fits KXRXXS*, and Ser236 fits RXRXXS*.
LC-MS/MS Analysis
[0243] The peptides that bound to and eluted from the phospho-Akt
substrate antibody were further analyzed by LC-MS/MS. A 25 .mu.l
portion of the peptide fraction was desalted and concentrated with
a reversed-phase ZipTip microcolumn and eluted with 2 .mu.l 0.1%
trifluoroacetic acid, 40% acetonitrile. An 0.4 .mu.l aliquot of the
eluted fraction was mixed with an ACHA matrix solution and analyzed
by MALDI-TOF mass spectrometry, and it gave a spectrum similar to
the one shown in FIG. 25. The remainder of the eluted fraction was
analyzed by LC-MS/MS.
[0244] LC-MS/MS analysis was performed as described above (Example
IV). The chromatogram obtained by analyzing this sample is shown in
FIG. 26. The first panel of FIG. 26 shows where survey MS scans
were collected, and the second panel shows where MS/MS spectra were
collected. The third, fourth, and fifth panels show where neutral
loss of 49, 32.7, and 24.5, respectively, was detected,
characteristic of ions with charges of +2, +3, and +4 that have
undergone neutral loss of phosphate. The occurrence and intensities
of neutral-loss ions are plotted in the third, fourth, and fifth
panels of FIG. 26 to help locate candidate phosphopeptides. The
neutral loss plots show that phosphopeptide candidates tend to
elute early in the chromatogram, as expected for phosphopeptides
due to the hydrophilicity of phosphate groups, and that neutral
loss is observed in many of the MS/MS spectra, suggesting this
sample is highly enriched with phosphopeptides.
[0245] As noted earlier, neutral loss during MS/MS is the same
process as metastable decomposition during MALDI-TOF mass
spectrometry. As expected, many of the phosphopeptides showing
neutral loss during LC-MS/MS (FIG. 26, panels 3-5) are the same
phosphopeptides that gave metastable decomposition during MALDI-TOF
mass spectrometry (FIG. 25, top panel), see FIG. 27. Most peptides
showing neutral loss during MS/MS contained more than one phosphate
group. The MS/MS spectra often gave a clear indication of the
number of phosphate groups present in each peptide ion, which
MALDI-TOF mass analysis did not provide even after treating the
peptides with phosphatase.
[0246] The phosphopeptide from the ribosomal protein S6 was
observed with one, two, and three phosphates, in good agreement
with the MALDI-TOF results described above. FIG. 28 shows a portion
of the MS/MS spectra for the S6 phosphopeptide with 1 (panel 1), 2
(panel 2), or 3 (panel 3) phosphates, the parent ions for all three
spectra have a charge of +3. Panel 1 shows the spectrum of a parent
ion with an m/z value of 752.51 undergoing neutral loss to give a
product ion with an m/z value of 719.86, a difference of 32.65,
close to the theoretical m/z difference of 32.66 for loss of 98
from an ion with a charge of +3. Only one loss of this m/z value is
detected, that is, a product ion corresponding to loss of two
phosphate groups (m/z of 687.20) is not detected. On this basis the
mass of the peptide is 2,254.5, and it contains 1 phosphate group.
Similarly panel 2 shows product ions that have lost this m/z value
once and twice, allowing assignment of two phosphate groups, and
panel 3 shows loss one, two, and three times, corresponding to 3
phosphate groups. In agreement with this the parent ion masses
increase by 80 for each additional phosphate group. As expected,
the phosphopeptides eluted earlier during reversed-phase HPLC as
the phosphate content increased: with 3, 2, or 1 phosphate groups
the peptide eluted at 7.3, 7.6, and 7.8 minutes respectively.
[0247] Like this group of ribosomal protein S6 phosphopeptides,
LC-MS/MS analysis showed there may be a second group of related
phosphopeptides, labeled "peptide A" in FIG. 27. The observed
masses are 2,324.5 (with two phosphate groups) and 2,404.6 (with
three phosphate groups).
[0248] All the MS/MS product ion spectra were analyzed with Sequest
in an attempt to assign a parent protein and phosphorylation site
to each peptide. This did not result in unambiguous assignments
because of the high level of neutral loss with very little residual
fragmentation along the peptide backbone. At present, this type of
multiply-phosphorylated sample cannot be analyzed effectively by
LC-MS.sup.3 using the currently available version of the software.
The current data-dependent acquisition software isolates and
fragments the most abundant neutral-loss ion; for multiply
phosphorylated peptides this corresponds to the peptide with one
phosphate removed by neutral loss, leaving one or more phosphate
groups to undergo neutral loss during MS.sup.3. The acquisition
software is being revised (per personal communication) to recognize
multiples of neutral loss and to isolate and fragment the ion with
the highest level of neutral loss, even if it is not the most
intense product ion. For example for the spectrum in panel 2 of
FIG. 28, the current software would select for MS.sup.3 analysis
the ion with an m/z value of 746.33 because it is the most intense
neutral-loss ion; but the revised software will select the ion with
an m/z value of 713.87 because it shows a higher level of neutral
loss. It is expected that further analysis of this sample with
revised acquisition software will allow the parent proteins and
phosphorylation sites of some of these peptides to be unambiguously
assigned.
[0249] Even without unambiguous fragmentation spectra, the
tentative assignment of some of these peptides to a phosphopeptide
from the ribosomal protein S6 is consistent with Western blotting
results, which suggest S6 is the major phosphoprotein detected by
phospho-Akt substrate antibody, and the observed masses and
phosphate contents agree with published reports on phosphorylation
of this protein after treatment with growth factors.
Example VII
Isolation of Peptides Containing the 14-3-3 Binding Motif from an
Extract of Cells Treated with a Cyclic AMP Analog and Insulin
[0250] The method of the invention was further employed to isolate
phosphopeptides containing a 14-3-3 binding motif from a complex
mixture of peptides existing in a digested cell lysate. The 14-3-3
proteins regulate several biological processes through
phosphorylation-dependent protein-protein interactions. A
phosphoserine-containing consensus sequence, motif #1, (R/K)SXS*XP,
is present in some binding partners of 14-3-3 proteins. Many
protein kinases such as Akt and cAMP-dependent protein kinase (PKA)
can phosphorylate this motif to initiate binding of 14-3-3
proteins.
[0251] Phospho-(Ser) 14-3-3 binding motif monoclonal antibody (4E2)
(Cell Signaling Technology, Inc., product number 9606) is a
motif-specific, context-independent antibody that recognizes
phosphopeptides containing consensus binding motif #1. This
antibody is highly specific for peptides and proteins that contain
the consensus motif (R/K)XXS*XP, where R is arginine, P is proline,
X is any amino acid, and S* indicates phosphoserine. This antibody
weakly cross-reacts with analogous sequences containing
phosphothreonine instead of phosphoserine in this motif. This
antibody was used to immunoaffinity purify phosphopeptides that
contain motif #1 from a proteinaceous preparation, so as to
identify proteins that may be previously unrecognized binding
partners of 14-3-3 proteins.
[0252] For this example, the model system was COS-1 cells, a cell
line derived from transformed monkey kidney cells, that had been
treated with insulin and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP).
Insulin induces the Akt protein kinase, and the membrane-permeable,
metabolically stable cAMP analog induces the PKA kinase. The
induced kinases will phosphorylate many protein sites, and among
these many will be 14-3-3 binding sites, that is, some proteins
will become binding partners of 14-3-3 proteins as a result of
phosphorylation by the Akt, PKA, and other induced protein kinases.
A culture of COS-1 cells was treated with 1 .mu.g/ml insulin and 1
mM 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) for 10 minutes. The cells
were washed, harvested, and lysed by sonication, proteins in the
lysate were denatured, and the lysate was cleared by
centrifugation.
[0253] To show that treatment with insulin and the cyclic AMP
analog had caused an increased level of protein phosphorylation at
potential 14-3-3 binding sites, the cell extract was analyzed by
SDS-PAGE and Western blotting (FIG. 29). Probing the treated cell
extract (lane 2) and the untreated cell extract (lane 1) with
phospho-(Ser) 14-3-3 binding motif antibody (Cell Signaling
Technology, Inc., product number 9606) showed that this treatment
altered the phosphorylation state of a significant number of
different proteins that contain the 14-3-3 binding motif #1.
[0254] Proteins in the extract were digested to peptides with
endoproteinase Glu-C immobilized to F7m, a polyvinyl matrix bead
(MoBiTec, part number P5101), and the immobilized Glu-C was removed
by centrifugation. Immobilized phospho-(Ser) 14-3-3 binding motif
monoclonal antibody (4E2) was prepared as described in Example IIB
and was found to contain 4 mg antibody per ml of resin.
[0255] Phosphopeptides containing the 14-3-3 binding motif were
purified from the Glu-C-digested crude cell extract with
phospho-(Ser) 14-3-3 binding motif monoclonal antibody bound
protein G-agarose resin. The digest (about 12 mg, 0.5 .mu.g/.mu.l
protein) was contacted with immobilized antibody-resin (40 .mu.l, 4
.mu.g/.mu.l) in batch format at 4.degree. C. for 16 hours, and
unbound peptides were removed by centrifugation. The antibody-resin
was washed extensively (three times with 1 ml ice-cold PBS and two
times with 1 ml ice-cold water). Bound peptides were then eluted
with 150 .mu.l 0.1% trifluoroacetic acid, and the eluted peptides
were separated from eluted antibody by centrifugation through a
Microcon YM-10 membrane (Millipore, product number 42407), which
retains molecules with molecular weights above 10,000. Before
analysis by MALDI-TOF mass spectrometry, a 9 .mu.l portion of the
YM-10 flow-through fraction was desalted and concentrated with a
reversed-phase ZipTip microcolumn.
MALDI-TOF Analysis
[0256] The masses of the peptides that bound to and eluted from the
phospho-(Ser) 14-3-3 binding motif antibody were measured by
MALDI-TOF mass spectrometry (FIG. 30). In FIG. 30, peaks labeled
with a star correspond to phosphopeptides, and peaks labeled with a
square are phosphopeptides that have undergone metastable
decomposition and neutral-loss of phosphate.
[0257] Metastable decomposition showed that the antibody-purified
peptide fraction contains several candidate phosphopeptides with
phosphoserine or phosphothreonine, as expected based on the
antibody's specificity. Metastable decomposition arises when
phosphopeptide ions undergo neutral-loss of phosphate while
traveling toward the instrument's detector and is indicated in
MALDI-TOF spectra by the presence of broad companion peaks about 84
lower in mass than intact phosphopeptide ions. As noted in Examples
V and VI, experience with synthetic peptides indicates metastable
decomposition is a specific and reliable indicator of peptides that
contain phosphoserine or phosphothreonine, so the appearance of
metastable decomposition in this spectrum fits the known
specificity of the antibody used for purification.
[0258] A comparison of the MALDI-TOF mass spectrum for this sample
(FIG. 30) and the Akt substrate sample described in Example VI
(FIG. 19) shows there may be some overlap between the two sample
sets. This is expected because, for both samples, the Akt protein
kinase was induced and for both samples the specificities of the
antibodies used for phosphopeptide purification overlap
[(R/K)XX(S*)XP for the phospho-(Ser) 14-3-3 binding motif
monoclonal antibody 4E2 versus (R/K)X(R/K)XX(S*/T*) for the
phospho-(Ser/Thr) Akt substrate motif polyclonal antibody]. Two
peptides that the sample sets may have in common correspond to the
Glu-C peptide from the ribosomal protein S6, QIAKR RRLSS LRAST SKSE
(SEQ ID NO: 41) with 1 phosphate group and with 2 phosphate groups
(see FIG. 27). Ser235 and Ser236, underlined in the peptide
sequence shown above, fit the Akt substrate motif fully and the
14-3-3 binding site motif partially.
LC-MS/MS Analysis
[0259] The peptides that bound to and eluted from the phospho-(Ser)
14-3-3 binding motif antibody were further analyzed by LC-MS/MS. A
20 .mu.l portion of the peptide fraction was desalted and
concentrated with a reversed-phase ZipTip microcolumn and eluted
with 2 .mu.l 0.1% trifluoroacetic acid, 40% acetonitrile. An 0.4
.mu.l aliquot of the eluted fraction was mixed with an ACHA matrix
solution and analyzed by MALDI-TOF mass spectrometry, and it gave a
spectrum similar to the one shown in FIG. 30. The remainder of the
eluted fraction was analyzed by LC-MS/MS.
[0260] LC-MS/MS analysis was performed as described above (Example
IV). The chromatogram obtained by analyzing this sample is shown in
FIG. 31. The first panel of FIG. 31 shows where survey MS scans
were collected, the second panel shows where MS/MS spectra were
collected, and the third, fourth, and fifth panels show where
neutral loss of 49, 32.7, and 24.5, respectively, was detected,
characteristic of ions with charges of +2, +3, and +4 that have
undergone neutral loss of phosphate. The occurrence and intensities
of neutral-loss ions are plotted in the third, fourth, and fifth
panels of FIG. 31 to help locate candidate phosphopeptides. The
neutral loss plots show that phosphopeptide candidates tend to
elute early in the chromatogram, as expected for phosphopeptides
due to the hydrophilicity of phosphate groups, and that neutral
loss is observed in many of the MS/MS spectra, suggesting this
sample is highly enriched with phosphopeptides.
[0261] Many of the phosphopeptides showing neutral loss during
LC-MS/MS (FIG. 31, panels 3-5) are the same phosphopeptides that
gave metastable decomposition during MALDI-TOF mass spectrometry
(FIG. 30), see FIG. 32. Like the Akt substrate sample described in
Example VI, most peptides showing neutral loss during MS/MS
contained more than one phosphate group. The LC-MS/MS analysis
results support the interpretation made on the basis of the
MALDI-TOF mass spectrum, that there is likely to be considerable
overlap between the set of peptides purified with the Akt substrate
antibody and the 14-3-3 binding motif antibody, including the
tentatively assigned multiply phosphorylated peptides from
ribosomal protein S6.
[0262] Another indication of overlap between the two sample sets is
provided by residual backbone fragmentation observed in some of the
MS/MS spectra, see FIG. 33. The left panels are MS/MS spectra from
the Akt substrate antibody sample set, and the right panels are the
corresponding spectra from the 14-3-3 binding motif antibody sample
set. The top panels are both assigned tentatively to an ion with a
charge of +4 corresponding to the S6 peptide with two phosphate
groups: in addition to neutral loss, both spectra show a product
ion with an m/z value of 668.2, possibly the b17-H.sub.3PO.sub.4
product ion with a charge of +3 (calculated m/z of 668.04). The
bottom panels are both assigned to an ion with a charge of +4 that
has a mass of 3,204 and contains two phosphate groups: both spectra
show a product ion with an m/z value of 990.7.
[0263] All the MS/MS product ion spectra were analyzed with Sequest
in an attempt to assign a phosphorylation site and parent protein
to each peptide. In many cases here (as also noted in Examples V
and VI) this did not result in unambiguous assignments because of
the high level of neutral loss with very little residual
fragmentation along the peptide backbone. MS/MS showed that many of
the most abundant phosphopeptides are multiply-phosphorylated and
will be amenable to MS.sup.3 analysis after the current data
acquisition software is revised to recognize multiples of neutral
loss and to isolate and fragment the ion with the highest level of
neutral loss (as noted in Example VI).
[0264] One peptide in this sample that was unambiguously identified
is a phosphoserine-containing peptide from heat shock 27 kDa
protein (FIG. 34). The residue identified as a phosphorylation site
by this method, Ser-78, is known to be phosphorylated by several
protein kinases, including S6 kinases and mitogen-activated protein
kinases, which are likely to have been activated by the treatments
used to prepare this sample. See e.g., Landry et al. J. Biol. Chem.
267: 794-803 (1992) and Bird et al. FEBS Lett. 338: 31-36 (1994).
This phosphopeptide corresponds to a prominent peak detected during
MALDI-TOF mass spectrometry, labeled "2,384.6" in FIG. 30. The
MS/MS spectrum for the peptide as an ion with a charge of +2 shows
a prominent neutral-loss product ion, consistent with loss of one
phosphate group (see FIG. 32). The spectrum that could be assigned
a parent protein and phosphorylation site by Sequest was produced
from the same peptide as an ion with a charge of +3. As expected,
the sequence context of Ser-78 (RALS*RQ) fits the known specificity
of the phospho-(Ser) 14-3-3 binding motif antibody used to purify
the phosphopeptide [underlined residues match the specificity motif
(R/K)XXS*XP].
Example VIII
Isolation of Peptides Containing the Phospho-PDK1 Docking Motif
from a Crude Cell Extract
[0265] Peptides containing the phospho-PDK1 docking motif
(FXXF(S*/T*)(F/Y) (SEQ ID NO: 42), where F is phenylalanine, X is
any amino acid, S*/T* indicates phosphoserine or phosphothreonine,
and F/Y indicates phenylalanine or tyrosine) may be selectively
isolated from a complex mixture of peptides, such as a digested
cell lysate. Many protein kinases contain this docking motif
sequence, and phosphorylation of this sequence is required for
these kinases to bind to 3-phosphoinositide-dependent kinase 1
(PDK1). PDK1 plays a central role in the activation of several
growth factor-induced protein kinases, including protein kinase B
(PKB), p70 S6 kinase, several PKC isotypes, and serum and
glucocorticoid-induced kinase (SGK). See, e.g. Belham et al., Curr.
Biol. 11: R93-R96 (1999).
[0266] The phospho-PDK1 docking motif 18A2 (bulky rings) monoclonal
antibody (Cell Signaling Technology, Inc., product number 9634) is
a motif-specific, context-independent antibody that recognizes
phosphopeptides with the consensus sequence FXXF(S*/T*)(F/Y), where
F is phenylalanine, X is any amino acid, S*/T* indicates
phosphoserine or phosphothreonine, and F/V indicates phenylalanine
or tyrosine. To identify other proteins with this PDK1 docking
motif or profile the activation states of known PDK1 substrates on
a cell-wide basis, immobilized PDK1 docking motif (bulky rings)
antibody may be employed to immunoaffinity purify phosphopeptides
containing the motif from a complex mixture of peptides, such as a
digested cell lysate. For example, a proteinaceous preparation may
be obtained from a COS cell line (monkey) that overexpresses Akt
protein, from 3T3 cells (mouse) treated with platelet derived
growth factor, or from Jurkat (human) cells. The extract is
prepared and proteins denatured as described above (see
"Proteinaceous preparations"), then digested with immobilized
trypsin or other proteases.
[0267] Phosphopeptides containing the PDK1 docking motif are
isolated from the complex mixture in the digested cell lysate with
the bulky rings monoclonal antibody (PDK1 docking motif)
immobilized to agarose resin by hydrazide chemistry, as described
above for P-Tyr-100 monoclonal and P-Thr polyclonal antibodies. The
digest is contacted with the antibody-resin in batch format at
4.degree. C. for 1 to 16 hours. Unbound peptides are then removed
by centrifugation, and the antibody-resin is extensively washed
before eluting bound peptides with 0.1 M glycine, pH 2.3. The
eluted peptides are concentrated and desalted with reversed-phase
ZipTip microcolumns. The masses of the eluted peptides are measured
before and after treating aliquots of the eluted peptides with
phosphatase, which can remove phosphate groups from the
phosphopeptides and reduce the phosphopeptide masses by 80 for each
phosphate present in the peptide. The mixture of phosphopeptides is
then analyzed by MS/MS, as described above, to obtain partial
peptide sequence information to facilitate identifying the parent
proteins from which each phosphopeptide originated. It is expected
that each phosphopeptide sequence will fit the PDK1 docking motif
consensus sequence described above.
Example IX
Isolation of Acetyl-lysine-Containing Peptides from a Crude Cell
Extract
[0268] Peptides containing acetylated residues, e.g.
acetylated-lysine, may be selectively isolated from a complex
mixture of peptides, such as a digested cell lysate, according to
the method of the invention. It is known that acetylation regulates
chromatin structure and gene activity through modification of
histones and transcription factors, and thus specific isolation of
acetylated peptides would provide important information on the
activation states of these biologically important proteins.
[0269] Acetylated-lysine monoclonal antibody (Cell Signaling
Technology, Inc., product number 9681) specifically recognizes
proteins that have been post-translationally modified by
acetylation at lysine epsilon-amino groups. To identify other sites
of acetylation, immobilized acetylated-lysine antibody may be used
to immunoaffinity purify modified (i.e. acetylated) peptides from a
proteinaceous preparation, according to the method of the
invention. For example, a digested cell lysate containing a complex
mixture of peptides may be prepared from a COS cell line (monkey)
that overexpresses the HIV Nef protein, which is acetylated at
lysine-4. The proteinaceous preparation is prepared and proteins
denatured as described above (see "Proteinaceous preparations"),
and digested with immobilized trypsin or other suitable immobilized
proteases that can be removed from the digest by
centrifugation.
[0270] Acetylated peptides may then be isolated from the digested
cell lysate with the acetyl-lysine specific antibody (a general
modification-specific antibody) linked to agarose resin using a
hydrazide chemistry, as was described above for the P-Tyr-100
monoclonal antibody and the P-Thr-polyclonal antibody. To isolate
acetylated peptides, the digested crude extract may then be
contacted with the immobilized acetylated-lysine monoclonal
antibody at 4.degree. C. overnight. The resin may then be recovered
by centrifugation and extensively washed as described above. The
bound peptides may then be eluted by treating the antibody-resin
with an eluting solvent such as 0.1% trifluoroacetic acid and
centrifugation through a plastic frit.
[0271] For this cell line, the overexpressed HIV Nef protein is the
most prominent acetylated protein in the cell, and it is expected
that acetylated HIV Nef peptides will be specifically isolated,
along with other acetylated peptides, according to the method of
the invention. These peptides and other acetylated peptides may be
further analyzed by MS/MS to obtain partial sequences that can be
used to identify the parent proteins. For the HIV Nef protein, for
example, a partial sequence will help confirm the peptide isolated
from the crude extract is indeed from the HIV Nef protein. Analysis
of other acetylated peptides purified by this method may identify
new, previously unknown acetylation sites, and in these cases the
partial sequence analysis is necessary to match each acetylated
peptide with its parent protein. Generally, a peptide's mass and a
partial sequence of that peptide is sufficient to identify the
parent protein for that peptide, as long as the parent protein's
sequence is stored in a public protein sequence database. See Mann
et al., Anal. Chem. 66: 4390-4399 (1994).
Example X
Profiling of Activated Pathways in Tumor Tissue by Isolation of
Modified Peptides from a Crude Tissue Extract
[0272] Activation status of important biological signaling pathways
in diseased tissue may be profiled by selective isolation of
modified peptides in accordance with the method of the invention.
Activation of specific cellular signaling pathways depends, for
example, upon the phosphorylation of specific proteins. Therefore,
protein phosphorylation states in target cell, e.g. tumor cells,
may be used to profile pathway activation by preparing cell
extracts from biopsy samples of tumor tissues from which modified
peptides may be selectively isolated.
[0273] Profiling of protein phosphorylation states in tumor cell,
e.g. a breast tumor cell, may be carried out by obtaining a
proteinaceous preparation, which contains a complex mixture of
peptides, from the target tumor cell. A proteinaceous preparation
may be obtained from a single needle biopsy from a breast tumor,
which provides sufficient cellular extract to profile the
activation status of multiple signaling pathways, including, e.g.,
the MAP kinase pathway, various growth factor receptor pathways,
including epidermal growth factor receptor, steroid receptors, such
as the estrogen receptor, and the PI-3-kinase Akt pathway. All of
these pathways have been shown to be involved in breast cancer and
are important targets for current and future drug development and
patient therapy.
[0274] To evaluate changes in the signaling pathways of specific
breast cancer biopsies, a proteinaceous preparation is obtained
from the biopsy sample and desired modified peptides, e.g.
phosphopeptides, from that fraction are immunoaffinity purified and
characterized by MS as described above. A protein fraction is
obtained from frozen biopsy tissue by sonication, and insoluble
material and cytoskeletal proteins are removed by centrifugation.
The supernatant fraction, containing the bulk of the cellular
proteins, is then denatured by heat treatment and digested with
immobilized trypsin or some other specific proteolytic enzyme. This
proteinaceous preparation contains modified phosphopeptides from
multiple different proteins. The proteinaceous preparation is
contacted with an immobilized general phospho-specific antibody,
e.g. a phosphotyrosine-specific antibody, to isolate
phosphopeptides from the complex mixture in the proteinaceous
preparation by immunoaffinity isolation. A single type of
antibody-resin or several types of antibody-resin in series may be
employed; e.g., the protein fraction is contacted with an
immobilized phosphotyrosine-specific antibody (e.g. in a column, as
previously described), and the unbound fraction from that step is
then treated with an immobilized Akt substrate motif-specific
antibody in a second support, etc. The immobilized antibody-resins
are washed extensively to remove unbound (e.g. nonphosphorylated)
peptides, and the bound peptide fraction is then recovered by
treating the antibody-resin with an eluting solvent such as 0.1%
trifluoroacetic acid.
[0275] The eluted phosphopeptides are then analyzed by MALDI-TOF
MS, and phosphorylation is confirmed by measuring the peptide mass
again after treating an aliquot of the bound fraction with
phosphatase, which should reduce each peptide mass by 80 for each
phosphate group. To assign the modified peptides to their parent
proteins, the bound peptide fraction is analyzed by MS/MS. The
partial sequence information obtained, along with the peptide mass,
is sufficient to unambiguously identify the parent protein of each
peptide. See Mann et al. (1994), supra. Ideally, this procedure is
performed with tumor and normal cell biopsies from the same
patient. However, if certain phosphorylation sites are known to be
diagnostic markers for a specific cancer, then the method can be
used to assay the presence of those markers only, without a normal
cell reference.
[0276] The amounts of phosphorylated peptides isolated from the
target cells from tumor tissues are compared to levels observed in
extracts from reference cells from normal tissues. Alterations in
phosphorylation of a given peptide (and thus, its parent protein),
when compared to the reference cell phosphorylation state, will
indicate activation of the corresponding signaling pathway.
Information obtained from this profiling may be used to determine
the best therapy for the patient, as well as to monitor the
specific effects of the therapy, e.g. drug treatment, on the
targeted signaling pathways. Profiling of phosphorylation states in
a target diseased cell, such as a breast tumor cell, also provides
information useful in drug development, e.g. to assess the effect
of a test drug, as well as for cancer research to identify which
signaling proteins and pathways are involved in specific cancers.
Other post-translational modifications of proteins that may be
relevant to disease states, such as cancer, may similarly be
examined by the methods disclosed herein.
Example XI
Purification and Identification of Phosphotyrosine Peptides from
Anaplastic Large Cell Lymphoma Cell Lines
[0277] The method of the invention was employed to identify
tyrosine phosphorylated peptides from two cancer cell lines that
express the same activated tyrosine kinase. Karpas 299 and SU-DHL-1
are derived from anaplastic large cell lymphomas (ALCL). The
majority of ALCL is characterized by the presence of the
t(2;5)(p23;q35) chromosomal translocation that causes the fusion of
the nucleophosmin and anaplastic lymphoma kinase (ALK) genes. See
Morris et al. Science 263: 1281-1284 (1994). Although the two cell
lines are derived from different patients, both express the
oncogenic fusion kinase NPM-ALK, which possesses constitutive
tyrosine kinase activity and can transform non-malignant cells.
[0278] Lysates were prepared from 2.times.10.sup.8 cells for both
cell lines and digested in 2 M urea with trypsin after treatment
with DTT and iodoacetamide to alkylate cysteine residues. Before
the immunoaffinity step, peptides were prefractionated by
reversed-phase solid phase extraction using Sep-Pak C.sub.18
columns (1 ml column volume per 2.times.10.sup.8 cells) to separate
peptides from other cellular components. The solid phase extraction
cartridges were eluted with steps of 5, 15, 25, and 40%
acetonitrile. Each lyophilized peptide fraction was redissolved in
1 ml PBS and treated with phosphotyrosine antibody (P-Tyr-100, CST
#9411) immobilized on protein G-Sepharose (Roche) (60 .mu.g
antibody, 15 .mu.l resin) overnight at 4.degree. C. Antibody-resin
was thoroughly washed, and immunoaffinity-purified peptides were
eluted with 75 .mu.l of 0.1% TFA. A portion of this fraction (40
.mu.l) was concentrated with Stage tips and analyzed by LC-MS/MS,
using a ThermoFinnigan LCQ Deca XP Plus ion trap mass spectrometer.
Peptides were eluted from a 10 cm.times.75 .mu.m reversed-phase
column with a 45-min linear gradient of acetonitrile delivered at
280 nl/min. MS/MS spectra were evaluated using the program Sequest
with the NCBI human protein database.
[0279] This revealed a total of 117 tyrosine phosphorylation sites
in SU-DHL-1 and 84 tyrosine phosphorylation sites in Karpas 299. As
expected there was large overlap (72%) between the phosphorylation
sites found in these two similar cell lines. Some phosphotyrosine
sites found in the ALCL cell lines that originate from known cell
signaling proteins are shown in Table 6.
TABLE-US-00006 TABLE 6 Phosphotyrosine peptides found in ALCL cell
lines Protein Name.dagger-dbl. K.sctn. S.sctn. Sequence Tyrosine
kinase activated p21cdc42Hs kinase .cndot. KPTpYDPVSEDQDPLSSDFK
anaplastic lymphoma kinase .cndot. HQELQAMQMELQSPEpYK anaplastic
lymphoma kinase .cndot. .cndot. TSTIMTDpYNPNpYCFAGK anaplastic
lymphoma kinase .cndot. GLGHGAFGEVpYEGQVSGMPNDPSPLQVAVK anaplastic
lymphoma kinase .cndot. .cndot. NKPTSLWNPTpYGSWFTEK anaplastic
lymphoma kinase .cndot. .cndot.
HFPCGNVNpYGYQQQGLPLEAATAPGAGHYEDTILK Janus kinase 3 .cndot. .cndot.
DLNSLISSDpYELLSDPTPGALAPR Ser/Thr kinase cdc2 .cndot. .cndot.
IGEGTpYGVVYK cdc2 .cndot. .cndot. IGEGTYGVVpYK DYRK1A .cndot.
.cndot. VYNDGYDDDNpYDYIVK * DYRK1A .cndot. .cndot. IYQpYIQSR DYRK3
.cndot. pYEVLKIIGKGSFGQVAR * ERK2 .cndot. VADPDHDHTGFLTEpYVATR *
GSK3 alpha .cndot. .cndot. GEPNVSpYICSR * HIPK 1 .cndot.
AVCSTpYLQSR * p38 alpha MAPK .cndot. .cndot. HTDDEMTGpYVATR * PRP4K
.cndot. .cndot. LCDFGSASHVADNDITPpYLVSR Adaptor CD2-associated
protein .cndot. ISTpYGLPAGGIQPHPQTK dok2 .cndot. GQEGEpYAVPFDAVAR
HGF reg. tyr. kinase subs. .cndot. VCEPCpYEQLNR insulin receptor
substrate 1 .cndot. LEpYYENEK insulin receptor substrate 1 .cndot.
VDPNGpYMMMSPSGGCSPDIGGGPSSSSSSSNAVPSGTSYGK intersectin 2 isoform 1
.cndot. REEPEALpYAAVNK Oncogene CBL2 .cndot. .cndot.
IKPSSSANAlpYSLAAR SHC .cndot. .cndot.
MAGFDGSAWDEEEEEPPDHQpYpYNDFPGK SHC .cndot. .cndot.
ELFDDPSpYVNVQNLDK SHP2 .cndot. .cndot. IQNTGDpYYDLYGGEK T
lymphocyte adaptor .cndot. SCQNLGpYTAASPQAPEAASSTGNAER T lymphocyte
adaptor .cndot. SQDPNPQpYSPIIK T lymphocyte adaptor .cndot.
GSPGEAPSNIpYVEVEDEGLPATLGHPVLR Wiskott-Aldrich syn. protein .cndot.
LIpYDFIEDQGGLEAVR .dagger-dbl.* indicates activation loop peptides.
.sctn."K" indicates peptide found in Karpas 299, "S" in SU-DHL-1.
.cndot. indicates phosphopeptides found in Karpas 299 or
SU-DHL-1.
[0280] To identify more phosphorylation sites, the same SU-DHL-1
cell extract was digested with trypsin, chymotrypsin,
endoproteinase GluC, or elastase, and tyrosine phosphorylated
peptides were purified and analyzed as described above, resulting
in the identification of 90 phosphotyrosine peptides from the
trypsin digest, 58 from chymotrypsin, 43 from endoproteinase GluC,
and 82 from elastase. A panel of proteases increased the number of
distinct tyrosine phosphorylation sites found from 88 using trypsin
alone to 197 using all four proteases. Most (35 of 54)
phosphorylation sites from the elastase digest were not found in
the tryptic digest. This small union between phosphorylation sites
from two digests shows that the use of different proteases produced
a more complete phosphorylation profile. Activation loop
phosphorylation at Tyr-1282 of ALK was found only after digestion
with chymotrypsin, as a 12-residue peptide; this residue is
predicted to be in a 5-residue tryptic peptide or a 57-residue
endoproteinase GluC peptide, both outside the range of peptide
lengths amenable to MS/MS-based identification. In addition, the
protease panel generated overlapping phosphopeptide sequences,
which confirmed phosphorylation site assignments. The ALK Tyr-1507
site was found in five peptides: three tryptic peptides, one
chymotryptic peptide, and one elastase peptide.
[0281] A total of 264 phosphopeptides representing 197 different
phosphotyrosine sites were identified in SU-DHL-1 cells. The only
tyrosine kinase showing activation loop phosphorylation in the ALCL
cell lines was ALK. Altogether nine different sites of ALK
phosphorylation, including five new sites, were observed. The four
known ALK sites included phosphotyrosine residues that allow ALK to
interact with other signaling proteins such as phospholipase
C-gamma, SHC, and IRS-1. Among human lymphomas, STAT3
phosphorylation is correlated with ALK expression, suggesting STAT3
phosphorylation may be a secondary marker for this malignancy, and
a recent study demonstrated a favorable clinical outcome for the
minority of ALCL patients who have tumors that express ALK but not
phosphorylated STAT3. These ALCL cell lines were found to contain
both STAT3 isoform 1 and STAT3 isoform 2 phosphorylated at Tyr-705,
which induces dimerization and nuclear translocation of this
transcription factor.
Example XII
Purification and Identification of Phosphotyrosine Peptides from
Pervanadate-Treated Jurkat Cells
[0282] Tyrosine phosphorylated peptides were identified from
pervanadate-treated Jurkat cells according to the method of the
invention. Jurkat cells are an established T cell line derived from
patients with acute lymphoblastic leukemia and leukemic transformed
non-Hodgkin lymphoma. Pervanadate produces a hyperphosphorylated
cell state by disabling protein tyrosine phosphatases, markedly
increasing the level of protein-associated phosphotyrosine. See
Srivastava and St-Louis, Mol. Cell. Biochem. 176: 47-51 (1997).
Proteins were extracted from 2.times.10.sup.8 cells under
denaturing conditions and digested with trypsin, and the resulting
complex peptide mixture was separated from non-peptide components,
concentrated, and partitioned into three fractions by
reversed-phase solid-phase extraction. Each fraction was then
treated with the phosphotyrosine-specific antibody P-Tyr-100
immobilized on agarose beads. After thorough washing, peptides were
eluted from the immobilized antibody with dilute acid and analyzed
by nanoflow LC-MS/MS using an ion trap mass spectrometer. MS/MS
spectra were assigned to peptide sequences using the program
Sequest, and each assignment was manually confirmed.
[0283] A total of 175 phosphorylation sites in 156 phosphotyrosine
peptides was found in this sample. Most of the purified peptides
were phosphorylated: 88 of the 100 top-scoring Sequest assignments
corresponded to phosphotyrosine peptides. Unphosphorylated peptides
were usually hydrophobic and from abundant proteins, and it is
likely they contaminated the phosphopeptide fractions because of a
non-specific affinity for agarose beads. Some of the
phosphopeptides originating from cell signaling proteins that were
found in this sample are listed in Table 7.
TABLE-US-00007 TABLE 7 Phosphotyrosine peptides found in
pervanadate-treated Jurkat cells Protein Name.dagger-dbl. TCR
Sequence Tyrosine kinase * ephrin receptor EphA4 VLEDDPEAApYTTR *
fer tyrosinekinase QEDGGVpYSSSGLK oncogene LCK Yes NLDNGGFpYISPR *
oncogene LCK Yes LIEDNEpYTAR oncogene LCK Yes SVLEDFFTATEGQpYQPQP
ZAP-70 Yes IDTLNSDGpYTPEPAR ZAP-70 Yes PMPMDTSVpYESPpYSDPEELK *
ZAP-70 Yes ALGADDSpYpYTAR Ser/Thr kinase cdc2 IGEGTpYGVVYK cdk6
ADQQpYECVAEIGEGApYGK * GSK3 alpha GEPNVSpYICSR myosin light chain
kinase 1 QEGSIEVpYEDAGSHpYLCLLK * p38 alpha MAPK Yes HTDDEMTGpYVATR
* PRP4K LCDFGSASHVADNDITPpYLVSR Adaptor abl-interactor 1
TLEPVKPPTVPNDpYMTSPAR Arrestin beta 2 GMKDDDpYDDQLC
erbb2-interacting protein SATLLpYDQPLQVFTGSSSSSDLISGTK
erbb2-interacting protein GPTSGPQSAPQ1pYGPPQYNIQpYSSSAAVK
erbb2-interacting protein AQIPEGDpYLSpYR intersectin 2 isoform 1
REEPEALpYAAVNK LAIR-1 ETDTSALAAGSSQEVTpYAQLDHWALTQR NCK adaptor
protein 1 LpYDLNMPAYVK p62dok1 Yes IAPCPSQDSLpYSDPLDSTSAQAGEGVQR
p62dok1 Yes EDPIpYDEPEGLAPVPPQGLpYDLPR p62dok1 Yes
VKEEGpYELPYNPATDDpYAVPPPR phosprot. assoc. GEM ENDpYESISDLQQGR
"phospholipase C, gamma 1" Yes IGTAEPDpYGALpYEGR "phospholipase C,
gamma 1" Yes NPGFpYVEANPMPTFK "phospholipase C, gamma 2"
DINSLpYDVSR "phospholipase C, gamma 2" RQEELNNQLFLpYDTHQNLR SHC Yes
ELFDDPSpYVNVQNLDK SHP2 interacting tm adaptor Yes
SGESVEEVPLpYGNLHpYLQTGR SHP2 interacting tm adaptor Yes
SQASGPEPELpYASVCAQTR SHP2 interacting tm adaptor Yes
ASFPDQApYANSQPAAS Wiskott-Aldrich syn. protein LIpYDFIEDQGGLEAVR
.dagger-dbl.* indicates activation loop peptides. TCR indicates
proteins with known roles is T cell receptor signaling.
[0284] About one-third (56 of 156) of the phosphotyrosine peptides
found in this sample originated from proteins with well-documented
roles in T cell receptor signaling. Among these were the tyrosine
kinases ZAP70 and Lck, which are activated by T cell receptor
stimulation. Six tyrosine phosphorylation sites were found in
ZAP70, including Tyr-493 in the activation loop, and three sites
were found in Lck, including the activation loop site Tyr-394. All
the tyrosine phosphorylation sites found in ZAP70 and Lck have been
reported previously. Phosphorylation sites in the activation loops
of two other tyrosine kinases and three serine/threonine kinases
were also found: the receptor tyrosine kinase EphA4 and the
cytoplasmic Fer tyrosine kinase, as well as activated
serine/threonine kinases p38 MAP kinase, GSK3 alpha, and PRP4. In
addition, many phosphotyrosine-binding adaptor proteins involved in
signal propagation were identified as tyrosine phosphorylated,
including p62dok1, NCK, SHC, SHP2, and the phospholipase C gamma 1
and gamma 2 isoforms.
[0285] This demonstrates the method of the invention can be used to
identify tyrosine kinases that are abnormally activated in cancer
cells as well as their substrates, including other protein kinases
and adaptor proteins.
Sequence CWU 1
1
163119PRTArtificial SequenceSynthetic Peptide 1Lys Ile Glu Lys Ile
Gly Glu Gly Thr Tyr Gly Val Val Tyr Lys Gly1 5 10 15Arg His
Lys219PRTArtificial SequenceSynthetic Peptide 2Lys Ile Glu Lys Ile
Gly Glu Gly Thr Tyr Gly Val Val Tyr Lys Gly1 5 10 15Arg His
Lys316PRTArtificial SequenceSynthetic Peptide 3Arg Leu Ile Glu Asp
Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Cys1 5 10
15416PRTArtificial SequenceSynthetic Peptide 4Arg Leu Ile Glu Asp
Asn Glu Tyr Thr Ala Arg Gln Gly Ala Lys Cys1 5 10
15512PRTArtificial SequenceSynthetic Peptide 5Leu Gln Glu Arg Arg
Lys Tyr Leu Lys His Arg Cys1 5 10612PRTArtificial SequenceSynthetic
Peptide 6Leu Gln Glu Arg Arg Lys Tyr Leu Lys His Arg Cys1 5
10714PRTArtificial SequenceSynthetic Peptide 7Arg Gln Gly Lys Asp
Tyr Val Gly Ala Ile Pro Val Asp Cys1 5 10814PRTArtificial
SequenceSynthetic Peptide 8Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile
Pro Val Asp Cys1 5 10912PRTArtificial SequenceSynthetic Peptide
9Gly Lys Asp Gly Arg Gly Tyr Val Pro Ala Thr Cys1 5
101012PRTArtificial SequenceSynthetic Peptide 10Gly Lys Asp Gly Arg
Gly Tyr Val Pro Ala Thr Cys1 5 101115PRTArtificial
SequenceSynthetic Peptide 11Asp Thr Gln Ile Lys Arg Asn Thr Phe Val
Gly Thr Pro Phe Cys1 5 10 151215PRTArtificial SequenceSynthetic
Peptide 12Asp Thr Gln Ile Lys Arg Asn Thr Phe Val Gly Thr Pro Phe
Cys1 5 10 151314PRTArtificial SequenceSynthetic Peptide 13Cys Lys
Glu Gly Leu Gly Pro Gly Asp Thr Thr Ser Thr Phe1 5
101414PRTArtificial SequenceSynthetic Peptide 14Cys Lys Glu Gly Leu
Gly Pro Gly Asp Thr Thr Ser Thr Phe1 5 10156PRTHomo
sapiensMISC_FEATURE(1)..(6)At positions 1 and 3, X = K or R; at
positions 2 and 4-5, X = any amino acid; at position 6, X =
phosphothreonine or phosphoserine 15Xaa Xaa Xaa Xaa Xaa Xaa1
51617PRTArtificial SequenceSynthetic Peptide 16Cys Ser Pro Arg Arg
Arg Ala Ala Ser Met Asp Asn Asn Ser Lys Phe1 5 10
15Ala1717PRTArtificial SequenceSynthetic Peptide 17Cys Ser Pro Arg
Arg Arg Ala Ala Ser Met Asp Asn Asn Ser Lys Phe1 5 10
15Ala1815PRTArtificial SequenceSynthetic Peptide 18Cys Leu Lys Asp
Arg Gln Gly Thr His Lys Asp Ala Glu Ile Leu1 5 10
151914PRTArtificial SequenceSynthetic Peptide 19Ser Arg Pro Arg Ser
Cys Thr Trp Pro Leu Pro Arg Glu Ile1 5 102015PRTArtificial
SequenceSynthetic Peptide 20Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu
Phe Ile Ile Gln Ala1 5 10 152115PRTArtificial SequenceSynthetic
Peptide 21Cys Leu Lys Asp Arg Gln Gly Thr His Lys Asp Ala Glu Ile
Leu1 5 10 152214PRTArtificial SequenceSynthetic Peptide 22Ser Arg
Pro Arg Ser Cys Thr Trp Pro Leu Pro Arg Glu Ile1 5
102315PRTArtificial SequenceSynthetic Peptide 23Cys Arg Ser Leu Thr
Gly Lys Pro Lys Leu Phe Ile Ile Gln Ala1 5 10 15246PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION; serine at position 4 is
phosphorylated 24Xaa Xaa Xaa Ser Xaa Pro1 52517PRTArtificial
SequenceSynthetic Peptide 25Cys Ser Pro Arg Arg Arg Ala Ala Ser Met
Asp Asn Asn Ser Lys Phe1 5 10 15Ala2617PRTArtificial
SequenceSynthetic Peptide 26Cys Ser Pro Arg Arg Arg Ala Ala Ser Met
Asp Asn Asn Ser Lys Phe1 5 10 15Ala2715PRTArtificial
SequenceSynthetic Peptide 27Phe Arg Gly Arg Ser Arg Ser Ala Pro Pro
Asn Leu Trp Ala Cys1 5 10 152814PRTArtificial SequenceSynthetic
Peptide 28Ser Arg Pro Arg Ser Cys Thr Trp Pro Leu Pro Arg Glu Ile1
5 102915PRTArtificial SequenceSynthetic Peptide 29Thr Arg Ser Arg
His Ser Ser Tyr Pro Ala Gly Thr Glu Glu Cys1 5 10
153015PRTArtificial SequenceSynthetic Peptide 30Cys Ala Glu Tyr Leu
Arg Ser Ile Ser Leu Pro Val Pro Val Leu1 5 10 153112PRTArtificial
SequenceSynthetic Peptide 31Leu Gln Glu Arg Arg Lys Tyr Leu Lys His
Arg Cys1 5 103214PRTArtificial SequenceSynthetic Peptide 32Ser Arg
Pro Arg Ser Cys Thr Trp Pro Leu Pro Arg Glu Ile1 5
103315PRTArtificial SequenceSynthetic Peptide 33Thr Arg Ser Arg His
Ser Ser Tyr Pro Ala Gly Thr Glu Glu Cys1 5 10 153415PRTArtificial
SequenceSynthetic Peptide 34Cys Ala Glu Tyr Leu Arg Ser Ile Ser Leu
Pro Val Pro Val Leu1 5 10 153514PRTArtificial SequenceSynthetic
Peptide 35Met Ser Gly Arg Pro Arg Thr Thr Ser Phe Ala Glu Ser Cys1
5 103614PRTArtificial SequenceSynthetic Peptide 36Arg Gln Gly Lys
Asp Tyr Val Gly Ala Ile Pro Val Asp Cys1 5 103714PRTArtificial
SequenceSynthetic Peptide 37Arg Gln Gly Lys Asp Tyr Val Gly Ala Ile
Pro Val Asp Cys1 5 10386PRTHomo sapiensMISC_FEATURE(1)..(6)At
positions 1, 2, and 6, X = R or K; at position 3, X= any amino
acid; at position 4, X = S or T; at position 5, X = any hydrophobic
amino acid. 38Xaa Xaa Xaa Xaa Xaa Xaa1 5395PRTHomo
sapiensMOD_RES(3)..(3)PHOSPHORYLATION serine at position 3 is
phosphorylated 39Xaa Xaa Ser Xaa Xaa1 5405PRTHomo
sapiensMOD_RES(1)..(1)PHOSPHORYLATION; serine at position 1 is
phosphorylated 40Ser Leu Xaa Xaa Xaa1 54119PRTHomo sapiens 41Gln
Ile Ala Lys Arg Arg Arg Leu Ser Ser Leu Arg Ala Ser Thr Ser1 5 10
15Lys Ser Glu426PRTHomo sapiensMOD_RES(5)..(5)PHOSPHORYLATION; at
position 5, X = phosphoserine or phosphothreonine 42Phe Xaa Xaa Phe
Xaa Xaa1 54310PRTHomo sapiensMOD_RES(7)..(7)PHOSPHORYLATION;
tyrosine at position 7 is phosphorylated 43Ile Ile Glu Asp Asn Glu
Tyr Thr Ala Arg1 5 104410PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 44Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg1 5
104516PRTHomo sapiensMOD_RES(10)..(10)PHOSPHORYLATION; tyrosine at
position 10 is phosphorylated 45Val Leu Glu Asp Asp Pro Glu Ala Thr
Tyr Thr Thr Ser Gly Gly Lys1 5 10 154613PRTHomo
sapiensMOD_RES(10)..(10)PHOSPHORYLATION; tyrosine at position 10 is
phosphorylated 46Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr
Arg1 5 104716PRTHomo sapiensMOD_RES(8)..(8)PHOSPHORYLATION;
tyrosine at position 8 is phosphorylated 47Val Tyr Ile Asp Pro Phe
Thr Tyr Glu Asp Pro Asn Glu Ala Val Arg1 5 10 154828PRTHomo
sapiensMOD_RES(12)..(12)PHOSPHORYLATION; tyrosine at position 12 is
phosphorylated 48Thr His Ala Val Ser Val Ser Glu Thr Asp Asp Tyr
Ala Glu Ile Ile1 5 10 15Asp Glu Glu Asp Thr Tyr Thr Met Pro Ser Thr
Arg 20 25499PRTHomo sapiensMOD_RES(7)..(8)PHOSPHORYLATION;
tyrosines at positions 7 and 8 are phosphorylated 49Tyr Met Glu Asp
Ser Thr Tyr Tyr Lys1 55033PRTHomo
sapiensMOD_RES(20)..(20)PHOSPHORYLATION; tyrosine at position 20 is
phosphorylated 50Gly Ser Ile Asp Arg Glu Asp Gly Ser Phe Gln Gly
Pro Thr Gly Asn1 5 10 15Gln His Ile Tyr Gln Pro Val Gly Lys Pro Asp
Pro Ala Ala Pro Pro 20 25 30Lys5110PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 51Ile Ile Glu Asp Asn Glu Tyr Thr Ala Arg1 5
105213PRTHomo sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at
position 7 is phosphorylated 52Gln Glu Asp Gly Gly Val Tyr Ser Ser
Ser Gly Leu Lys1 5 105310PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 53Leu Ile Glu Asp Asn Glu Tyr Thr Ala Arg1 5
105411PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 54Ile Gly Glu Gly Thr Tyr Gly Val Val
Tyr Lys1 5 10558PRTHomo sapiensMOD_RES(4)..(4)PHOSPHORYLATION;
tyrosine at position 4 is phosphorylated 55Ile Tyr Gln Tyr Ile Gln
Ser Arg1 55617PRTHomo sapiensMOD_RES(1)..(1)PHOSPHORYLATION;
tyrosine at position 1 is phosphorylated 56Tyr Glu Val Leu Lys Ile
Ile Gly Lys Gly Ser Phe Gly Gln Val Ala1 5 10 15Arg5711PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 57Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg1 5
105810PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 58Ala Val Cys Ser Thr Tyr Leu Gln Ser
Arg1 5 105910PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION;
tyrosine at position 6 is phosphorylated 59Thr Val Cys Ser Thr Tyr
Leu Gln Ser Arg1 5 106019PRTHomo
sapiensMOD_RES(15)..(15)PHOSPHORYLATION; tyrosine at position 15 is
phosphorylated 60Val Ala Asp Pro Asp His Asp His Thr Gly Phe Leu
Thr Glu Tyr Val1 5 10 15Ala Thr Arg6113PRTHomo
sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at position 9 is
phosphorylated 61His Thr Asp Asp Glu Met Thr Gly Tyr Val Ala Thr
Arg1 5 106222PRTHomo sapiensMOD_RES(18)..(18)PHOSPHORYLATION;
tyrosine at position 18 is phosphorylated 62Leu Cys Asp Phe Gly Ser
Ala Ser His Val Ala Asp Asn Asp Ile Thr1 5 10 15Pro Tyr Leu Val Ser
Arg 206324PRTHomo sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine
at position 11 is phosphorylated 63Leu Pro Asp Phe Gln Asp Ser Ile
Phe Glu Tyr Phe Asn Thr Ala Pro1 5 10 15Leu Ala His Asp Leu Thr Phe
Arg 206420PRTHomo sapiensMOD_RES(14)..(14)PHOSPHORYLATION; tyrosine
at position 14 is phosphorylated 64Thr Leu Glu Pro Val Lys Pro Pro
Thr Val Pro Asn Asp Tyr Met Thr1 5 10 15Ser Pro Ala Arg
206514PRTHomo sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at
position 9 is phosphorylated 65Tyr Val Asp Ser Glu Gly His Leu Tyr
Thr Val Pro Ile Arg1 5 106625PRTHomo
sapiensMOD_RES(5)..(5)PHOSPHORYLATION; tyrosine at position 5 is
phosphorylated 66Ala Ser Gln Asp Tyr Asp Gln Leu Pro Ser Ser Ser
Asp Gly Ser Gln1 5 10 15Ala Pro Ala Arg Pro Pro Lys Pro Arg 20
256726PRTHomo sapiensMOD_RES(18)..(18)PHOSPHORYLATION; tyrosine at
position 18 is phosphorylated 67Thr Val Pro Pro Pro Val Pro Gln Asp
Pro Leu Gly Ser Pro Pro Ala1 5 10 15Leu Tyr Ala Glu Pro Leu Asp Ser
Leu Arg 20 256829PRTHomo sapiensMOD_RES(11)..(11)PHOSPHORYLATION;
tyrosine at position 11 is phosphorylated 68Ile Pro Pro Gly Pro Ser
Gln Asp Ser Val Tyr Ser Asp Pro Leu Gly1 5 10 15Ser Thr Pro Ala Gly
Ala Gly Glu Gly Val His Ser Lys 20 256922PRTHomo
sapiensMOD_RES(10)..(10)PHOSPHORYLATION; tyrosine at position 10 is
phosphorylated 69Leu Thr Asp Ser Lys Glu Asp Pro Ile Tyr Asp Glu
Pro Glu Gly Leu1 5 10 15Ala Pro Ala Pro Pro Arg 207023PRTHomo
sapiensMOD_RES(17)..(17)PHOSPHORYLATION; tyrosine at position 17 is
phosphorylated 70Leu Lys Glu Glu Gly Tyr Glu Leu Pro Tyr Asn Pro
Ala Thr Asp Asp1 5 10 15Tyr Ala Val Pro Pro Pro Arg 207114PRTHomo
sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at position 9 is
phosphorylated 71Gly Phe Ser Ser Asp Thr Ala Leu Tyr Ser Gln Val
Gln Lys1 5 107212PRTHomo sapiensMOD_RES(8)..(8)PHOSPHORYLATION;
tyrosine at position 8 is phosphorylated 72Asp Ala Ser Ser Gln Asp
Cys Tyr Asp Ile Pro Arg1 5 107321PRTHomo
sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at position 6 is
phosphorylated 73Thr Gln Gln Gly Leu Tyr Gln Ala Pro Gly Pro Asn
Pro Gln Phe Gln1 5 10 15Ser Pro Pro Ala Lys 207421PRTHomo
sapiensMOD_RES(17)..(17)PHOSPHORYLATION; tyrosine at position 17 is
phosphorylated 74Val Gly Gln Gly Tyr Val Tyr Glu Ala Ala Gln Thr
Glu Gln Asp Glu1 5 10 15Tyr Asp Thr Pro Arg 207512PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION; tyrosine at position 4 is
phosphorylated 75Glu Glu Thr Tyr Asp Val Pro Pro Ala Phe Ala Lys1 5
107611PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 76Glu Tyr Asp Gln Leu Tyr Glu Glu Tyr
Thr Arg1 5 107714PRTHomo sapiensMOD_RES(7)..(7)PHOSPHORYLATION;
tyrosine at position 7 is phosphorylated 77Val Thr Ile Ala Asp Asp
Tyr Ser Asp Pro Phe Asp Ala Lys1 5 107816PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 78Glu Leu Phe Asp Asp Pro Ser Tyr Val Asn Ile Gln
Asn Leu Asp Lys1 5 10 157917PRTHomo
sapiensMOD_RES(3)..(3)PHOSPHORYLATION; tyrosine at position 3 is
phosphorylated 79Leu Asp Tyr Cys Gly Gly Gly Gly Gly Gly Asp Pro
Gly Gly Gly Gln1 5 10 15Arg808PRTHomo
sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at position 6 is
phosphorylated 80Arg Val Pro Cys Ala Tyr Asp Lys1 58120PRTHomo
sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at position 9 is
phosphorylated 81Gly Pro Leu Asn Gly Asp Thr Asp Tyr Phe Gly Gln
Gln Phe Asp Gln1 5 10 15Leu Ser Asn Arg 208217PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION; tyrosine at position 4 is
phosphorylated 82Lys Pro Leu Tyr Trp Asp Leu Tyr Gly His Val Gln
Gln Gln Leu Leu1 5 10 15Lys8310PRTHomo
sapiensMOD_RES(3)..(3)PHOSPHORYLATION; tyrosine at position 3 is
phosphorylated 83Gly Leu Tyr Asp Leu Pro Gln Glu Pro Arg1 5
108423PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 84Leu Lys Glu Glu Gly Tyr Glu Leu Pro
Tyr Asn Pro Ala Thr Asp Asp1 5 10 15Tyr Ala Val Pro Pro Pro Arg
208511PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 85Val Cys Glu Pro Cys Tyr Glu Gln Leu
Asn Lys1 5 108612PRTHomo sapiensMOD_RES(7)..(7)PHOSPHORYLATION;
tyrosine at position 7 is phosphorylated 86Gly Glu Pro Glu Ala Leu
Tyr Ala Ala Val Thr Lys1 5 108721PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 87Val Gly Gln Gly Tyr Val Tyr Glu Ala Ala Gln Thr
Glu Gln Asp Glu1 5 10 15Tyr Asp Thr Pro Arg 208836PRTHomo
sapiensMOD_RES(19)..(19)PHOSPHORYLATION; tyrosine at position 19 is
phosphorylated 88His Pro Leu Ile Leu Ala Ala Pro Pro Pro Asp Ser
Pro Ala Ala Glu1 5 10 15Asp Val Tyr Asp Val Pro Pro Pro Ala Pro Asp
Leu Tyr Asp Val Pro 20 25 30Pro Gly Leu Arg 358925PRTHomo
sapiensMOD_RES(17)..(17)PHOSPHORYLATION; tyrosine at position 17 is
phosphorylated 89Val Leu Pro Pro Glu Val Ala Asp Gly Ser Val Val
Asp Asp Gly Val1 5 10 15Tyr Ala Val Pro Pro Pro Ala Glu Arg 20
259029PRTHomo sapiensMOD_RES(14)..(14)PHOSPHORYLATION; tyrosine at
position 14 is phosphorylated 90Leu Asn Glu Trp Leu Gly Asn Glu Asn
Thr Glu Asp Gln Tyr Ser Leu1 5 10 15Val Glu
Asp Asp Glu Asp Leu Pro His His Asp Glu Lys 20 259115PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 91Ile Gly Thr Ala Glu Pro Asp Tyr Gly Ala Leu Tyr
Glu Gly Arg1 5 10 159228PRTHomo
sapiensMOD_RES(21)..(22)PHOSPHORYLATION; tyrosines at positions 21
and 22 are phosphorylated 92Met Ala Gly Phe Asp Gly Ser Ala Trp Asp
Glu Glu Glu Glu Glu Pro1 5 10 15Pro Asp His Gln Tyr Tyr Asn Asp Phe
Pro Gly Lys 20 259336PRTHomo
sapiensMOD_RES(19)..(19)PHOSPHORYLATION; tyrosine at position 19 is
phosphorylated 93Ser Val Tyr Leu Gln Glu Phe Gln Asp Lys Gly Asp
Ala Glu Asp Gly1 5 10 15Asp Glu Tyr Asp Asp Pro Phe Ala Gly Pro Ala
Asp Thr Ile Ser Leu 20 25 30Ala Ser Glu Arg 359410PRTHomo
sapiensMOD_RES(2)..(2)PHOSPHORYLATION; tyrosine at position 2 is
phosphorylated 94Ile Tyr Gln Phe Thr Ala Ala Ser Pro Lys1 5
109523PRTHomo sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at
position 9 is phosphorylated 95Ser Gln Pro Ile Asp Asp Glu Ile Tyr
Glu Glu Leu Pro Glu Glu Glu1 5 10 15Glu Asp Thr Ala Ser Val Lys
209613PRTHomo sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at
position 11 is phosphorylated 96Leu Val Asn Glu Ala Pro Val Tyr Ser
Val Tyr Ser Lys1 5 10978PRTHomo
sapiensMOD_RES(3)..(3)PHOSPHORYLATION; tyrosine at position 3 is
phosphorylated 97Val Ile Tyr Asp Phe Ile Glu Lys1 59819PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION; tyrosine at position 4 is
phosphorylated 98Lys Pro Thr Tyr Asp Pro Val Ser Glu Asp Gln Asp
Pro Leu Ser Ser1 5 10 15Asp Phe Lys9917PRTHomo
sapiensMOD_RES(16)..(16)PHOSPHORYLATION; tyrosine at position 16 is
phosphorylated 99His Gln Glu Leu Gln Ala Met Gln Met Glu Leu Gln
Ser Pro Glu Tyr1 5 10 15Lys10017PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 100Thr Ser Thr Ile Met Thr Asp Tyr Asn Pro Asn Tyr
Cys Phe Ala Gly1 5 10 15Lys10130PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 101Gly Leu Gly His Gly Ala Phe Gly Glu Val Tyr Glu
Gly Gln Val Ser1 5 10 15Gly Met Pro Asn Asp Pro Ser Pro Leu Gln Val
Ala Val Lys 20 25 3010218PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 102Asn Lys Pro Thr Ser Leu Trp Asn Pro Thr Tyr Gly
Ser Trp Phe Thr1 5 10 15Glu Lys10335PRTHomo
sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at position 9 is
phosphorylated 103His Phe Pro Cys Gly Asn Val Asn Tyr Gly Tyr Gln
Gln Gln Gly Leu1 5 10 15Pro Leu Glu Ala Ala Thr Ala Pro Gly Ala Gly
His Tyr Glu Asp Thr 20 25 30Ile Leu Lys 3510424PRTHomo
sapiensMOD_RES(10)..(10)PHOSPHORYLATION; tyrosine at position 10 is
phosphorylated 104Asp Leu Asn Ser Leu Ile Ser Ser Asp Tyr Glu Leu
Leu Ser Asp Pro1 5 10 15Thr Pro Gly Ala Leu Ala Pro Arg
2010511PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 105Ile Gly Glu Gly Thr Tyr Gly Val Val
Tyr Lys1 5 1010611PRTHomo sapiensMOD_RES(10)..(10)PHOSPHORYLATION;
tyrosine at position 10 is phosphorylated 106Ile Gly Glu Gly Thr
Tyr Gly Val Val Tyr Lys1 5 1010716PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 107Val Tyr Asn Asp Gly Tyr Asp Asp Asp Asn Tyr Asp
Tyr Ile Val Lys1 5 10 151088PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION; tyrosine at position 4 is
phosphorylated 108Ile Tyr Gln Tyr Ile Gln Ser Arg1 510917PRTHomo
sapiensMOD_RES(1)..(1)PHOSPHORYLATION; tyrosine at position 1 is
phosphorylated 109Tyr Glu Val Leu Lys Ile Ile Gly Lys Gly Ser Phe
Gly Gln Val Ala1 5 10 15Arg11019PRTHomo
sapiensMOD_RES(15)..(15)PHOSPHORYLATION; tyrosine at position 15 is
phosphorylated 110Val Ala Asp Pro Asp His Asp His Thr Gly Phe Leu
Thr Glu Tyr Val1 5 10 15Ala Thr Arg11111PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 111Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg1 5
1011210PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 112Ala Val Cys Ser Thr Tyr Leu Gln Ser
Arg1 5 1011313PRTHomo sapiensMOD_RES(9)..(9)PHOSPHORYLATION;
tyrosine at position 9 is phosphorylated 113His Thr Asp Asp Glu Met
Thr Gly Tyr Val Ala Thr Arg1 5 1011422PRTHomo
sapiensMOD_RES(18)..(18)PHOSPHORYLATION; tyrosine at position 18 is
phosphorylated 114Leu Cys Asp Phe Gly Ser Ala Ser His Val Ala Asp
Asn Asp Ile Thr1 5 10 15Pro Tyr Leu Val Ser Arg 2011518PRTHomo
sapiensMOD_RES(4)..(4)PHOSPHORYLATION; tyrosine at position 4 is
phosphorylated 115Ile Ser Thr Tyr Gly Leu Pro Ala Gly Gly Ile Gln
Pro His Pro Gln1 5 10 15Thr Lys11615PRTHomo
sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at position 6 is
phosphorylated 116Gly Gln Glu Gly Glu Tyr Ala Val Pro Phe Asp Ala
Val Ala Arg1 5 10 1511711PRTHomo
sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at position 6 is
phosphorylated 117Val Cys Glu Pro Cys Tyr Glu Gln Leu Asn Arg1 5
101188PRTHomo sapiensMOD_RES(3)..(3)PHOSPHORYLATION; tyrosine at
position 3 is phosphorylated 118Leu Glu Tyr Tyr Glu Asn Glu Lys1
511941PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at
position 6 is phosphorylated 119Val Asp Pro Asn Gly Tyr Met Met Met
Ser Pro Ser Gly Gly Cys Ser1 5 10 15Pro Asp Ile Gly Gly Gly Pro Ser
Ser Ser Ser Ser Ser Ser Asn Ala 20 25 30Val Pro Ser Gly Thr Ser Tyr
Gly Lys 35 4012013PRTHomo sapiensMOD_RES(8)..(8)PHOSPHORYLATION;
tyrosine at position 8 is phosphorylated 120Arg Glu Glu Pro Glu Ala
Leu Tyr Ala Ala Val Asn Lys1 5 1012116PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 121Ile Lys Pro Ser Ser Ser Ala Asn Ala Ile Tyr Ser
Leu Ala Ala Arg1 5 10 1512228PRTHomo
sapiensMOD_RES(21)..(22)PHOSPHORYLATION; tyrosines at positions 21
and 22 are phosphorylated 122Met Ala Gly Phe Asp Gly Ser Ala Trp
Asp Glu Glu Glu Glu Glu Pro1 5 10 15Pro Asp His Gln Tyr Tyr Asn Asp
Phe Pro Gly Lys 20 2512316PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 123Glu Leu Phe Asp Asp Pro Ser Tyr Val Asn Val Gln
Asn Leu Asp Lys1 5 10 1512415PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 124Ile Gln Asn Thr Gly Asp Tyr Tyr Asp Leu Tyr Gly
Gly Glu Lys1 5 10 1512526PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 125Ser Cys Gln Asn Leu Gly Tyr Thr Ala Ala Ser Pro
Gln Ala Pro Glu1 5 10 15Ala Ala Ser Ser Thr Gly Asn Ala Glu Arg 20
2512613PRTHomo sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at
position 8 is phosphorylated 126Ser Gln Asp Pro Asn Pro Gln Tyr Ser
Pro Ile Ile Lys1 5 1012729PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 127Gly Ser Pro Gly Glu Ala Pro Ser Asn Ile Tyr Val
Glu Val Glu Asp1 5 10 15Glu Gly Leu Pro Ala Thr Leu Gly His Pro Val
Leu Arg 20 2512816PRTHomo sapiensMOD_RES(3)..(3)PHOSPHORYLATION;
tyrosine at position 3 is phosphorylated 128Leu Ile Tyr Asp Phe Ile
Glu Asp Gln Gly Gly Leu Glu Ala Val Arg1 5 10 1512913PRTHomo
sapiensMOD_RES(10)..(10)PHOSPHORYLATION; tyrosine at position 10 is
phosphorylated 129Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr
Arg1 5 1013013PRTHomo sapiensMOD_RES(7)..(7)PHOSPHORYLATION;
tyrosine at position 7 is phosphorylated 130Gln Glu Asp Gly Gly Val
Tyr Ser Ser Ser Gly Leu Lys1 5 1013112PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 131Asn Leu Asp Asn Gly Gly Phe Tyr Ile Ser Pro Arg1
5 1013210PRTHomo sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at
position 7 is phosphorylated 132Leu Ile Glu Asp Asn Glu Tyr Thr Ala
Arg1 5 1013318PRTHomo sapiensMOD_RES(14)..(14)PHOSPHORYLATION;
tyrosine at position 14 is phosphorylated 133Ser Val Leu Glu Asp
Phe Phe Thr Ala Thr Glu Gly Gln Tyr Gln Pro1 5 10 15Gln
Pro13415PRTHomo sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at
position 9 is phosphorylated 134Ile Asp Thr Leu Asn Ser Asp Gly Tyr
Thr Pro Glu Pro Ala Arg1 5 10 1513520PRTHomo
sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at position 9 is
phosphorylated 135Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro
Tyr Ser Asp Pro1 5 10 15Glu Glu Leu Lys 2013612PRTHomo
sapiensMOD_RES(8)..(9)PHOSPHORYLATION; tyrosines at positions 8 and
9 are phosphorylated 136Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala
Arg1 5 1013711PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION;
tyrosine at position 6 is phosphorylated 137Ile Gly Glu Gly Thr Tyr
Gly Val Val Tyr Lys1 5 1013818PRTHomo
sapiensMOD_RES(5)..(5)PHOSPHORYLATION; tyrosine at position 5 is
phosphorylated 138Ala Asp Gln Gln Tyr Glu Cys Val Ala Glu Ile Gly
Glu Gly Ala Tyr1 5 10 15Gly Lys13911PRTHomo
sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at position 7 is
phosphorylated 139Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg1 5
1014020PRTHomo sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at
position 8 is phosphorylated 140Gln Glu Gly Ser Ile Glu Val Tyr Glu
Asp Ala Gly Ser His Tyr Leu1 5 10 15Cys Leu Leu Lys 2014113PRTHomo
sapiensMOD_RES(9)..(9)PHOSPHORYLATION; tyrosine at position 9 is
phosphorylated 141His Thr Asp Asp Glu Met Thr Gly Tyr Val Ala Thr
Arg1 5 1014222PRTHomo sapiensMOD_RES(18)..(18)PHOSPHORYLATION;
tyrosine at position 18 is phosphorylated 142Leu Cys Asp Phe Gly
Ser Ala Ser His Val Ala Asp Asn Asp Ile Thr1 5 10 15Pro Tyr Leu Val
Ser Arg 2014320PRTHomo sapiensMOD_RES(14)..(14)PHOSPHORYLATION;
tyrosine at position 14 is phosphorylated 143Thr Leu Glu Pro Val
Lys Pro Pro Thr Val Pro Asn Asp Tyr Met Thr1 5 10 15Ser Pro Ala Arg
2014412PRTHomo sapiensMOD_RES(7)..(7)PHOSPHORYLATION; tyrosine at
position 7 is phosphorylated 144Gly Met Lys Asp Asp Asp Tyr Asp Asp
Gln Leu Cys1 5 1014527PRTHomo
sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at position 6 is
phosphorylated 145Ser Ala Thr Leu Leu Tyr Asp Gln Pro Leu Gln Val
Phe Thr Gly Ser1 5 10 15Ser Ser Ser Ser Asp Leu Ile Ser Gly Thr Lys
20 2514629PRTHomo sapiensMOD_RES(13)..(13)PHOSPHORYLATION; tyrosine
at position 13 is phosphorylated 146Gly Pro Thr Ser Gly Pro Gln Ser
Ala Pro Gln Ile Tyr Gly Pro Pro1 5 10 15Gln Tyr Asn Ile Gln Tyr Ser
Ser Ser Ala Ala Val Lys 20 2514712PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 147Ala Gln Ile Pro Glu Gly Asp Tyr Leu Ser Tyr Arg1
5 1014813PRTHomo sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at
position 8 is phosphorylated 148Arg Glu Glu Pro Glu Ala Leu Tyr Ala
Ala Val Asn Lys1 5 1014928PRTHomo
sapiensMOD_RES(17)..(17)PHOSPHORYLATION; tyrosine at position 17 is
phosphorylated 149Glu Thr Asp Thr Ser Ala Leu Ala Ala Gly Ser Ser
Gln Glu Val Thr1 5 10 15Tyr Ala Gln Leu Asp His Trp Ala Leu Thr Gln
Arg 20 2515011PRTHomo sapiensMOD_RES(2)..(2)PHOSPHORYLATION;
tyrosine at position 2 is phosphorylated 150Leu Tyr Asp Leu Asn Met
Pro Ala Tyr Val Lys1 5 1015127PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 151Ile Ala Pro Cys Pro Ser Gln Asp Ser Leu Tyr Ser
Asp Pro Leu Asp1 5 10 15Ser Thr Ser Ala Gln Ala Gly Glu Gly Val Gln
20 2515224PRTHomo sapiensMOD_RES(5)..(5)PHOSPHORYLATION; tyrosine
at position 5 is phosphorylated 152Glu Asp Pro Ile Tyr Asp Glu Pro
Glu Gly Leu Ala Pro Val Pro Pro1 5 10 15Gln Gly Leu Tyr Asp Leu Pro
Arg 2015323PRTHomo sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine
at position 6 is phosphorylated 153Val Lys Glu Glu Gly Tyr Glu Leu
Pro Tyr Asn Pro Ala Thr Asp Asp1 5 10 15Tyr Ala Val Pro Pro Pro Arg
2015414PRTHomo sapiensMOD_RES(4)..(4)PHOSPHORYLATION; tyrosine at
position 4 is phosphorylated 154Glu Asn Asp Tyr Glu Ser Ile Ser Asp
Leu Gln Gln Gly Arg1 5 1015515PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 155Ile Gly Thr Ala Glu Pro Asp Tyr Gly Ala Leu Tyr
Glu Gly Arg1 5 10 1515615PRTHomo
sapiensMOD_RES(5)..(5)PHOSPHORYLATION; tyrosine at position 5 is
phosphorylated 156Asn Pro Gly Phe Tyr Val Glu Ala Asn Pro Met Pro
Thr Phe Lys1 5 10 1515710PRTHomo
sapiensMOD_RES(6)..(6)PHOSPHORYLATION; tyrosine at position 6 is
phosphorylated 157Asp Ile Asn Ser Leu Tyr Asp Val Ser Arg1 5
1015819PRTHomo sapiensMOD_RES(12)..(12)PHOSPHORYLATION; tyrosine at
position 12 is phosphorylated 158Arg Gln Glu Glu Leu Asn Asn Gln
Leu Phe Leu Tyr Asp Thr His Gln1 5 10 15Asn Leu Arg15916PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 159Glu Leu Phe Asp Asp Pro Ser Tyr Val Asn Val Gln
Asn Leu Asp Lys1 5 10 1516021PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 160Ser Gly Glu Ser Val Glu Glu Val Pro Leu Tyr Gly
Asn Leu His Tyr1 5 10 15Leu Gln Thr Gly Arg 2016119PRTHomo
sapiensMOD_RES(11)..(11)PHOSPHORYLATION; tyrosine at position 11 is
phosphorylated 161Ser Gln Ala Ser Gly Pro Glu Pro Glu Leu Tyr Ala
Ser Val Cys Ala1 5 10 15Gln Thr Arg16216PRTHomo
sapiensMOD_RES(8)..(8)PHOSPHORYLATION; tyrosine at position 8 is
phosphorylated 162Ala Ser Phe Pro Asp Gln Ala Tyr Ala Asn Ser Gln
Pro Ala Ala Ser1 5 10 1516316PRTHomo
sapiensMOD_RES(3)..(3)PHOSPHORYLATION; tyrosine at position 3 is
phosphorylated 163Leu Ile Tyr Asp Phe Ile Glu Asp Gln Gly Gly Leu
Glu Ala Val Arg1 5 10 15
* * * * *