U.S. patent application number 09/839884 was filed with the patent office on 2002-06-20 for rapid quantitative analysis of proteins or protein function in complex mixtures.
This patent application is currently assigned to University of Washington. Invention is credited to Aebersold, Rudolf Hans, Gelb, Michael H., Gerber, Scott A., Gygi, Steven P., Rist, Beate, Scott, C. Ronald, Turecek, Frantisek.
Application Number | 20020076739 09/839884 |
Document ID | / |
Family ID | 26793647 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020076739 |
Kind Code |
A1 |
Aebersold, Rudolf Hans ; et
al. |
June 20, 2002 |
Rapid quantitative analysis of proteins or protein function in
complex mixtures
Abstract
Analytical reagents and mass spectrometry-based methods using
these reagents for the rapid, and quantitative analysis of proteins
or protein function in mixtures of proteins. The methods employ
affinity labeled protein reactive reagents having three portions:
an affinity label (A) covalently linked to a protein reactive group
(PRG) through a linker group (L). The linker may be differentially
isotopically labeled, e.g., by substitution of one or more atoms in
the linker with a stable isotope thereof. These reagents allow for
the selective isolation of peptide fragments or the products of
reaction with a given protein (e.g., products of enzymatic
reaction) from complex mixtures. The isolated peptide fragments or
reaction products are characteristic of the presence of a protein
or the presence of a protein function in those mixtures. Isolated
peptides or reaction products are characterized by mass
spectrometric (MS) techniques. The reagents also provide for
differential isotopic labeling of the isolated peptides or reaction
products which facilitates quantitative determination by mass
spectrometry of the relative amounts of proteins in different
samples. The methods of this invention can be used for qualitative
and quantitative analysis of global protein expression profiles in
cells and tissues, to screen for and identify proteins whose
expression level in cells, tissue or biological fluids is affected
by a stimulus or by a change in condition or state of the cell,
tissue or organism from which the sample originated.
Inventors: |
Aebersold, Rudolf Hans;
(Mercer Island, WA) ; Gelb, Michael H.; (Seattle,
WA) ; Gygi, Steven P.; (Seattle, WA) ; Scott,
C. Ronald; (Seattle, WA) ; Turecek, Frantisek;
(Seattle, WA) ; Gerber, Scott A.; (Seattle,
WA) ; Rist, Beate; (Seattle, WA) |
Correspondence
Address: |
GREENLEE WINNER and SULLIVAN, P.C.
5370 Manhattan Circle, Suite 201
Boulder
CO
80303
US
|
Assignee: |
University of Washington
|
Family ID: |
26793647 |
Appl. No.: |
09/839884 |
Filed: |
April 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09839884 |
Apr 20, 2001 |
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09383062 |
Aug 25, 1999 |
|
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60097788 |
Aug 25, 1998 |
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60099113 |
Sep 3, 1998 |
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Current U.S.
Class: |
435/7.92 ;
530/403 |
Current CPC
Class: |
Y10T 436/25375 20150115;
G01N 33/58 20130101; G01N 33/6848 20130101; Y10T 436/24 20150115;
G01N 33/6842 20130101; Y10T 436/25 20150115; G01N 33/6803 20130101;
Y10T 436/25125 20150115; C12Q 1/25 20130101; Y10T 436/182 20150115;
Y10S 530/812 20130101 |
Class at
Publication: |
435/7.92 ;
530/403 |
International
Class: |
G01N 033/53; G01N
033/537; G01N 033/543; C07K 014/435 |
Goverment Interests
[0002] This invention was made through funding from the National
Science Foundation Science and Technology Center for Molecular
Biotechnology (grants 5T32HG and BIR9214821) and the National
Institutes of Health (NIH grants RR11823, T32HG00035, HD-02274 and
GM60184). The United States government has certain rights in this
invention.
Claims
We claim:
1. A reagent for mass spectrometric analysis of proteins which has
the general formula:A-L-PRGwhere A is an affinity label that
selectively binds to a capture reagent, L is a linker group which
can be differentially labelled with stable isotopes and PRG is a
protein reactive group that selectively that selectively reacts
with certain protein functional groups.
2. The reagent of claim 1 wherein PRG is a sulfhydryl reactive
group or an amine reactive group.
3. The reagent of claim 1 wherein PRG is an enzyme substrate.
4. The reagent of claim 1 wherein the A-L-PRG is soluble in a
sample liquid to be analyzed.
5. The reagent of claim 1 wherein the linker is a cleavable
linker.
6. The reagent of claim 1 which has the general
formula:A-B.sup.1--X.sup.1-
--(CH.sub.2).sub.n--[X.sup.2--(CH.sub.2).sub.m].sub.x--X.sup.3--(CH.sub.2)-
.sub.p--X.sup.4--B.sup.2-PRGwhere: A is an affinity label; PRG is a
protein reactive group; and
B.sup.1--X.sup.1--(CH.sub.2).sub.n--[X.sup.2--
-(CH.sub.2).sub.m]x--X.sup.3--(CH.sub.2)p --X.sup.4--B.sup.2 is a
linker group wherein: X.sup.1, X.sup.2, X.sup.3 and X.sup.4,
independently of one another, and X.sup.2 independently of other
X.sup.2 , can be selected from O, S, NH, NR, NRR'+, CO, COO, COS,
S--S, SO, SO.sub.2, CO--NR', CS--NR', Si--O, aryl or diaryl groups
or X.sup.1 -X.sup.4 may be absent; B.sup.1 and B.sup.2,
independently of one another, are optional groups selected from
COO, CO, CO--NR', CS--NR', (CH.sub.2).sub.q--CONR',
(CH.sub.2).sub.q--CS--NR', or (CH.sub.2).sub.q; n, m, p, q and x
are whole numbers that can take values from 0 to about 100, where
the sum of n+xm+p+q is less than about 100; R is an alkyl, alkenyl,
alkynyl, alkoxy or an aryl group that is optionally substituted
with one or more alkyl, alkenyl, alkynyl, or alkoxy groups; and R'
is a hydrogen, an alkyl, alkenyl, alkynyl, alkoxy or an aryl group
that is optionally substituted with one or more alkyl, alkenyl,
alkynyl, or alkoxy groups wherein one or more of the CH.sub.2
groups in the linker can be optionally substituted with alkyl,
alkenyl, alkoxy groups, an aryl group that is optionally
substituted with one or more alkyl, alkenyl, alkynyl, or alkoxy
groups, an acidic group, a basic group or a group carrying a
permanent positive or negative charge; wherein one or more single
bonds linking non-adjacent CH2 groups in the linker can be replaced
with a double or a triple bond and wherein one or more of the atoms
in the linker can be substituted with a stable isotope.
7. The reagent of claim 1 wherein the affinity label is biotin or a
modified biotin.
8. The reagent of claim 1 wherein the affinity label is selected
from the group consisting of a 1,2-diol, glutathione, maltose, a
nitrilotriacetic acid group, or an oligohistidine.
9. The reagent of claim 1 wherein the affinity label is a
hapten.
10. The reagent of claim 1 wherein PRG is a sulfhydryl-reactive
group.
11. The reagent of claim 1 wherein PRG is an iodoacetylamide group,
an epoxide, an .alpha.-haloacyl group, a nitrites, a sulfonated
alkyl, an aryl thiols or a maleimide.
12. The reagent of claim 1 wherein PRG is an amine reactive group,
a group that reacts with a homoserine lactone or a group that
reacts with carboxylic acid group.
13. The reagent of claim 1 wherein PRG is selected from the groups
consisting of a amine reactive pentafluorophenyl ester group, an
amine reactive N-hydroxy succinimide ester group, sulfonyl halide,
isocyanate, isothiocyanante, active ester, tertafluorophenyl ester,
an acid halide, and an acid anyhydride; a homoserine lactone
reactive primary amine group, and an carboxylic acid reactive
amine, alcohols or 2,3,5,6-tetrafluorophenyl trifluoroacetate.
14. The reagent of claim 1 wherein PRG is a substrate for an
enzyme.
15. The reagent of claim 1 wherein PRG is a substrate for an enzyme
the deficiency of which is associated with a birth defect.
16. The reagent of claim 1 wherein PRG is a substrate for an enzyme
the deficiency of which is associated with a lysosomal storage
disease.
17. The reagent of claim 1 wherein PRG is a substrate for
.beta.-galactosidase, acetyl-.alpha.-D-glucosaminidase, heparan
sulfamidase, acetyl-CoA-.alpha.-D-glucosaminide N-acetyltransferase
or N-acetylglucosamine-6-sulfatase.
18. The reagent of claim 1 wherein at least one of B1 or B2 is
CO--NR' or CS--NR.
19. The reagent of claim 1 wherein X.sup.1 and X.sup.4 are selected
from NH, NR, and NRR'.sup.+, X.sup.3 is O and all X.sup.2 groups
are O.
20. The reagent of claim 1 wherein the linker contains a disulfide
group.
21. The reagent of claim 1 wherein any atom of the linker may be
substituted with a heavy isotope.
22. A reagent kit for the analysis of proteins by mass spectral
analysis that comprises a reagent of claim 1.
23. The reagent kit of claim 22 that comprises one or more reagents
of claim 1.
24. The reagent kit of claim 22 further comprising one or more
proteolytic enzymes for use in digestion of affinity tagged
proteins.
25. The reagent kit of claim 22 which comprises a set of
substantially chemically identical differentially labelled affinity
tagged reagents.
26. The reagent kit of claim 22 wherein the reagent is an affinity
tagged enzyme substrate reagent.
27. The reagent kit of claim 26 which comprises a set of
substantially chemically identical differentially labeled affinity
tagged enzyme substrates.
28. The reagent kit of claim 27 further comprising a set of
substantially chemically identical differentially labeled affinity
tagged enzyme products.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 09/388,062, filed Aug. 25, 1999, which takes priority
under 35 U.S.C. .sctn.119(e) from U.S. Provisional application Ser.
No. 60/097,788, filed Aug. 25, 1998, and Ser. No. 60/099,113, filed
Sep. 3, 1998, all of which are incorporated in their entirety by
reference herein.
BACKGROUND OF THE INVENTION
[0003] Genomic technology has advanced to a point at which, in
principle, it has become possible to determine complete genomic
sequences and to quantitatively measure the mRNA levels for each
gene expressed in a cell. For some species the complete genomic
sequence has now been determined, and for one strain of the yeast
Saccharomyces cervisiae, the mRNA levels for each expressed gene
have been precisely quantified under different growth conditions
(Velculescu et al., 1997). Comparative cDNA array analysis and
related technologies have been used to determine induced changes in
gene expression at the mRNA level by concurrently monitoring the
expression level of a large number of genes (in some cases all the
genes) expressed by the investigated cell or tissue (Shalon et al.,
1996). Furthermore, biological and computational techniques have
been used to correlate specific function with gene sequences. The
interpretation of the data obtained by these techniques in the
context of the structure, control and mechanism of biological
systems has been recognized as a considerable challenge. In
particular, it has been extremely difficult to explain the
mechanism of biological processes by genomic analysis alone.
[0004] Proteins are essential for the control and execution of
virtually every biological process. The rate of synthesis and the
half-life of proteins and thus their expression level are also
controlled post-transcriptionally. Furthermore, the activity of
proteins is frequently modulated by post-translational
modifications, in particular protein phosphorylation, and dependent
on the association of the protein with other molecules including
DNA and proteins. Neither the level of expression nor the state of
activity of proteins is therefore directly apparent from the gene
sequence or even the expression level of the corresponding mRNA
transcript. It is therefore essential that a complete description
of a biological system include measurements that indicate the
identity, quantity and the state of activity of the proteins which
constitute the system. The large-scale (ultimately global) analysis
of proteins expressed in a cell or tissue has been termed proteome
analysis (Pennington et al., 1997).
[0005] At present no protein analytical technology approaches the
throughput and level of automation of genomic technology. The most
common implementation of proteome analysis is based on the
separation of complex protein samples most commonly by
two-dimensional gel electrophoresis (2DE) and the subsequent
sequential identification of the separated protein species (Ducret
et al., 1998; Garrels et al., 1997; Link et al., 1997; Shevchenko
et al., 1996; Gygi et al. 1999; Boucherie et al., 1996). This
approach has been revolutionized by the development of powerful
mass spectrometric techniques and the development of computer
algorithms which correlate protein and peptide mass spectral data
with sequence databases and thus rapidly and conclusively identify
proteins (Eng et al., 1994; Mann and Wilm, 1994; Yates et al.,
1995). This technology has reached a level of sensitivity which now
permits the identification of essentially any protein which is
detectable by conventional protein staining methods including
silver staining (Figeys and Aebersold, 1998; Figeys et al., 1996;
Figeys et al., 1997; Shevchenko et al., 1996). However, the
sequential manner in which samples are processed limits the sample
throughput, the most sensitive methods have been difficult to
automate and low abundance proteins, such as regulatory proteins,
escape detection without prior enrichment, thus effectively
limiting the dynamic range of the technique. In the 2DE/(MS)N
method, proteins are quantified by densitometry of stained spots in
the 2DE gels.
[0006] The development of methods and instrumentation for
automated, data-dependent electrospray ionization (ESI) tandem mass
spectrometry (MS.sup.n) in conjunction with microcapillary liquid
chromatography (.mu.LC) and database searching has significantly
increased the sensitivity and speed of the identification of
gel-separated proteins. As an alternative to the 2DE/MS.sup.n
approach to proteome analysis, the direct analysis by tandem mass
spectrometry of peptide mixtures generated by the digestion of
complex protein mixtures has been proposed (Dongr'e et al., 1997).
.mu.LC-Ms/MS has also been used successfully for the large-scale
identification of individual proteins directly from mixtures
without gel electrophoretic separation (Link et al., 1999; Opitek
et al., 1997) While these approaches dramatically accelerate
protein identification, the quantities of the analyzed proteins
cannot be easily determined, and these methods have not been shown
to substantially alleviate the dynamic range problem also
encountered by the 2DE/MS/MS approach. Therefore, low abundance
proteins in complex samples are also difficult to analyze by the
.mu.LC/MS/MS method without their prior enrichment.
[0007] It is therefore apparent that current technologies, while
suitable to identify the components of protein mixtures, are
neither capable of measuring the quantity nor the state of activity
of the protein in a mixture. Even evolutionary improvements of the
current approaches are unlikely to advance their performance
sufficiently to make routine quantitative and functional proteome
analysis a reality.
[0008] This invention provides methods and reagents that can be
employed in proteome analysis which overcome the limitations
inherent in traditional techniques. The basic approach described
can be employed for the quantitative analysis of protein expression
in complex samples (such as cells, tissues, and fractions thereof),
the detection and quantitation of specific proteins in complex
samples, and the quantitative measurement of specific enzymatic
activities in complex samples.
[0009] In this regard, a multitude of analytical techniques are
presently available for clinical and diagnostic assays which detect
the presence, absence, deficiency or excess of a protein or protein
function associable with a normal or disease state. While these
techniques are quite sensitive, they do not necessarily provide
chemical speciation of products and may, as a result, be difficult
to use for assaying several proteins or enzymes simultaneously in a
single sample. Current methods may not distinguish among aberrant
expression of different enzymes or their malfunctions which lead to
a common set of clinical symptoms. The methods and reagents herein
can be employed in clinical and diagnostic assays for simultaneous
(multiplex) monitoring of multiple proteins and protein
reactions.
SUMMARY OF THE INVENTION
[0010] This invention provides analytical reagents and mass
spectrometry-based methods using these reagents for the rapid, and
quantitative analysis of proteins or protein function in mixtures
of proteins. The analytical method can be used for qualitative and
particularly for quantitative analysis of global protein expression
profiles in cells and tissues, i.e. the quantitative analysis of
proteomes. The method can also be employed to screen for and
identify proteins whose expression level in cells, tissue or
biological fluids is affected by a stimulus (e.g., administration
of a idrug or contact with a potentially toxic material), by a
change in environment (e.g., nutrient level, temperature, passage
of time) or by a change in condition or cell state (e.g., disease
state, malignancy, site-directed mutation, gene knockouts) of the
cell, tissue or organism from which the sample originated. The
proteins identified in such a screen can function as markers for
the changed state. For example, comparisons of protein expression
profiles of normal and malignant cells can result in the
identification of proteins whose presence or absence is
characteristic and diagnostic of the malignancy.
[0011] In an exemplary embodiment, the methods herein can be
employed to screen for changes in the expression or state of
enzymatic activity of specific proteins. These changes may be
induced by a variety of chemicals, including pharmaceutical
agonists or antagonists, or potentially harmful or toxic materials.
The knowledge of such changes may be useful for diagnosing
enzyme-based diseases and for investigating complex regulatory
networks in cells.
[0012] The methods herein can also be used to implement a variety
of clinical and diagnostic analyses to detect the presence,
absence, deficiency or excess of a given protein or protein
function in a biological fluid (e.g., blood), or in cells or
tissue. The method is particularly useful in the analysis of
complex mixtures of proteins, i.e., those containing 5 or more
distinct proteins or protein functions.
[0013] The inventive method employs affinity-labeled protein
reactive reagents that allow for the selective isolation of peptide
fragments or the products of reaction with a given protein (e.g.,
products of enzymatic reaction) from complex mixtures. The isolated
peptide fragments or reaction products are characteristic of the
presence of a protein or the presence of a protein function, e.g.,
an enzymatic activity, respectively, in those mixtures. Isolated
peptides or reaction products are characterized by mass
spectrometric (MS) techniques. In particular, the sequence of
isolated peptides can be determined using tandem MS (MS.sup.n)
techniques, and by application of sequence database searching
techniques, the protein from which the sequenced peptide originated
can be identified. The reagents also provide for differential
isotopic labeling of the isolated peptides or reaction products
which facilitates quantitative determination by mass spectrometry
of the relative amounts of proteins in different samples. Also, the
use of differentially isotopically-labeled reagents as internal
standards facilitates quantitative determination of the absolute
amounts of one or more proteins or reaction products present in the
sample.
[0014] In general, the affinity labeled protein reactive reagents
of this invention have three portions: an affinity label (A)
covalently linked to a protein reactive group (PRG) through a
linker group (L):
A-L-PRG
[0015] The linker may be differentially isotopically labeled, e.g.,
by substitution of one or more atoms in the linker with a stable
isotope thereof. For example, hydrogens can be substituted with
deuteriums or C.sup.12 with C.sup.13.
[0016] The affinity label A functions as a molecular handle that
selectively binds covalently or non-covalently, to a capture
reagent (CR). Binding to CR facilitates isolation of peptides,
substrates or reaction products tagged or labeled with A. In
specific embodiments, A is a strepavidin or avidinn. After affinity
isolation of affinity tagged materials, some of which may be
isotopically labeled, the interaction between A and the capture
reagent is disrupted or broken to allow MS analysis of the isolated
materials. The affinity label may be displaced from the capture
reagent by addition of displacing ligand, which may be free A or a
derivative of A, or by changing solvent (e.g., solvent type or pH)
or temperature conditions or the linker may be cleaved chemically,
enzymatically, thermally or photochemically to release the isolated
materials for MS analysis.
[0017] Two types of PRG groups are specifically provided herein:
(a) those groups that selectively react with a protein functional
group to form a covalent or non-covalent bond tagging the protein
at specific sites, and (b) those that are transformed by action of
the protein, e.g., that are substrates for an enzyme. In specific
embodiments, PRG is a group having specific reactivity for certain
protein groups, such as specificity for sulfhydryl groups, and is
useful in general for selectively tagging proteins in complex
mixtures. A sulfhydryl specific reagent tags proteins containing
cysteine. In other specific embodiments, PRG is an enzyme substrate
that is selectively cleaved (leaving A-L) or modified (giving
A-L-PRG') by the action of an enzyme of interest.
[0018] Exemplary reagents have the general formula:
A-B.sup.1--X.sup.1--(CH.sub.2).sub.n--[X.sup.2--(CH.sub.2).sub.m].sub.x--X-
.sup.3--(CH.sub.2).sub.p--X.sup.4--B.sup.2-PRG
[0019] where:
[0020] A is the affinity label;
[0021] PRG is the protein reactive group;
[0022] X.sup.1, X.sup.2, X.sup.3 and X.sup.4, independently of one
another, and X.sup.2 independently of other X.sup.2 in the linker
group, can be selected from O, S, NH, NR, NRR'.sup.+, CO, COO, COS,
S--S, SO, SO.sub.2, CO--NR', CS--NR', Si--O, aryl or diaryl groups
or X.sup.1 -X.sup.4 may be absent, but preferably at least one of
X.sup.1 -X.sup.4 is present;
[0023] B.sup.1 and B.sup.2, independently of one another, are
optional moieties that can faciliate bonding of the A or PRG group
to the linker or prevent undesired cleavage of those groups from
the linker and can be selected, for example, from COO, CO, CO--NR',
CS--NR'and may contain one or more CH.sub.2 groups alone or in
combination with other groups, e.g.(CH.sub.2).sub.q--CONR',
(CH.sub.2).sub.q--CS--NR', or (CH.sub.2).sub.q;
[0024] n, m, p and q are whole numbers that can have values from 0
to about 100, preferably one of n, m, p or q is not 0 and x is also
a whole number that can range from 0 to about 100 where the sum of
n+xm+p+q is preferably less than about 100 and more preferably less
than about 20;
[0025] R is an alkyl, alkenyl, alkynyl, alkoxy or aryl group;
and
[0026] R' is a hydrogen, an alkyl, alkenyl, alkynyl, alkoxy or aryl
group.
[0027] One or more of the CH.sub.2 groups of the linker can be
optionally substituted with small (C1-C6) alkyl, alkenyl, or alkoxy
groups, an aryl group or can be substituted with functional groups
that promote ionization, such as acidic or basic groups or groups
carrying permanent positive or negative charge. One or more single
bonds connecting CH.sub.2 groups in the linker can be replaced with
a double or a triple bond. Preferred R and R' alkyl, alkenyl,
alkynyl or alkoxy groups are small having 1 to about 6 carbon
atoms.
[0028] One or more of the atoms in the linker can be substituted
with a stable isotope to generate one or more substantially
chemically identical, but isotopically distinguishable reagents.
For example, one or more hydrogens in the linker can be substituted
with deuterium to generate isotopically heavy reagents.
[0029] In an exemplary embodiment the linker contains groups that
can be cleaved to remove the affinity tag. If a cleavable linker
group is employed, it is typically cleaved after affinity tagged
peptides, substrates or reaction products have been isolated using
the affinity label together with the CR. In this case, any isotopic
labeling in the linker preferably remains bound to the protein,
peptide, substrate or reaction product.
[0030] Linker groups include among others: ethers, polyethers,
ether diamines, polyether diamines, diamines, amides, polyamides,
polythioethers, disulfides, silyl ethers, alkyl or alkenyl chains
(straight chain or branched and portions of which may be cyclic),
aryl, diaryl or alkyl-aryl groups. Aryl groups in linkers can
contain one or more heteroatoms (e.g., N, O or S atoms).
[0031] In one aspect, the invention provides a mass spectrometric
method for identification and quantitation of one or more proteins
in a complex mixture which employs affinity labeled reagents in
which the PRG is a group that selectively reacts with certain
groups that are typically found in peptides (e.g.,sulfhydryl,
amino, carboxy, homoserine lactone groups). One or more affinity
labeled reagents with different PRG groups are introduced into a
mixture containing proteins and the reagents react with certain
proteins to tag them with the affinity label. It may be necessary
to pretreat the protein mixture to reduce disulfide bonds or
otherwise facilitate affinity labeling. After reaction with the
affinity labeled reagents, proteins in the complex mixture are
cleaved, e.g., enzymatically, into a number of peptides. This
digestion step may not be necessary, if the proteins are relatively
small. Peptides that remain tagged with the affinity label are
isolated by an affinity isolation method, e.g., affinity
chromatography, via their selective binding to the CR. Isolated
peptides are released from the CR by displacement of A or cleavage
of the linker, and released materials are analyzed by liquid
chromatography/mass spectrometry (LC/MS). The sequence of one or
more tagged peptides is then determined by MS.sup.n techniques. At
least one peptide sequence derived from a protein will be
characteristic of that protein and be indicative of its presence in
the mixture. Thus, the sequences of the peptides typically provide
sufficient information to identify one or more proteins present in
a mixture.
[0032] Quantitative relative amounts of proteins in one or more
different samples containing protein mixtures (e.g., biological
fluids, cell or tissue lysates, etc.) can be determined using
chemically identical, affinity tagged and differentially
isotopically labeled reagents to affinity tag and differentially
isotopically label proteins in the different samples. In this
method, each sample to be compared is treated with a different
isotopically labeled reagent to tag certain proteins therein with
the affinity label. The treated samples are then combined,
preferably in equal amounts, and the proteins in the combined
sample are enzymatically digested, if necessary, to generate
peptides. Some of the peptides are affinity tagged and in addition
tagged peptides originating from different samples are
differentially isotopically labeled. As described above, affinity
labeled peptides are isolated, released from the capture reagent
and analyzed by (LC/MS). Peptides characteristic of their protein
origin are sequenced using MS.sup.n techniques allowing
identification of proteins in the samples. The relative amounts of
a given protein in each sample is determined by comparing relative
abundance of the ions generated from any differentially labeled
peptides originating from that protein. The method can be used to
assess relative amounts of known proteins in different samples.
Further, since the method does not require any prior knowledge of
the type of proteins that may be present in the samples, it can be
used to identify proteins which are present at different levels in
the samples examined. More specifically, the method can be applied
to screen for and identify proteins which exhibit differential
express in cells, tissue or biological fluids. It is also possible
to determine the absolute amounts of specific proteins in a complex
mixture. In this case, a known amount of internal standard, one for
each specific protein in the mixture to be quantified, is added to
the sample to be analyzed. The internal standard is an affinity
tagged peptide that is identical in chemical structure to the
affinity tagged peptide to be quantified except that the internal
standard is differentially isotopically labeled, either in the
peptide or in the affinity tag portion, to distinguish it from the
affinity tagged peptide to be quantified. The internal standard can
be provided in the sample to be analyzed in other ways. For
example, a specific protein or set of proteins can be chemically
tagged with an isotopically-labeled affinity tagging reagent. A
known amount of this material can be added to the sample to be
analyzed. Alternatively, a specific protein or set of proteins may
be labeled with heavy atom isotopes and then derivatized with an
affinity tagging reagent.
[0033] Also, it is possible to quantify the levels of specific
proteins in multiple samples in a single analysis (multiplexing).
In this case, affinity tagging reagent s used to derivatize
proteins present in different affinity tagged peptides from
different samples can be selectively quantified by mass
spectrometry.
[0034] In this aspect of the invention, the method provides for
quantitative measurement of specific proteins in biological fluids,
cells or tissues and can be applied to determine global protein
expression profiles in different cells and tissues. The same
general strategy can be broadened to achieve the proteome-wide,
qualitative and quantitative analysis of the state of modification
of proteins, by employing affinity reagents with differing
specificity for reaction with proteins. The method and reagents of
this invention can be used to identify low abundance proteins in
complex mixtures and can be used to selectively analyze specific
groups or classes of proteins such as membrane or cell surface
proteins, or proteins contained within organelles, sub-cellular
fractions, or biochemical fractions such as immunoprecipitates.
Further, these methods can be applied to analyze differences in
expressed proteins in different cell states. For example, the
methods and reagents herein can be employed in diagnostic assays
for the detection of the presence or the absence of one or more
proteins indicative of a disease state, such as cancer.
[0035] In a second aspect, the invention provides a MS method for
detection of the presence or absence of a protein function, e.g.,
an enzyme activity, in a sample. The method can also be employed to
detect a deficiency or excess (over normal levels) of protein
function in a sample. Samples that can be analyzed include various
biological fluids and materials, including tissue and cells. In
this case, the PRG of the affinity labeled reagent is a substrate
for the enzyme of interest. Affinity labeled substrates are
provided for each enzyme of interest and are introduced into a
sample where they react to generate affinity labeled products, if
the enzyme of interest is present in the sample. Products or
unreacted substrate that are tagged with the affinity label are
isolated by an affinity isolation method, e.g., affinity
chromatography, via their selective binding to the CR. The isolated
tagged substrates and products are analyzed by mass spectrometry.
Affinity labeled products include those in which the substrate is
entirely cleaved from the linker or in which the substrate is
modified by reaction with a protein of interest. Detection of the
affinity-labeled product indicates the protein function is present
in the sample. Detection of little or no affinity labeled product
indicates deficiency or absence, respectively, of the protein
function in the sample.
[0036] The amount of selected protein, e.g., measured in terms of
enzyme activity, present in a sample can be measured by introducing
a known amount of an internal standard which is an isotopically
labeled analog of the expected product of the enzymatic reaction of
the reagent substrate. The internal standard is substantially
chemically identical to the expected enzymatic reaction product,
but is isotopically distinguishable therefrom. The level of protein
function (e.g., enzymatic activity) in a given sample can be
compared with activity levels in other samples or controls (either
negative or positive controls). The procedure therefore can detect
the presence, absence, deficiency or excess of a protein function
in a sample. The method is capable of quantifying the velocity of
an enzymatic reaction since it enables the amount of product formed
over a known time period to be measured. This method can be
multiplexed, by simultaneous use of a plurality of affinity labeled
substrates selective for different protein functions and if
quantitation is desired by inclusion of the corresponding internal
standards for expected products, to analyze for a plurality of
protein functions in a single sample.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The methods of this invention employ affinity tagged protein
reactive reagents in which the affinity tag is covalently attached
to a protein reactive group by a linker. The linker can be
isotopically labeled to generate pairs or sets of reagents that are
substantially chemically identical, but which are distinguishable
by mass. For example a pair of reagents, one of which is
isotopically heavy and the other of which is isotopically light can
be employed for the comparison of two samples one of which may be a
reference sample containing one or more known proteins in known
amounts. For example, any one or more of the hydrogen, nitrogen,
oxygen or sulfur atoms in the linker may be replaced with their
isotopically stable isotopes: .sup.2H, .sup.13C, .sup.15N ,
.sup.17O, .sup.18O or .sup.34S.
[0038] Suitable affinity tags bind selectively either covalently or
non-covalently and with high affinity to a capture reagent (CR).
The CR-A interaction or bond should remain intact after extensive
and multiple washings with a variety of solutions to remove
non-specifically bound components. The affinity tag binds minimally
or preferably not at all to components in the assay system, except
CR, and does not significantly bind to surfaces of reaction
vessels. Any non-specific interaction of the affinity tag with
other components or surfaces should be disrupted by multiple washes
that leave CR-A intact. Further, it must be possible to disrupt the
interaction of A and CR to release peptides, substrates or reaction
products, for example, by addition of a displacing ligand or by
changing the temperature or solvent conditions. Preferably, neither
CR or A react chemically with other components in the assay system
and both groups should be chemically stable over the time period of
an assay or experiment. The affinity tag preferably does not
undergo peptide-like fragmentation during (MS).sup.n analysis. The
affinity label is preferably soluble in the sample liquid to be
analyzed and the CR should remain soluble in the sample liquid even
though attached to an insoluble resin such as Agarose. In the case
of CR term soluble means that CR is sufficiently hydrated or
otherwise solvated such that it functions properly for binding to
A. CR or CR-containing conjugates should not be present in the
sample to be analyzed, except when added to capture A.
[0039] Examples of A and CR pairs include:
[0040] d-biotin or structurally modified biotin-based reagents,
including d-iminobiotin, which bind to proteins of the
avidin/streptavidin, which may, for example, be used in the forms
of strepavidin-Agarose, oligomeric-avidin-Agarose, or
monomeric-avidin-Agarose;
[0041] any 1,2-diol, such as 1,2-dihydroxyethane
(HO--CH.sub.2--CH.sub.2--- OH), and other 1,2-dihydroxyalkanes
including those of cyclic alkanes, e.g., 1,2-dihydroxycyclohexane
which bind to an alkyl or aryl boronic acid or boronic acid esters
, such as phenyl-B(OH).sub.2 or hexyl-B(OEthyl).sub.2 which may be
attached via the alkyl or aryl group to a solid support material,
such as Agarose;
[0042] maltose which binds to maltose binding protein (as well as
any other sugar/sugar binding protein pair or more generally to any
ligand/ligand binding protein pairs that has properties discussed
above);
[0043] a hapten, such as dinitrophenyl group, for any antibody
where the hapten binds to an anti-hapten antibody that recognizes
the hapten, for example the dinitrophenyl group will bind to an
anti-dinitrophenyl-lgG;
[0044] a ligand which binds to a transition metal, for example, an
oligomeric histidine will bind to Ni(II), the transition metal CR
may be used in the form of a resin bound chelated transition metal,
such as nitrilotriacetic acid-chelated Ni(II) or iminodiacetic
acid-chelated Ni(II);
[0045] glutathione which binds to glutathione-S-transferase.
[0046] In general, any A-CR pair commonly used for affinity
enrichment which meets the suitability criteria discussed above.
Biotin and biotin-based affinity tags are preferred. Of particular
interest are structurally modified biotins, such as d-iminobiotin,
which will elute from avidin or strepavidin columns under solvent
conditions compatible with ESI-MS analysis, such as dilute acids
containing 10-20% organic solvent. It is expected that
d-iminobiotin tagged compounds will elute in solvents below pH 4.
d-Iminobiotin tagged protein reactive reagents can be synthesized
by methods described herein for the corresponding biotin tagged
reagents.
[0047] A displacement ligand, DL, is optionally used to displace A
from CR. Suitable DLs are not typically present in samples unless
added. DL should be chemically and enzymatically stable in the
sample to be analyzed and should not react with or bind to
components (other than CR) in samples or bind non-specifically to
reaction vessel walls. DL preferably does not undergo peptide-like
fragmentation during MS analysis, and its presence in sample should
not significantly suppress the ionization of tagged peptide,
substrate or reaction product conjugates.
[0048] DL itself preferably is minimally ionized during mass
spectrometric analysis and the formation of ions composed of DL
clusters is preferably minimal. The selection of DL, depends upon
the A and CR groups that are employed. In general, DL is selected
to displace A from CR in a reasonable time scale, at most within a
week of its addition, but more preferably within a few minutes or
up to an hour. The affinity of DL for CR should be comparable or
stronger than the affinity of the tagged compounds containing A for
CR. Furthermore, DL should be soluble in the solvent used during
the elution of tagged compounds containing A from CR. DL preferably
is free A or a derivative or structural modification of A. Examples
of DL include, d-biotin or d-biotin derivatives, particularly those
containing groups that suppress cluster formation or suppress
ionization in MS.
[0049] The linker group (L) should be soluble in the sample liquid
to be analyzed and it should be stable with respect to chemical
reaction, e.g., substantially chemically inert, with components of
the sample as well as A and CR groups. The linker when bound to A
should not interfere with the specific interaction of A with CR or
interfere with the displacement of A from CR by a displacing ligand
or by a change in temperature or solvent. The linker should bind
minimally or preferably not at all to other components in the
system, to reaction vessel surfaces or CR. Any non-specific
interactions of the linker should be broken after multiple washes
which leave the A-CR complex intact. Linkers preferably do not
undergo peptide-like fragmentation during (MS).sup.n analysis. At
least some of the atoms in the linker groups should be readily
replaceable with stable heavy-atom isotopes. The linker preferably
contains groups or moieties that facilitate ionization of the
affinity tagged reagents, peptides, substrates or reaction
products.
[0050] To promote ionization, the linker may contain acidic or
basic groups, e.g., COOH.sub.1 SO.sub.3H, primary, secondary or
tertiary amino groups, nitrogen-heterocycles, ethers, or
combinations of these groups. The linker may also contain groups
having a permanent charge, e.g., phosphonium groups, quaternary
ammonium groups, sulfonium groups, chelated metal ions, tetralky or
tetraryl borate or stable carbanions.
[0051] The covalent bond of the linker to A or PRG should typically
not be unintentionally cleaved by chemical or enzymatic reactions
during the assay. In some cases it may be desirable to cleave the
linker from the affinity tag A or from the PRG, for example to
facilitate release from an affinity column. Thus, the linker can be
cleavable, for example, by chemical, thermal or photochemical
reaction. Photocleavable groups in the linker may include the
1-(2-nitrophenyl)-ethyl group. Thermally labile linkers may, for
example, be a double-stranded duplex formed from two complementary
strands of nucleic acid, a strand of a nucleic acid with a
complementary strand of a peptide nucleic acid, or two
complementary peptide nucleic acid strands which will dissociate
upon heating. Cleavable linkers also include those having disulfide
bonds, acid or base labile groups, including among others,
diarylmethyl or trimethylarylmethyl groups, silyl ethers,
carbamates, oxyesters, thiesters, thionoesters, and
.alpha.-fluorinated amides and esters. Enzymatically cleavable
linkers can contain, for example, protease-sensitive amides or
esters, .beta.-lactamase-sensitive .beta.-lactam analogs and
linkers that are nuclease-cleavable, or glycosidase-cleavable.
[0052] The protein reactive group (PRG) can be a group that
selectively reacts with certain protein functional groups or is a
substrate of an enzyme of interest. Any selectively reactive
protein reactive group should react with a functional group of
interest that is present in at least a portion of the proteins in a
sample. Reaction of PRG with functional groups on the protein
should occur under conditions that do not lead to substantial
degradation of the compounds in the sample to be analyzed. Examples
of selectively reactive PRGs suitable for use in the affinity
tagged reagents of this invention, include those which react with
sulfhydryl groups to tag proteins containing cysteine, those that
react with amino groups, carboxylate groups, ester groups,
phosphate reactive groups, and aldehyde and/or ketone reactive
groups or, after fragmentation with CNBr, with homoserine
lactone.
[0053] Thiol reactive groups include epoxides, .alpha.-haloacyl
group, nitriles, sulfonated alkyl or aryl thiols and maleimides.
Amino reactive groups tag amino groups in proteins and include
sulfonyl halides, isocyanates, isothiocyanantes, active esters,
including tetrafluorophenyl esters, and N-hydroxysuccinimidyl
esters, acid halides, and acid anyhydrides. In addition, amino
reactive groups include aldehydes or ketones in the presence or
absence of NaBH.sub.4 or NaCNBH.sub.3.
[0054] Carboxylic acid reactive groups include amines or alcohols
in the presence of a coupling agent such as
dicyclohexylcarbodiimide, or 2,3,5,6-tetrafluorophenyl
trifluoroacetate and in the presence or absence of a coupling
catalyst such as 4-dimethylaminopyridine; and transition
metal-diamine complexes including Cu(II)phenanthroline
[0055] Ester reactive groups include amines which, for example,
react with homoserine lactone.
[0056] Phosphate reactive groups include chelated metal where the
metal is, for example Fe(III) or Ga(III), chelated to, for example,
nitrilotriacetiac acid or iminodiacetic acid.
[0057] Aldehyde or ketone reactive groups include amine plus
NaBH.sub.4 or NaCNBH.sub.3, or these reagents after first treating
a carbohydrate with periodate to generate an aldehyde or
ketone.
[0058] PRG groups can also be substrates for a selected enzyme of
interest. The enzyme of interest may, for example, be one that is
associated with a disease state or birth defect or one that is
routinely assayed for medical purposes. Enzyme substrates of
interest for use with the methods of this invention include, acid
phosphatase, alkaline phosphatase, alanine aminotransferase,
amylase, angiotensin converting enzyme, aspartate aminotransferase,
creatine kinase, gamma-glutamyltransferase, lipase, lactate
dehydrogenase, and glucose-6-phosphate dehydrogenase which are
currently routinely assayed by other methods.
[0059] The requirements discussed above for A, L, PRG, extend to
the corresponding to the segments of A-L-PRG and the reaction
products generated with this reagent.
[0060] Internal standards, which are appropriately isotopically
labeled, may be employed in the methods of this invention to
measure absolute quantitative amounts of proteins in samples.
Internal standards are of particular use in assays intended to
quantitate affinity tagged products of enzymatic reactions. In this
application, the internal standard is chemically identical to the
tagged enzymatic product generated by the action of the enzyme on
the affinity tagged enzyme substrate, but carries isotope labels
which may include .sup.2H, .sup.13C, .sup.15N, .sup.17O, .sup.18O,
or .sup.34S, that allow it to be independently detected by MS
techniques. Internal standards for use in method herein to
quantitative one or several proteins in a sample are prepared by
reaction of affinity labeled protein reactive reagents with a known
protein to generate the affinity tagged peptides generated from
digestion of the tagged protein. Affinity tagged peptides internal
standards are substantially chemically identical to the
corresponding affinity tagged peptides generated from digestion of
affinity tagged protein, except that they are differentially
isotopically labeled to allow their independent detection by MS
techniques.
[0061] The method of this invention can also be applied to
determine the relative quantities of one or more proteins in two or
more protein samples, the proteins in each sample are reacted with
affinity tagging reagents which are substantially chemically
identical but differentially isotopically labeled. The samples are
combined and processed as one. The relative quantity of each tagged
peptide which reflects the relative quantity of the protein from
which the peptide originates is determined by the measurement of
the respective isotope peaks by mass spectrometry.
[0062] The methods of this invention can be applied to the analysis
or comparison of multiple different samples. Samples that can be
analyzed by methods of this invention include cell homogenates;
cell fractions; biological fluids including urine, blood, and
cerebrospinal fluid; tissue homogenates; tears; feces; saliva;
lavage fluids such as lung or peritoneal ravages; mixtures of
biological molecules including proteins, lipids, carbohydrates and
nucleic acids generated by partial or complete fractionation of
cell or tissue homogenates.
[0063] The methods of this invention employ MS and (MS).sup.n
methods. While a variety of MS and (MS).sup.n are available and may
be used in these methods, Matrix Assisted Laser Desorption
Ionization MS (MALDI/MS) and Electrospray Ionization MS (ESI/MS)
methods are preferred.
[0064] Quantitative Proteome Analysis
[0065] This method is schematically illustrated in Scheme 1 using a
biotin labeled sulfhydryl-reactive reagent for quantitative protein
profile measurements in a sample protein mixture and a reference
protein mixture. The method comprises the following steps:
[0066] Reduction. Disulfide bonds of proteins in the sample and
reference mixtures are reduced to free SH groups. The preferred
reducing agent is tri-n-butylphosphine which is used under standard
conditions. Alternative reducing agents include mercaptoethylamine
and dithiothreitol. If required, this reaction can be performed in
the presence of solubilizing agents including high concentrations
of urea and detergents to maintain protein solubility. The
reference and sample protein mixtures to be compared are processed
separately, applying identical reaction conditions;
[0067] Derivatization of SH groups with an affinity tag. Free SH
groups are derivatized with the biotinylating reagent
biotinyl-iodoacetylamidyl-- 4,7,10 trioxatridecanediamine the
synthesis of which is described below. The reagent is prepared in
different isotopically labeled forms by substitution of linker
atoms with stable isotopes and each sample is derivatized with a
different isotopically labeled form of the reagent. Derivatization
of SH groups is preferably performed under slightly basic
conditions (pH 8.5) for 90 min at RT. For the quantitative,
comparative analysis of two samples, one sample each (termed
reference sample and sample) are derivatized as illustrated in
Scheme 1 with the isotopically light and the isotopically heavy
form of the reagent, respectively. For the comparative analysis of
several samples one sample is designated a reference to which the
other samples are related to. Typically, the reference sample is
labeled with the isotopically heavy reagent and the experimental
samples are labeled with the isotopically light form of the
reagent, although this choice of reagents is arbitrary. These
reactions are also compatible with the presence of high
concentrations of solubilizing agents;
[0068] Combination of labeled samples. After completion of the
affinity tagging reaction defined aliquots of the samples labeled
with the isotopically different reagents (e.g., heavy and light
reagents) are combined and all the subsequent steps are performed
on the pooled samples. Combination of the differentially labeled
samples at this early stage of the procedure eliminates variability
due to subsequent reactions and manipulations. Preferably equal
amounts of each sample are combined;
[0069] Removal of excess affinity tagged reagent. Excess reagent is
adsorbed, for example, by adding an excess of SH-containing beads
to the reaction mixture after protein SH groups are completely
derivatized. Beads are added to the solution to achieve about a
5-fold molar excess of SH groups over the reagent added and
incubated for 30 min at RT. After the reaction the beads are be
removed by centrifugation;
[0070] Protein digestion. The proteins in the sample mixture are
digested, typically with trypsin. Alternative proteases are also
compatible with the procedure as in fact are chemical fragmentation
procedures. In cases in which the preceding steps were performed in
the presence of high concentrations of denaturing solubilizing
agents the sample mixture are diluted until the denaturant
concentration is compatible with the activity of the proteases
used. This step may be omitting in the analysis of small
proteins;
[0071] Affinity isolation of the affinity tagged peptides by
interaction with a capture reagent. The biotinylated peptides are
isolated on avidin-agarose. After digestion the pH of the peptide
samples is lowered to 6.5 and the biotinylated peptides are
immobilized on beads coated with monomeric avidin (Pierce). The
beads are extensively washed. The last washing solvent includes 10%
methanol to remove residual SDS. Biotinylated peptides are eluted
from avidin-agarose, for example, with 0.3% formic acid at pH
2;
[0072] Analysis of the isolated, derivatized peptides by
.mu.LC-MS.sup.n or CE-MS.sup.n with data dependent fragmentation.
Methods and instrument control protocols well-known in the art and
described, for example, in Ducret et al., 1998; Figeys and
Aebersold, 1998; Figeys et al., 1996; or Haynes et al., 1998 are
used.
[0073] In this last step, both the quantity and sequence identity
of the proteins from which the tagged peptides originated can be
determined by automated multistage MS. This is achieved by the
operation of the mass spectrometer in a dual mode in which it
alternates in successive scans between measuring the relative
quantities of peptides eluting from the capillary column and
recording the sequence information of selected peptides. Peptides
are quantified by measuring in the MS mode the relative signal
intensities for pairs of peptide ions of identical sequence that
are tagged with the isotopically light or heavy forms of the
reagent, respectively, and which therefore differ in mass by the
mass differential encoded within the affinity tagged reagent.
Peptide sequence information is automatically generated by
selecting peptide ions of a particular mass-to-charge (m/z) ratio
for collision-induced dissociation (CID) in the mass spectrometer
operating in the MS.sup.n mode. (Link, A. J. et al., 1997; Gygi, S.
P., et al. 1999; and Gygi, S. P. et al., 1999). The resulting CID
spectra are then automatically correlated with sequence databases
to identify the protein from which the sequenced peptide
originated. Combination of the results generated by MS and MS.sup.n
analyses of affinity tagged and differentially labeled peptide
samples therefore determines the relative quantities as well as the
sequence identities of the components of protein mixtures in a
single, automated operation.
[0074] Results of this applying this method using the biotinylated
sulfhydryl reagent and to the quantitative analysis of synthetic
peptide samples, to the relative quantitation of the peptides in a
protein digest an the tandem mass spectral analysis of a
derivatized peptide are shown in FIG. 1, Table 1, and FIG. 2,
respectively.
[0075] This method can also be practiced using other affinity tags
and other protein reactive groups, including amino reactive groups,
carboxyl reactive groups, or groups that react with homoserine
lactones.
[0076] The approach employed herein for quantitative proteome
analysis is based on two principles. First, a short sequence of
contiguous amino acids from a protein (5-25 residues) contains
sufficient information to uniquely identify that protein. Protein
identification by MS.sup.n is accomplished by correlating the
sequence information contained in the CID mass spectrum with
sequence databases, using sophisticated computer searching
algorithms (Eng, J. et al., 1994; Mann, M. et al., 1994; Qin, J. et
al., 1997; Clauser, K. R. et al., 1995). Second, pairs of identical
peptides tagged with the light and heavy affinity tagged reagents,
respectively, (or in analysis of more than two samples, sets of
identical tagged peptides in which each set member is
differentially isotopically labeled) are chemically identical and
therefore serve as mutual internal standards for accurate
quantitation. The MS measurement readily differentiates between
peptides originating from different samples, representing for
example different cell states, because of the difference between
isotopically distinct reagents attached to the peptides. The ratios
between the intensities of the differing weight components of these
pairs or sets of peaks provide an accurate measure of the relative
abundance of the peptides (and hence the proteins) in the original
cell pools because the MS intensity response to a given peptide is
independent of the isotopic composition of the reagents (De
Leenheer, A. P. et al (1992). The use of isotopically labeled
internal standards is standard practice in quantitative mass
spectrometry and has been exploited to great advantage in, for
example, the precise quantitation of drugs and metabolites in
bodily fluids (De Leenheer, A. P. et al., 1992).
[0077] In another illustration of the method, two mixtures
consisting of the same six proteins at known, but different,
concentrations were prepared and analyzed. The protein mixtures
were labeled, combined and treated as schematically illustrated in
Scheme 1. The isolated, tagged peptides were quantified and
sequenced in a single combined .mu.LC-MS and .mu.LC-MS.sup.n
experiment on an ESI ion trap mass spectrometer. All six proteins
were unambiguously identified and accurately quantified (Table 2).
Multiple tagged peptides were encountered for each protein. The
differences between the observed and expected quantities for the
six proteins ranged between 2 and 12%.
[0078] The process is further illustrated for a single peptide pair
in FIGS. 3A-C. A single scan of the mass spectrometer operated in
MS mode is shown in FIG. 3A. Four pairs of peptide ions
characterized by the mass differential encoded in the affinity
tagged reagent are detected in this scan and indicated with their
respective m/z values. The scan shown was acquired in 1.3 s. Over
the course of the one-hour chromatographic elution gradient, more
than 1200 such scans were automatically recorded. FIG. 3B shows an
expanded view of the mass spectrum around the ion pair with m/z
ratios of 993.8 and 977.7, respectively. Co-elution and a detected
mass differential of four units potentially identifies the ions as
a pair of doubly charged affinity tagged peptides of identical
sequence (mass difference of eight and a charge state of two). FIG.
3C shows the reconstructed ion chromatograms for these two species.
The relative quantities were determined by integrating the contour
of the respective peaks. The ratio (light/heavy) was determined as
0.54 (Table 1). The peaks in the reconstructed ion chromatograms
appear serrated because in every second scan the mass spectrometer
switched between the MS and the MS.sup.n modes to collect sequence
information (CID mass spectrum) of a selected peptide ion. These
CID spectra were used to identify the protein from which the tagged
peptides originated. FIG. 4A shows the CID spectrum recorded from
the peptide ion with m/z=998 (marked with an arrow in FIG. 3A).
Database searching with this CID spectrum identified the protein as
glyceraldehyde-3-phosphate dehydrogenase (FIG. 4B) which was a
member of the protein mixture.
[0079] Several beneficial features of the this method are apparent.
First, at least two peptides were detected from each protein in the
mixture. Therefore, both quantitation and protein identification
can be redundant. Second, the identified peptides all contained at
least one tagged cysteinyl residue. The presence of the relatively
rare cysteinyl residue in a peptide adds an additional powerful
constraint for database searching (Sechi, S. et al., 1998). Third,
tagging and selective enrichment of cysteine-containing peptides
significantly reduced the complexity of the peptide mixture
generated by the concurrent digestion of six proteins. For this
protein mixture, the complexity was reduced from 293 potential
tryptic peptides to 44 tryptic peptides containing at least one
cysteinyl residue. Fourth, the peptide samples eluted from the
avidin affinity column are directly compatible with analysis by
.mu.LC-MS.sup.n.
[0080] Quantitative Analysis of Protein Expression in Different
Cell States
[0081] The protein reactive affinity reagent strategy was applied
to study differences in steady-state protein expression in the
yeast, S. cerevisiae, in two non-glucose repressed states (Table
3). Cells were harvested from yeast growing in log-phase utilizing
either 2% galactose or 2% ethanol as the carbon source. One-hundred
.mu.g of soluble yeast protein from each cell state were labeled
independently with the isotopically different affinity tagged
reagents. The labeled samples were combined and subjected to the
strategy described in FIG. 1. One fiftieth (the equivalent of
approximately 2 .mu.g of protein from each cell state) of the
sample was analyzed.
[0082] Glucose repression causes large numbers of proteins with
metabolic functions significant to growth on other carbon sources
to be minimally expressed (Ronne, H., 1995; Hodges, P. E. et al.,
1999). Growth on galactose or ethanol with no glucose present
results in the expression of glucose repressed genes. Table 3
presents a selection of 34 yeast genes encountered in the analysis,
but it contains every known glucose-repressed genes that was
identified (Mann, M. et al., 1994). Each of these genes would have
been minimally expressed in yeast grown on glucose. Genes specific
to both growth on galactose (GAL1, GALL 10) as well as growth on
ethanol (ADH2, ACH1) were detected and quantitated.
[0083] The quantitative nature of the method is apparent in the
ability to accurately measure small changes in relative protein
levels. Evidence of the accuracy of the measurements can be seen by
the excellent agreement found by examining ratios for proteins for
which multiple peptides were quantified. For example, the five
peptides found from PCK1 had a mean ratio .+-.95% confidence
intervals of 1.57.+-.0.15, and the percent errorwas <10%. In
addition, the observed changes fit the expected changes from the
literature (Ronne, H., 1995; Hodges, P. E. et al., 1999). Finally,
the observed changes are in agreement with the changes in staining
intensity for these same proteins examined after two-dimensional
gel electrophoresis (data not shown).
[0084] The alcohol dehydrogenase family of isozymes in yeast
facilitates growth on either hexose sugars (ADH1) and ethanol
(ADH2). The gene ADH2 encodes an enzyme that is both glucose- and
galactose-repressed and permits a yeast cell to grow entirely on
ethanol by converting it into acetaldehyde which enters the TCA
cycle (FIG. 5A). In the presence of sugar, ADH1 performs the
reverse reaction converting acetaldehyde into ethanol. The
regulation of these isozymes is key to carbon utilization in yeast
(Ronne, H., 1995). The ability to accurately measure differences in
gene expression across families of isozymes is sometimes difficult
using cDNA array techniques because of cross hybridization (DeRisi,
J. L. et al., 1997). The method of this invention applied as
illustrated in Fig. 1 succeeded in measuring gene expression for
each isozyme even though ADH1 and ADH2 share 93% amino acid (88%
nucleotide) sequence similarity. This was because the affinity
tagged peptides from each isozyme differed by a single amino acid
residue (valine to threonine) which shifted the retention time by
more than 2 min and the mass by 2 daltons for the ADH2 peptides
(FIG. 5B). ADH1 was expressed at approximately 2-fold high levels
when galactose was the carbon source compared with ethanol.
Ethanol-induction of ADH2 expression resulted in more than 200-fold
increases compared with galactose-induction.
[0085] The results described above illustrate that the method of
this invention provides quantitative analysis of protein mixtures
and the identification of the protein components therein in a
single, automated operation.
[0086] The method as applied using a sulfhydryl reactive reagent
significantly reduces the complexity of the peptide mixtures
because affinity tagged cysteine-containing peptides are
selectively isolated. For example, a theoretical tryptic digest of
the entire yeast proteome (6113 proteins) produces 344,855
peptides, but only 30,619 of these peptides contain a cysteinyl
residue. Thus, the complexity of the mixture is reduced, while
protein quantitation and identification are still achieved. The
chemical reaction in of the sulfhydryl reagent with protein can be
performed in the presence of urea, sodium dodecyl sulfate (SDS),
salts and other chemicals that do not contain a reactive thiol
group. Therefore, proteins can be kept in solution with powerful
stabilizing agents until they are enzymatically digested. The
sensitivity of the .mu.LC-MS.sup.n system is dependent of the
sample quality. In particular, commonly used protein solubilizing
agents are poorly compatible or incompatible with MS. Affinity
purification of the tagged peptides completely eliminates
contaminants incompatible with MS. The quantitation and
identification of low abundance proteins by conventional methods
requires large amounts (milligrams) of starting protein lysate and
involves some type of enrichment for these low abundance proteins.
Assays described above, start with about 100 .mu.g of protein and
used no fractionation techniques. Of this, approximately {fraction
(1/50)} of the protein was analyzed in a single .mu.LC-MS.sup.n
experiment. This system has a limit of detection of 10-20 fmol per
peptide (Gygi, S. P. et al., 1999). For this reason, in the assays
described which employ .mu.LC-MS.sup.n only abundant proteins are
detected. However, the methods of this invention are compatible
with any biochemical, immunological or cell biological
fractionation methods that reduce the mixture complexity and enrich
for proteins of low abundance while quantitation is maintained.
This method can be redundant in both quantitation and
identification if multiple cysteines are detected. There is a
dynamic range associated with the ability of the method to
quantitate differences in expression levels of affinity tagged
peptides which is dependent on both the intensity of the peaks
corresponding the peptide pair (or set) and the overall mixture
complexity. In addition, this dynamic range will be different for
each type of mass spectrometer used. The ion trap was employed in
assays described herein because of its ability to collect
impressive amounts of sequencing information (thousands of proteins
can potentially be identified) in a data-dependent fashion even
though it offers a more limited dynamic quantitation range. The
dynamic range of the ion trap (based on signal-to-noise ratios)
varied depending on the signal intensity of the peptide pair and
complexity of the mixture, but differences of up to 100-fold were
generally detectable and even larger differences could be
determined for more abundant peptides. In addition, protein
expression level changes of more than 100-200-fold still identify
those proteins as major potential contributors to the phenotypic
differences between the two original cell states. The method can be
extended to include reactivity toward other functional groups. A
small percentage of proteins (8% for S. cerevisiae) contain no
cysteinyl residues and are therefore missed by analysis using
reagents with sulfhydryl group specificity (i.e.,thiol group
specificity). Affinity tagged reagents with specificities toward
functional groups other than sulfhydryl groups will also make
cysteine-free proteins susceptible to analysis.
[0087] The methods of this invention can be applied to analysis of
low abundance proteins and classes of proteins with particular
physico-chemical properties including poor solubility, large or
small size and extreme p/values.
[0088] The prototypical application of the chemistry and method is
the establishment of quantitative profiles of complex protein
samples and ultimately total lysates of cells and tissues following
the preferred method described above. In addition the reagents and
methods of this invention have applications which go beyond the
determination of protein expression profiles. Such applications
include the following:
[0089] Application of amino-reactive or sulfhydryl-reactive,
differentially isotopically labeled affinity tagged reagents for
the quantitative analysis of proteins in immuno precipitated
complexes. In the preferred version of this technique protein
complexes from cells representing different states (e.g., different
states of activation, different disease states, different states of
differentiation) are precipitated with a specific reagent,
preferably an antibody. The proteins in the precipitated complex
are then derivatized and analyzed as above.
[0090] Application of amino-reactive, differentially isotopically
labeled affinity tagged reagents to determine the sites of induced
protein phosphorylation. In a preferred version of this method
purified proteins (e.g., immunoprecipitated from cells under
different stimulatory conditions) are fragmented and derivatized as
described above. Phosphopeptides are identified in the resulting
peptide mixture by fragmentation in the ion source of the ESI-MS
instrument and their relative abundances are determined by
comparing the ion signal intensities of the experimental sample
with the intensity of an included, isotopically labeled
standard.
[0091] Amino-reactive, differentially isotopically labeled affinity
tagged reagents are used to identify the N-terminal ion series in
MS.sup.n spectra. In a preferred version of this application, the
peptides to be analyzed are derivatized with a 50:50 mixture of an
isotopically light and heavy reagent which is specific for amino
groups. Fragmentation of the peptides by CID therefore produce two
N-terminal ion series which differ in mass precisely by the mass
differential of the reagent species used. This application
dramatically reduces the difficulty in determining the amino acid
sequence of the derivatized peptide.
[0092] Quantitative Analysis of Surface Proteins in Cells and
Tissue
[0093] The cell exterior membrane and its associated proteins (cell
surface proteins) participate in sensing external signals and
responding to environmental cues. Changes in the abundance of cell
surface proteins can reflect a specific cellular state or the
ability of a cell to respond to its changing environment. Thus, the
comprehensive, quantitative characterization of the protein
components of the cell surface can identify marker proteins or
constellations of marker proteins characteristic for a particular
cellular state, or explain the molecular basis for cellular
responses to external stimuli. Indeed, changes in expression of a
number of cell surface receptors such as Her2/neu, erbB, IGFI
receptor, and EGF receptor have been implicated in carcinogenesis
and a current immunological therapeutic approach for breast cancer
is based on the infusion of an antibody (Herceptin, Genentech, Palo
Alto, Calif.) that specifically recognizes Her2/neu receptor.
[0094] Cell surface proteins are also experimentally accessible.
Diagnostic assays for cell classification and preparative isolation
of specific cells by methods such as cell sorting or panning are
based on cell surface proteins. Thus, differential analysis of cell
surface proteins between normal and diseased (e.g., cancer) cells
can identify important diagnostic or therapeutic targets. While the
importance of cell surface proteins for diagnosis and therapy of
cancer has been recognized, membrane proteins have been difficult
to analyze. Due to their generally poor solubility they tend to be
under-represented in standard 2D gel electrophoresis patterns and
attempts to adapt 2D electrophoresis conditions to the separation
of membrane proteins have met limited success. The method of this
invention can overcome the limitations inherent in the traditional
techniques.
[0095] The analysis of membrane proteins is challenging because
they generally are difficult to maintain in solution under
conditions that are compatible with high sensitivity analytical
instruments such as mass spectrometers. The application of the
methods of the present invention to the analysis of membrane
proteins is exemplified using human T cell lymphoma cell line
Jurkat for membrane protein labeling and extraction and the well
characterized human prostate epithelial cell line P69SV40T and two
P69SV40T sublines which differ in IGF-1 receptor expression by
factor of 10 to exemplify quantitative, differential analysis of
membrane proteins.
[0096] Jurkat cells are an appropriate model system because the
cells are easy to grow in large numbers and because the modulation
of cell surface proteins in response to different stimuli and
experimental conditions has been well characterized in T
lymphocytes. Commercially available biotinylating reagents or more
generally affinity tagging reagents are employed to derivatize
lysine residues and the free N-termini. Water soluble biotinylating
reagents such as Sulfo-NHS (N-hydroxy succinimide) biotin and
analogs (Sulfosuccinimidyl-6-(biotinamido)-hexanoate, Pierce,
Rockford, Ill.) which have been used extensively for labeling cell
surface proteins can be employed. The reaction of NHS esters with
primary amines is best at neutral pH values and above and is
compatible with the presence of organic solvent such as DMSO or
DMF. Biotinylation of cell surface proteins from the Jurkat cells
is carried out in PBS buffer at pH 7.2. Cells (1.times.10.sup.7)
are washed with PBS buffer to remove contaminating serum and other
proteins from the culture medium. The cells are resuspended at
25.times.10.sup.6 cell/ml and reacted with 0.5 mg/ml of
Sulfo-NHS-Biotin (Pierce, Rockford, Ill.) for 30 min at RT. The
labeled cells are washed twice with cold PBS to remove unreacted
biotinylating reagent. Biotinylated cells are solubilized at
5.times.10.sup.7 cells/ml in lysis buffer containing 1% Triton
X-114. Triton X-114 has the property of phase-partitioning into
detergent phase and aqueous phase at 30.degree. C. Following the
phase partitioning, detergent phase is removed from the aqueous
phase by centrifugation at 300 xg. Phase partitioning has
previously been successfully used to enrich cell membrane. Also,
this technique was found to enrich membrane proteins from Jurkat
cell lysates . Triton phase is diluted 1:5 (v/v) using 50 mM
ammonium bicarbonate buffer, pH 8.5, and high-purity, modified
porcine-trypsin is added to digest the proteins at a concentration
of 12.5 ng/ml for overnight at 37.degree. C. Trypsin is neutralized
by the addition of a cocktail of serine protease inhibitors and
tryptic peptides are isolated by the avidin affinity chromatography
techniques. Eluted peptides are separated e.g., by .mu.LC methods
and identified by searching peptide sequence databases, using for
example, the Sequest program.
[0097] The human prostate epithelial cell line P69SV40T which was
immortalized with SV 40 T antigen has been well characterized .
This cell line is immortal but not tumorigenic and expresses type 1
insulin like growth factor receptor (IGF-1 R) at 2.times.10.sup.4
receptors per cell. A subline, called M12, was derived from
P69SV40T by sequential passage in male athymic nude mice . This
cell line is highly tumorigenic and metastatic and expresses
1.1.times.10.sup.3 IGF-1 R per cell. The relative difference in the
abundance of IGF-1R in the cell lines P69SV40T and M12 can be
quantitatively determined using methods of this invention adapted
for application to membrane proteins. Since the number of IGF-1R
for these cell lines has already been determined, this well
characterized system can provide a reference to validate the
efficiency of the quantitative methods of this invention
[0098] P69SV40T cells (1.times.10.sup.7) are biotinylated with an
isotopically heavy biotin tagged amino reactive reagent and the M12
cells (1.times.10.sup.7) are biotinylated with a corresponding
isotopically light amine reactive biotin tagged amino reactive
reagent. IGF-1 R is then immunoprecipitated from the combined
lysate of both cell lines using an antibody against human IGF-1 R
and the total mass of immunoprecipitated proteins is digested with
trypsin. Trypsin is then neutralized, e.g., by the addition of
inhibitors and tagged peptides are purified by biotin-avidin
affinity chromatography. The eluted peptides are analyzed by LC-MS
and LC-MS.sup.N for peptide quantitation and identification,
respectively, as has been described above. Quantitation in this
experiment is facilitated by the option to use selective ion
monitoring in the MS. In this mode only the masses of tagged
peptide ions expected to derive from IGF-1R need be monitored.
[0099] The described technique can be applied to compare the
differences in relative abundance of cell surface proteins between
parental prostate cell line (P69SV40T) and M12 cells to detect and
identify those cell surface proteins whose expression level is
different in the two cell lines and which may be characteristic of
the different cell states. Using the methods described herein
global, relative quantitation of the cell surface proteins in any
two or more cell lines can be analyzed to detect and identify those
cell surface proteins characteristic of the different cell states.
Results can be independent confirmed using procedure such as 1D or
2D gels, if applicable, or quantitative western blotting to confirm
quantitation results.
[0100] It is expected that the experimental variability of
quantitation of cell surface proteins will be considerably better
than the accuracy of quantitation achieved by currently available
cDNA array technology. In addition to relative protein quantity and
identity, the method can also be used to reveal the orientation of
the protein in the membrane, based on the presumption that intact,
alive cells will exclude the biotinylating reagent.
[0101] Alternative methods can be applied to enhance the
selectivity for tagged peptides derived from cell surface proteins.
For example, tagged cell surface proteins can be trypsinized
directly on the intact cells to generate tagged peptides, purified
and analyzed as discussed. In addition, traditional cell membrane
preparations may be used as an initial step to enrich cell surface
proteins. These methods can include gentle cell lysis with a dounce
homogenizer and series of density gradient centrifugations to
isolate membrane proteins prior to proteolysis. This method can
provide highly enriched preparations of cell surface proteins.
Affinity tagged proteins may also be isolated by affinity
chromatography prior to proteolysis as well as after proteolysis.
This chromatography can be performed in the presence of surfactants
such as TX-100, NP-40 or Tween-20 to maintain protein solubility.
The sequential application of affinity chromatography steps (one
for the intact protein and one for the tagged peptide fragments)
provides a high degree of selectivity. These alternative methods
are easily scalable for the detection of low abundance membrane
proteins and the relative quantity of tagged peptides tagged is
maintained through the selective enrichment steps.
[0102] In the application of the methods of this invention to cell
surface proteins, once the tagged proteins are fragmented, the
tagged peptides behave no differently from the peptides generated
from more soluble samples.
[0103] Synthesis of Affinity Tagged Protein Reactive Reagents That
are Selective for Certain Proteins Groups
[0104] Synthetic routes exemplary affinity tagged reagents suitable
for use in the methods of this invention are provided in Schemes
2-3 where well-known synthetic techniques are employed in synthesis
of the non-deuterated and deuterated reagents.
[0105] Biotinyl-iodoacetylamidyl-4,7,10 trioxatridecanediamine 4
(Scheme 3) consists of a biotin group, a chemically inert spacer of
capable of being isotopically labeled with stable isotopes and a
iodoacetamidyl group, respectively. The biotin group is used for
affinity enrichment of peptides derivatized with the reagent, the
ethylene glycol linker is differentially isotopically labeled for
mass spectral analysis and the iodoacetamidyl group provides
specificity of the reagent for sulfhydryl-containing peptides. The
reagent an be synthesized in an all hydrogen form (isotopically
light form) with and with 1-20, and preferably 4-8 deuterium atoms
in the linker (isotopically heavy forms).
[0106] Analysis of Velocities of Multiple Enzymes in Cell
Lysates
[0107] Monitoring enzyme functions by biochemical assays is an
essential diagnostic tool that employs a multitude of analytical
techniques including spectrophotometric, fluorometric, and
radiometric detection of products. However, current methods are
difficult to use for assaying several enzymes simultaneously in a
single sample. Mass spectrometry for quantification of a collection
of metabolites in biological fluids has emerged as a powerful
approach for the analysis of birth defects (Morris et al., 1994),
but this analytical technique has not been developed for the direct
analysis of rates of individual enzymatic steps. The analytical
method described herein for monitoring and quantification of
enzymatic activities in cell homogenates and other biological
samples permits simultaneous (multiplex) monitoring of multiple
reactions, and can be readily automated.
[0108] A feature of the method of this invention as applied to
enzyme assays is the use of electrospray ionization mass
spectrometry (ESI-MS) (Cole et al., 1997) for the simultaneous
detection of enzymatic products and chemically identical internal
standards, which are distinguished by stable isotope (deuterium)
labeling. A second feature is the use of affinity tagged reagents
containing an enzyme substrate which when combined with affinity
purification provide for facile capture of enzymatic products from
crude biological fluids. The affinity tagged reagents are designed
to contain a target substrate for an enzyme of interest that is
covalently attached to an affinity tag via a linker. Action of the
enzyme of interest on the substrate conjugate causes cleavage or
other modification that changes its molecular mass (Scheme 4). The
change of mass is detected by ESI-MS. The linker and affinity tag
used preferably facilitate ionization by ESI, block action of other
enzymes in the biological fluid, and allow highly selective capture
from the complex matrix for facile purification.
[0109] An example of this approach is the design and synthesis of
affinity tagged enzyme substrate reagents 1 and 2 (Scheme 5) to
simultaneously assay lysosomal .beta.-galactosidase and
N-acetyl-.alpha.-D-glucosaminida- se, respectively. Deficiency of
the former enzyme results in one of the lysosomal storage diseases,
GM.sub.1-gangliosidosis, a condition that occurs in the population
with a frequency of about 1 in 50,000 and leads to early death of
affected children. Deficiency of N-acetyl-R-D-glucosaminidase
results in the rare lysosomal storage disorder Sanfilippo syndrome
type B. This example has been described in Gerber et al., 1999,
which is incorporated by reference herein in its entirety.
[0110] Conjugates 1 and 2 consist of biotin as an affinity tag,
which is coupled to sarcosine. Biotin allows highly specific
capture of the substrate conjugate through non-covalent binding to
streptavidin immobilized on agarose beads (Bayer et al., 1990).
Sarcosine provides an N-methylated amide linkage to biotin to block
the enzyme biotinidase, which is often present in the cellular
fluids and could cause cleavage of the conjugate molecule during
the assay (Wilbur et al., 1997). In addition, it was found that
biotinyl-sarcosine conjugates can be displaced from streptavidin by
addition of biotin. The N-biotinylsarcosine block is linked to a
polyether diamine, the length of which can be varied to avoid
mass/charge overlaps of products and internal standards. The linker
also allows facile introduction of multiple deuterium atoms (i.e.,
8 deuteriums in 5 and 4 in 6, Scheme 5) to permit the synthesis of
internal standards. The d8-linker was made by reacting
DOCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OD with CD.sub.2=CDCN in benzene
with catalytic NaOD (Ashikaga, K., et al., 1988) and the resulting
dinitrile was reduced to the diamine with Ra--Ni. The d4-linker was
made in the same way using ethylene glycol and CD2=dCDCN in
CH.sub.3CN and catalytic NaOH.
[0111] In addition, the linker is hydrophilic to ensure good water
solubility of the substrate conjugate, and it has basic groups
which are efficiently protonated by ESI and thus ensure sensitive
detection by mass spectrometry. The target carbohydrate substrates
are attached to the polyether linker by a .beta.-alanine unit
(Scheme 5). The enzymatic product conjugates 3 and 4 are also shown
Scheme 5. Conjugates 1 and 2 were prepared as shown in Scheme 5.
All reagents were purified to homogeneity by reverse-phase HPLC and
characterized by high-field 1 H NMR and ESI-MS. The substrate was
linked to the diamine spacer by Michael addition of the latter onto
the p-acryloylamidophenyl glycoside, (Romanowska et al., 1994) and
the intermediate was coupled with the tetrafluorophenyl ester of
N-biotinylsarcosine (Wilbur et al., 1997).
[0112] The ESI-MS assay of .beta.-galactosidase and
N-acetyl-R-D-glucosaminidase is based on enzymatic cleavage of the
glycosidic bond to release monosaccharide and conjugates 3 and 4
(mass differences are 162 and 203 Da, respectively). In a typical
procedure, 0.2 mM 1 and 0.3 mM 2 were ineubated with sonicated
cultured fibroblasts from individual patients with
.beta.-galactosidase deficiency and with fibroblasts cultured from
unaffected people. After ineubation, labeled internal standards 5
and 6 were added, and the biotinylated components were captured on
streptavidin-agarose beads. Quantitative strepavidin capture
efficiency from a cell homogenate was observed with model reagents.
After purification by multiple washings to remove nonspecifically
bound components, the biotinylated products were released by free
biotin, and the eluant was analyzed by ESI-MS. About 85% release of
the biotinylated products was observed after ineubation with excess
biotin for 90 min. A blank was obtained by quenching the assay with
all components present at time zero.
[0113] A typical procedure, cell protein (75 .mu.g) in 15 .mu.L of
water was added to 15 .mu.L buffer (0.1 M Na citrate, pH 4.25)
containing 2 (0.3 mM) and 1 (0.2 mM, added 5 h after addition of
cell protein). After ineubation for 5.5 h at 37.degree. C., the
reaction was quenched by addition of 200 .mu.L of 0.2 M glycine
carbonate buffer, pH 10.3, and 5 and 6 (1 nmol each) were added.
After centrifugation to remove cell debris, the supernatant was
loaded onto a bed of streptavidin-agarose (7 nmol biotin binding
capacity, Pierce) in a small filtration device (micro BioSpin,
Bio-Rad). After 5 min, filtration was effected by centrifugation,
and the gel bed was washed with 0.1% Triton X-100 ( about 1 min
ineubation, then spin) and then six times with purified water
(Milli-Q, Millipore). Elution was carried out in 25 .mu.L of 50%
methanol containing 56 nmol of free biotin (1-10 h ineubation, then
spin). Filtrate was diluted 4-fold with 50% methanol/water, and 1
.mu.L was analyzed by ESI-MS.
[0114] The ESI-MS spectrum of the blank (FIG. 6A) is remarkably
simple, showing peaks of the (M+H).sup.+ ions from reagents 1 and 2
(m/z 843 and 840), internal standards 5 and 6 (m/z 689 and 641),
and trace amounts of products 3 and 4 (m/z 681 and 637). Ions due
to clusters of biotin also appear in the spectrum but did not
interfere with the analysis. The presence of nondeuterated products
in the blank may be due to nonenzymatic substrate reagent
hydrolysis during sample work up or to collision-induced
dissociation of the substrate ion in the gas phase. A MS/MS
spectrum of the (conjugate 1+H).sup.+ ion at m/z 843 gave a
prominent fragment of (conjugate 3+H).sup.+ at m/z 681 (spectrum
not shown). The ESI-MS spectrum of a sample ineubated with cell
homogenate from a healthy individual clearly shows the
p-galactosidase product at m/z 681 and the
N-acetyl-.alpha.-D-glucosaminidase product at m/z 637 (FIG. 6B).
Triplicate enzymatic reactions using cells from a healthy patient
yielded a .beta.-galactosidase specific activity of 51.+-.3
nmol/h/(mg cell protein) and an N-acetyl-.alpha.-D-glucosaminidase
specific activity of 1.4.+-.0.3 nmol/h/mg. Time course studies
confirmed that the initial reaction velocities were being measured.
Values obtained with cells from six healthy individuals ranged from
36.+-.4 to 68.+-.3 nmol/h/mg for .beta.-galactosidase and
0.9.+-.0.05 to 2.3.+-.0.4 nmol/h/mg for
N-acetyl-.alpha.-D-glucosaminidase. In contrast, very little
enzymatic product above the blank level (0.9.+-.0.9 and 0.8.+-.0.6
nmol/h/mg) was observed when cells from two patients with
galactosidase deficiency were used, whereas
N-acetyl-.alpha.-D-glucosaminidase activity is clearly detected
(FIG. 6C). These spectra were obtained with 0.75 .mu.g of cell
protein, corresponding to .about.1000 fibroblasts. Thus the ESI-MS
method has very high sensitivity for biomedical applications.
[0115] ESI-MS was carried out on a Finnigan LCQ ion trap
instrument. Data were collected in full scan mode from m/z 625 to
875 by direct infusion at 1.5 .mu.L/min. Specific activities were
obtained from the ratio of product to internal standard ion peak
areas (averaged over 30 scans).
[0116] The approach described for assaying enzymes using substrate
reagents and ESI-MS can be broadly applied. The multiplex technique
can be expanded to assay dozens or more enzymes simultaneously in a
single reaction, obviating the need for multiple assays to assist
in confirming diagnoses of rare disorders. The method can be used
to measure several enzymes simultaneously when evaluating the rate
of chemical flux through a specific biochemical pathway or for
monitoring biochemical signaling pathways. The affinity tag-capture
reagent method for isolation of affinity tagged reaction products
and substrates from complex mixtures is technically simple and can
be readily automated, particular when biotin-strepavidin is
employed. Because of the high sensitivity of the ESI-MS detection
employed, which requires only sub-microgram quantities of the
substrate reagents per assay, the synthesis of several hundred
substrate reagents on a low-gram scale becomes practical and
economical. Since most enzyme active sites are exposed to solvent,
it is possible to attach an affinity tagged linker to most enzyme
substrates while preserving enzymatic activity. Scheme 6 provides
the structures of several additional enzyme substrates, suitable
for use in this method, indicating by arrows allowable positions
for tag attachment sites. Allowable tag sites for additional enzyme
substrates can be determined by review of X-ray crystal structures
of enzyme-substrate or enzyme-substrate analog structures. Using a
standard computer graphics program, available X-ray data and by
attaching an extended chain butyl group (as a model for the
affinity tagged linker) to potential tag attachment sites, suitable
attachment sites that show there are no enzyme-atoms in van der
Waals overlap with the model tag can be predicted.
[0117] Analogous methods to those described above can be applied to
the analysis of enzymes associated with other Sanfillipo Syndromes
(A, C and D). SFA is associated with heparin sulfamidase, SFC is
associated with acetyl-CoA-alpha-glucosaminide N-acetyltransferase
and SFD is associated with N-acetylglucosamine 6-sulfatase.
Exemplary affinity tagged enzyme substrate reagents useful in the
analysis of these enzymes and the diagnosis of these disorders are
provided below. The methods can also be applied of the diagnosis of
Niemann-Pick Type A and B disease by assaying for acid
sphingomyelinase and to the diagnosis of Krabbe disease by assaying
for galactocerebroside beta-galacatosidase. These enzymes are
currently assayed employing fluorophore-derivatized reagents as
indicated in Scheme 7. Enzyme substrate reagents for assay of these
enzymes in the methods herein can be readily prepared by
replacement of the fluorophore with an A-L group herein. This
approach to preparation of affinity tagged enzyme substrates is
generally applicable to any known fluorophore-derivatized enzyme
substrate or substrate analog.
[0118] Table 4 provides exemplary enzymes that are associates with
certain birth defects or disease states. These enzymes can be
assayed by the methods described herein.
[0119] Assaying Enzymatic Pathways for Carbohydrate-Deficient
Glycoprotein Syndromes (CDGS)
[0120] The methods and reagents of this invention can be employed
to quantify the velocities of multiple enzymes pertinent to
diagnosis of CDGS diseases.
[0121] CDGS Type Ia and Ib are caused by the deficiency or absence
of the enzymes phosphomannoisomerase (PMIb) (Type Ib) and
phosphomannomutase (PMM2) (Type Ia) which are part of a multistep
pathway (Scheme 8) for conversion of glucose to mannose-1-phosphate
(Freeze, 1998). The monosaccharide substrates involved in the
pathway are fructose-6-phosphate, mannose-6-phosphate, and
mannose-1 -phosphate. These monosaccharides can be somewhat
difficult to convert to substrate conjugates because it is not a
priori clear which atom on the sugar should be conjugated with the
linker without impairing enzyme activity. PMlb and PMM2 can,
however, be assayed indirectly. Mammalian cell microsomes contain
dolichol-P-mannose synthase which catalyzes the reaction of
dolichol-phosphate with GDP-mannose to form dolichol-P-mannose and
GDP (Scheme 8, Chapman et al. 1980). This synthase can be assayed
using the methods of this invention, specifically with a
biotin-linker substrate. Microbial PMM and the enzyme which makes
GDP-mannose from GTP and mannose-1-P, GDP-mannose
pyrophosphorylase, are readily purified from bacteria and yeast
(Glaser, 1966, Preiss, 1966), and these enzymes can be supplied
exogenously to the enzyme assay. If PMIb activity is deficient,
little or no mannose-6-P will be made when the reaction sequence is
started by addition of fructose-6-P. Without mannose-6-P,
mannose-1-P and GDP-mannose will not be made, and thus no
conjugated-dolichol-.beta.-mannose will be detected by ESI-MS.
Exogenous GTP is supplied as a requirement for the GDP-mannose
pyrophosphorylase step, and ATP is omitted so that mannose-6-P
cannot be made from mannose. To assay PMM2, the reaction sequence
is initiated with mannose-6-P, and PMM2 deficiency results in the
failure to make conjugated-dolichol-P-mann- ose.
[0122] The carrier dolichol is a .about.60- to 105-carbon
isoprenoid. Evidence is accumulating that many enzymes that operate
on carbohydrates attached to dolichol chains are tolerant to
significant shortening of the dolichol chain; even 10- and
15-carbon dolichols are tolerated (Rush and Wachter, 1995). It
appears that such enzymes act on the water-soluble carbohydrate
portion of the dolichol conjugate and thus have little or no
requirement to bind the dolichol anchor. Based on this, an affinity
labeled substrate for the direct assay of dolichol-P-mannose
synthase and the indirect assay of PMIb and PMM2 is prepared by
attaching an affinity labeled linker to the non-polar end of a
short dolichol, such as the 10-carbon dolichol analog
citronellol.
[0123] The synthesis of a biotinylated dolichol.sub.10-substrate
conjugate containing a sarcosinyl linker (B-S-Dol.sub.10-P) is
shown in Scheme 9. Protected citronellol R=t-BuSiMe.sub.2) is
regioselectively oxidized at the terminal alyllic methyl group
(McMurry and Kocovsky, 1984), and the allylic alcohol is coupled
with biotinylsarcosine active ester R=CH.sub.3). The citronellol
1-hydroxy group is subsequently deprotected and phosphorylated with
POCl.sub.3 (Rush and Wachter, 1995). In a parallel synthesis,
d.sub.5-sarcosine, CD.sub.3NHCD.sub.2COOH, is used to prepare the
isotopically labeled (heavy) reagent for use as an internal
standard. d.sub.5- Sarcosine is readily prepares form commercially
available materials (BrCD.sub.2COOD and CD.sub.3NH.sub.2) using
standard synthetic techniques.
[0124] The deuterated internal standard,
B-d.sub.5-S-Dol.sub.10-P-Mannose, is synthesized enzymatically by
ineubating hen oviduct microsomes with GDP-mannose and the
synthetic B-d.sub.5-S-Dol.sub.10-P substrate conjugate (Rush and
Waechler, 1995). An added advantage of the B-S-conjugate is that it
allows for a facile affinity purification of the microsomal
mannosylated product by specific capture on agarose-streptavidin
beads followed by elution with free biotin.
[0125] This method employing affinity tagged short dolichol
analogues is generally applicable for assaying other enzymes that
operated on dolichol anchored carbohydrates. Such an approach is
useful for the subsequent identification of enzyme deficiencies
present in other types of CDGS that have not been yet
identified.
[0126] CDGS Type II results from defective GlcNAc transferase II
(GlcNAc-T II) which transfers GlcNAc from UDP-GlcNAc to the
2-position of a mannose residue in the intermediate branched
oligosaccharide (the Core Region) in the process of building up the
disialo-biantennary chain (Scheme 10) (Schachter, 1986; Brockhausen
et al, 1989). GlcNAc transferase II is one of the six known enzymes
that mediate highly regiospecific glycosylation of the mannose
residues in the Core Region. The Core Region is anchored at the
reducing end to chitobiosylasparagine, where the asparagine residue
is part of the peptide chain of the glycosylated protein. The
latter structure unit in the substrate can be replaced by a
hydrophobic chain without loss of enzyme activity (Kaur et al.,
1991). Thus, the substrate conjugate for CDGS Type II is assembled
by linking a affinity-labeled linker group to the reducing end to
chitobiosylasparagine. However, the latter structure unit in the
substrate can be replaced by a hydrophobic chain without loss of
enzyme activity (Kaur et al., 1991). For example, commercially
available .alpha.-D-manno-pyranosylphenylisothiocyanate can be
coupled to a biotin-labeled linker and the 5,6-hydroxyls are
selectively protected as illustrated in Scheme 11 (Paulsen and
Meinjohanns, 1992). Coupling of the equatorial 3-OH with
per-O-acetylmannosyl-1-trichloroacetamidate (Paulsen et al, 1993)
will provide a disaccharide conjugate (Scheme 12). If a minor
amount of coupling occurs at the axial 2-OH group the products can
be separated by HPLC. After deprotection, the primary 6-OH is
coupled with a second equivalent of
per-O-acetylmannosyl-1-trichloroacetamidate to yield the Core
Region conjugate. Deprotection of the 0-acetyl groups yields the
substrate conjugate for GlcNAc transferase I which can be converted
to the GlcNAc-T II substrate by enzymatic glycosyl transfer using a
Triton X-100 rabbit liver extract, a reaction that has been carried
out on a preparative scale (Kaur et al., 1981).
[0127] The synthesis of the deuterium labeled derivative needed for
the internal standard is performed in parallel by using a labeled
PEG-diamine building block (Gerber et al., 1999). The biotinylated
trisaccharide is converted to the tetrasaccharide (product of
GlcNAc-T II) by ineubation with UD.beta.-GlcNAc and transferase II
( (Kaur and Hindsgaul, 1991;Tan et al., 1996) and utilizing the B-S
handle for affinity purification of the enzymatic products.
[0128] CDGS Type V
[0129] The lipid-linked oligosaccharide (LLO) that is transferred
to the Asn residue of the glycosylated protein is composed of 2
GlcNAc, 9 mannoses, and 3 glucoses. It has recently been shown that
microsomes from CDGS type V patients are greatly deficient in the
enzyme that transfers one or more glucose residues during LLO
biosynthesis (Korner et al, 1998). Since the transferase that
attaches the carbohydrate unit of LLO to the Asn residue
discriminates against the glucose-deficient LLO, CDGS Type V
patients have fewer numbers of carbohydrate units attached to
glycoproteins, such as transferrin (Korner et al., 1998). However,
the few carbohydrate units that are present are full-length,
demonstrating that residual glucosyl transfer occurs in type V CDGS
patients (Korner et al., 1998). Thus, quantification of the rate of
Asn glycosylation by ESI-MS would constitute a viable assay of CDGS
Type V syndrome.
[0130] Synthetic peptides with 3-7 amino acid residues containing
the Asn-Xaa-Ser/Thr sequence have been shown to be good substrates
for glycosylation (Ronin et al., 1981). The strategy for the ESI-MS
assay of the oligosaccharide transferase relies on a B-S conjugate
of an appropriate peptide containing the Asn-Xaa-Ser/Thr sequence
(Scheme 13). A heptapeptide,
NH.sub.2-Tyr-Gln-Ser-Asn-Ser-Thr-Met-NH.sub.2 (SEQ ID NO:1) has
shown high activity in a previous study (Ronin et al., 1981). The
peptide is readily available by standard peptide synthesis using an
in-house automatic synthesizer. The heptapeptide and its
glycoconjugates can be ionized by ESI to provide stable
singly-charged ions. Coupling of BS-tetrafluorophenyl ester with
NH.sub.2-Tyr-Gln-Ser-Asn-Ser-Thr-Met-NH.s- ub.2will directly yield
the substrate for the transferase. Several products are expected
from the enzymatic glycosylation and subsequent modifications of
the oligosaccharide antenna. The products can be prepared
enzymatically by ineubating thyroid rough microsomes with
BS-Tyr-Gln-Ser-Asn-Ser-Thr-Met-NH.sub.2 and Dol-P-Glu (Ronin et
al., 1981a), followed by affinity purification of the biotinylated
products. Product distribution due to different degrees of
glycosylation can be monitored by ESI-MS, and the major components
can be purified by HPLC. An analogous procedure using a
B-N(CD.sub.3)CD.sub.2CO-conjugate is used to prepare deuterated
internal standards.
[0131] The molecular masses of the ionized substrate conjugates for
the set of enzymes assayed for CDGS Ia, Ib, II, and V syndromes, as
well as products and internal standards are compiled in Table 5,
which shows that no isobaric overlaps among the (M+H).sup.+ species
occur. The close spacing between the (M+Na).sup.+ ion from the Type
Ia,b product and the (M+H).sup.+ ion of the demannosylated
B-(N-C.sub.2D.sub.5)-2,2-D.sub.2-Gl- y-Dol.sub.10-P internal
standard can be readily avoided by adjusting the ESI-MS conditions
by addition of Na' ions to generate the gas phase ions as
Na-adducts.
[0132] All three of the targeted enzymes can be analyzed
simultaneously in a single biological sample, such as a cell
lysate. The PMM2 and PMIb cannot be assayed simultaneously because
they require the addition of different exogenous substrates.
Nevertheless, two assays using identical ESI-MS techniques can be
used for diagnosing the various CDGS types instead of relying on a
battery of different methods.
[0133] Clinical Enzymology Assays
[0134] A fibroblast cell pellet is thawed on ice. Sufficient 0.9%
NaCl is added to give a final protein concentration in the lysate
of .about.5mg/mL (typically 100 mcL), and the cell pellet is
sonicated in ice water 5 times for 2 seconds each at moderate
power. Total protein is determined spectrophotometrically using the
BCA reagent (BCA Protein Assay kit, Pierce).
[0135] The total enzyme reaction volume is 20- 30 mcL. The
substrate stock solutions are maintained at concentrations of 3 mM
(SFB) and 2 mM (GM1). These concentrations were measured by 1H-NMR
signal integration versus an internal standard (formamide proton of
DMF). Final concentration of substrates is 0.3 and 0.2 mM,
respectively. A volume of reaction buffer (e.g. 200 mM sodium
citrate, pH 4.5) equal to the difference of the substrate addition
(2-3 mcL) plus sufficient cell sample volume to equal 50 75mcg
total protein from 20-30 mcL is added to a 0.5 mL Eppendorf tube,
followed by substrate. The sample is cooled on ice, and patient
cell sample is added. The reaction is initiated by ineubation at
37.degree. C.
[0136] For SFB: The reaction is allowed to proceed for 4.5-6 hours,
after which GM1 substrate can be added or the reaction can be
quenched with 100 mcL of 200 mM glycine-carbonate buffer, pH
10.5.
[0137] For GM1: The reaction is allowed to proceed for 0.5 hours.
Quenching is as for SFB.
[0138] After quenching, the samples are placed on ice. Internal
standards are added (1 nmol each, i.e. 50 mcL of a 0.02 mM
solution). The samples are microfuged at .about.15,000 rpm for 2
min at room temperature to pellet cell debris. Streptavidin-Agarose
beads (Immunopure immobilized streptavidin, Pierce) are placed in a
micro bio-spin chromatography column (Bio-Rad). Sufficient beads
are added to give a total biotin binding capacity of 5 nmol
(typical binding capacity 100 pmol per mcL of beads as determined
by Pierce). The sample supernatant is transferred to the bio-spin
tube and allowed to bind for 10 minutes at room temperature. The
sample is spun at .about.3,000 rpm to remove excess supernatant,
then washed once with 0.01% Triton X-100 and at least five times
with purified water, spinning the tube in-between to remove
solution. For each wash, sufficient wash solution is added to fill
the bio-spin tube.
[0139] The purified beads are then treated with 30 mcL purified
water, followed by 10 mcL of a 4 mM biotin solution. The tubes are
capped at the bottom to prevent leakage and allowed to ineubate at
2-8.degree. C. for 2-12 hours. The samples are spun at .about.3,000
rpm to elute the sample into a clean Eppendorf tube.
[0140] The sample is then diluted with 60 mcL of 50% methanol/water
and infused into the ion-trap mass spectrometer. The ESI-MS
spectrum is tuned to reduce non-specific cleavage of the samples by
first analyzing a blank sample (cell lysate added after reaction
quench). The infused sample is analyzed by ion chromatogram
integration of a 1 amu-wide window about the (M+H+).sup.+ ions of
product and internal standard.
[0141] Results are reported in nmol product formed/hour of
ineubation/milligrams total protein in reaction mixtures.
[0142] Clinical Analysis of Patient Samples for GM1 and SFB
[0143] Patient skin fibroblasts were obtained as frozen pellets,
and stored at 20.degree. C. until use. Two GM1 affected samples and
six normal controls were analyzed.
[0144] 50 mcL of 0.9% NaCl was added to each patient cell pellet.
The pellets were lysed by sonication in ice water 5.times. for 2
seconds each at moderate sonication power, chilling the microtip in
ice water in between sonications.
[0145] Samples were quantitated by BCA (Pierce) assay as
follows:
[0146] Reagent A and B were mixed in 50:1 ratio as described. A
protein standard curve was prepared using bovine serum albumin as a
standard at concentrations of 2, 1, 0.5, 0.2, and 0.05 mg/mL. A
portion of the patient sonicates were diluted 1:15 in water, and 5
mcL of each diluted patient sample and standard curve point was
added to separate glass culture tubes containing 200 mcL water, in
duplicate. The samples were then diluted with 1 mL of the mixed BCA
reagent, vortexed to mix, and ineubated at 37.degree. C. for 60
minutes. The samples were allowed to cool to room temperature, and
analyzed against a blank containing only 200 mcL water. The samples
were analyzed by monitoring absorbance at 562 nm in polystyrene
cuvettes. Average patient absorbance values were blank corrected
and compared to standards via linear regression.
[0147] The patient protein concentrations were determined to
be:
[0148] 1.(Affected) 12.2 mg/mL, 2. (Normal) 10.8 mg/mL, 3. (Normal)
11.9mg/mL, 4. (Normal) 12.1 mg/mL, 5. (Normal) 10.3 mg/mL, 6.
(Normal) 7.79 mg/mL, 7. (Normal) 15.7 mg/mL, 8. (Affected) 11.4
mg/mL Incubations were performed in a total of 30 mcL of total
volume. The amount of reaction buffer (200 mM sodium citrate, pH
4.25) added to blank Eppendorf tubes was the difference of the
substrate volume (3 mcL of each substrate stock solution, 2 mM for
GM1 and 3 mM for SFB, for a total of 6 mcL) plus the volume of cell
lysate required to equal 75 mcg total protein, from 30 mcL. For
example, patient 1. incubation mixture initially contained 3 mcL of
SFB substrate solution, 6.14 mcL patient cell lysate, and 17.86 mcL
reaction buffer. The GM1 substrate was added later in the
incubation (see below).
[0149] Each patient sample was analyzed in triplicate. The reaction
mixtures were kept on ice during preparation, and the reaction was
initiated by transfer to a 37.degree. C. water bath. 5.5 hr into
the ineubation, 3 mcL GM1 substrate was added to each reaction, and
after an additional 0.5 hours the reactions were placed on ice and
quenched with 200 mcL of a 200 mM glycine-carbonate buffer, pH
10.25.
[0150] The purification and analysis procedures are as described in
Clinical Enzymology Assay (Typical).
[0151] The resultant enzyme activities, as an average standard
deviation nmol product/hour ineubation/mg total protein:
1 B-Gal SFB RATE +/- SD RATE +/- SD Normals Patient 2 68.0 2.6 0.90
0.05 Patient 3 35.5 3.9 1.54 0.38 Patient 4 51.1 2.7 1.36 0.26
Patient 5 38.8 8.3 1.01 0.12 Patient 6 51.4 9.9 2.25 0.36 Patient 7
40.9 3.7 1.12 0.20 Affecteds GM.sub.1 (#1) 0.9 0.9 0.80 0.21
GM.sub.1 (#8) 0.8 0.6 0.70 0.20
[0152] The following synthetic methods refer to Schemes 14-23.
Synthesis for GM1-gangliosidosis (beta-D-galactosidase
deficiency)
[0153] 1. 2,3,5,6-Tetrafluorophenyl trifluoroacetate (1) 25 g (0.15
mol) 2,3,5,6-tetrafluorophenol, 35 mL (0.2 mol) trifluoroacetic
anhydride and 0.5 mL boron trifluoride etherate were refluxed for
18 hours under argon atmosphere. Trifluoroacetic anhydride and
trifluoroacetic acid were removed by distillation at room
temperature. The trifluoroacetic anhydride fraction was returned to
the mixture, and the reaction was refluxed for 24 hours. This was
repeated twice. After final distillation at room temperature, the
desired product 1 was distilled at reduced pressure (62.degree.
C./45 mmHg) to produce a colorless liquid (30 g, 82%). 1 H-NMR.
(Gamper, H. B., 1993). Biotin-2,3,5,6-tetrafluorophenyl ester (2) A
2.5 g (10.3 mmol) quantity of d-biotin in 20 mL anhydrous DMF under
argon atmosphere was warmed to 60.degree. C. with stirring to
effect dissolution. 1.7 mL (12.5 mmol) triethylamine was added,
followed by 3.4 g (12.5 mmol) 1. The mixture was stirred for 2
hours, after which the solvent was removed by rotary evaporation.
The resultant semi-solid was triturated with 15 mL ether twice to
produce a white solid (2.6 g, 65%). 1 H-NMR. (Wilbur, D. S., et
al., 1997). N-methylglycylbiotinamide-m- ethyl ester (3) A 2.5 g
(6.4 mmol) quantity of biotin tetrafluorophenyl ester in 30 mL
anhydrous DMF under argon atmosphere was added to a mixture of 1.1
g (7.7 mmol) N-methylglycine methyl ester hydrochloride dissolved
in 10 mL anhydrous DMF and 1.25 mL (9.0 mmol) triethylamine. The
reaction mixture was stirred at room temperature for 2 hours, then
the solvent was removed by rotary evaporation. The residue was
extracted with chloroform (2.times.100 mL), washed with water
(2.times.20 mL), and dried with anhydrous sodium sulfate. The
solvent was removed under vacuum to yield 2.1 g (98%) of methyl
ester of N-methylglycine biotinamide as an oil. 1H-NMR. (Wilbur, D.
S., et al., 1997).
[0154] 4. N-methylglycylbiotinamide acid (4)
N-Methylglycylbiotinamide methyl ester was hydrolyzed in a mixture
of 31 mL MeOH and 10 mL of 1N NaOH at room temperature with
stirring for 1 hour. The mixture was diluted with 50 mL 50%
MeOH/water and neutralized with cation exchange resin, hydrogen
form (AG M.beta.-50, BioRad). The solution was filtered, the resin
washed (3.times.50 mL) with 50% MeOH/water, and the solvents
removed by rotary evaporation to yield 1.6 g (90%) of
N-methylglycylbiotinamide acid as an off-white solid. 1H-NMR.
(Wilbur, D. S., et al., 1997).
[0155] 5. p-Acrylamidophenyl-.beta.-D-galactopyranoside (5) 40 mg
(0.1 5 mmol) p-aminophenyl .beta.-D-galactopyranoside was added to
25 mL methanol and 200 mcL triethylamine with stirring. The
solution was chilled in an ice bath. 53.3 mg (0.6 mmol) acryloyl
chloride was dissolved in 5 mL dry methylene chloride and added
dropwise to the stirred solution over 5 minutes. The reaction was
allowed to return to room temperature, followed by 2 hours of
stirring. The solution was then treated with successive anion and
cation exchange resins (AG MP-1 and AG MP-50, respectively, BioRad)
until neutral pH was obtained with moist pH paper. Solvent was
removed by rotary evaporation to yield a solid (43 mg, 90%). 1
H-NMR. (Romanowska, A., et al., 1994). Michael addition product of
4,7,10-trioxa-1,13-tridecanediamine and 5 (6) 20 mg (0.07 mmol) 5
was added to a stirred solution of 80 mg (0.35 mmol)
4,7,10-trioxa-1,13-tride- canediamine in 5 mL 0.2M sodium
carbonate, pH 10.5 at 37.degree. C. The reaction was allowed to
proceed for 3 days, after which the solution was neutralized with
dilute trifluoroacetic acid and purified by reverse-phase HPLC
(Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H.sub.2O
(0.08% TFA)/ACN (0.08% TFA)) to give 7.3 mg of product.
(Romanowska, A., et al., 1994).
[0156] 7. GM1 substrate conjugate of 4 and 6 (7) A 2.5 mg (7.4
mcmol) quantity of 4 was dissolved in 1.5 mL anhydrous DMF with
stirring, under argon atmosphere. 5 mcL triethylamine was added,
followed by 2.3 mg (8.8 mcmol) 1. The formation of active ester was
monitored by silica TLC (5:1 CHCl.sub.3/CH.sub.3OH, Rf 0.5, UV) by
briefly drying the spotted TLC plate with a stream of air. After 25
minutes, the mixture was added to 3.2 mg (5.9 mcmol) 6 in 1 mL
anhydrous DMF. After 2 hours, the solvent was removed by vacuum
centrifugation and the final product was purified by reverse-phase
HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H2O
(0.08% TFA)/ACN (0.08% TFA)). Yield 4.6 mg. (For analogous
chemistry, see Wilbur, D. S., et al., 1997). 8.
1,2,10,11-octadeutero-3,6- ,9-trioxa-1,11-undecanedinitrile (8) 1 g
(9.4 mmol) of diethylene glycol was dissolved in 2 mL D2O in a 10
mL round bottom flask under argon atmosphere. The D.sub.2O was
removed by rotary evaporation and the process was repeated 4 times.
The d-2 diethylene glycol was additioned with 25 mL dry benzene,
followed by 1.6 g (28.2 mmol) d-3 acrylonitrile with stirring under
argon atmosphere. After 12 h, the solvent was removed under reduced
pressure and the resultant semisolid was extracted with chloroform
(2.times.5 mL). The solvent was removed by rotary evaporation to
yield 1.85 g (89%) product. (Ashikaga, K., et al., 1988).
[0157] 9. 2,3,11,12-octadeutero-4,7,10-trioxa-1,13-tridecanediamine
(9) Raney nickel (Aldrich) was washed five times with anhydrous
methanol by inversion and decantation. 50 mg of the washed catalyst
was placed in 20 mL anhydrous methanol, followed by 1 g (4.6 mmol)
8 in a 50 mL screw-cap vial fitted with a Teflon-lined rubber
septum. The vial headspace was flushed for a few min with H.sub.2
gas via an 18-gauge needle piercing the septum. The cap was screwed
on tightly and the entire assembly was charged to 40 psi H.sub.2
and placed in a hot water bath (80.degree. C.) for 4 hours, after
which the solid catalyst was removed by filtration and the methanol
evaporated. The final product was purified by reverse-phase HPLC
(Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H.sub.2O
(0.08% TFA)/ACN (0.08% TFA)). Yield 180 mg. (Ashikaga, K., et al.,
1988).
[0158] 10. Deuterated analog of 6 (10) 25 mg (0.09 mmol) 5 was
added to a stirred solution of 90 mg (0.4 mmol) 9 in 5 mL 0.2M
sodium carbonate, pH 10.5 at 37.degree. C. The reaction was allowed
to proceed for 3 days, after which the solution was neutralized
with dilute trifluoroacetic acid and purified by reverse-phase HPLC
(Vydac C-1 8 prep-scale column, 6 mL/min. Mobile phase: H.sub.2O
(0.08% TFA)/ACN (0.08% TFA)). Yield 6 mg.
[0159] 11. Deuterated analog of 7 (11) A 3 mg (8.4mcmol) quantity
of 4 was dissolved in 0.7 mL anhydrous DMF with stirring, under
argon atmosphere. 5 mcL triethylamine was added, followed by 2.4 mg
(8.9 mcmol) 1. The formation of active ester was monitored by
silica TLC (5:1 CHCl.sub.3/CH.sub.3OH, Rf 0.5, UV) by briefly
drying the spotted TLC plate with a stream of air. After 25
minutes, the mixture was added to 6 mg (11 mcmol) 10 in 1 mL
anhydrous DMF. After2 hours, the solvent was removed by vacuum
centrifugation and the final product was purified by reverse-phase
HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase:
H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 1.8 mg.
[0160] 12. GM1 internal standard conjugate (12) 1.8 mg 11 was added
to 2 mL of 100 mM Tris/10 mM MgCl2, pH 7.3 buffer with stirring. 15
units recombinant .beta.-D-galactosidase (Sigma) was added, and
after 12 hours the mixture was purified by reverse-phase HPLC
(Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H.sub.2O
(0.08% TFA)/ACN (0.08% TFA)). Yield 1.5 mg.
[0161] Polyether Diamine Linker Synthesis (Second Generation)
[0162] Synthesis is based on chemistry previously described
(Kataky, R. et. al., 1990), with minor modifications and an
additional two steps. As an example, deviations from the
established procedure as well as exact details for the additional
steps are outlined below for the starting material diethylene
glycol.
[0163] 1,11 -Dicyano-3,6,9-trioxaundecane (13) To a stirred
solution of 2% (w/v) sodium hydroxide (5 mL) and diethylene glycol
(5.3 g, 50 mmol) was added acrylonitrile (7.95 g, 150 mmol). The
mixture was stirred at room temperature overnight and additioned
with 50 mL dichloromethane. The organic layer was washed 2.times.
with brine and dried (MgSO.sub.4). The solvent was removed by
rotary evaporation. The oily residue was treated with 200 proof
ethanol, and the solvent was removed by rotary evaporation. This
was repeated 2.times. to remove excess unreacted acrylonitrile. The
product was used without further purification
[0164] Diethyl 4,7,10-trioxatridecane-1,13-dioate (14) 2 g (9.4
mmol) 13, was dissolved in 5 mL ethanol. 1 g conc. sulfuric acid
was added slowly, over 5 minutes. The reaction was heated to reflux
overnight. The reaction was extracted with 40 mL methylene
chloride, washed once with 10 mL water and 3.times. with 10 mL
dilute brine solution. The organic layer was dried (MgSO.sub.4) and
solvent was removed to yield an oil. The final product was purified
by silica chromatography (methylene chloride/ethyl acetate).
[0165] 1,13-dihydroxy-4,7,10-trioxatridecane (15) Prepared exactly
as described, using tetrahydrofuran as solvent. (1.7 g, 5.5 mmol
14, 50 mL distilled [CaH.sub.2] THF, 0.66 g, 16.5 mmol lithium
aluminum hydride). Once addition was complete, excess LAH was
quenched with ethanol, and the salts precipitated by dropwise
addition of saturated sodium sulfate solution until a white
precipitate formed. The solvent was removed, the precipitate washed
6.times.30 mL with THF and the combined organic extracts were
evaporated to yield an oil. Final product is purified by silica
chromatography (first with methylene chloride then with ethyl
acetate and finally with acetone).
[0166] 1,13-dichloro-4,7,10-trioxatridecane (analog using P2) (16)
1.1 g, (4.9 mmol) 15 was added to 1.15 g (14.6 mmol) distilled
pyridine in 30 mL dry benzene with stirring, followed by 1.8 g
(14.6 mmol) thionyl chloride. The mixture was heated to reflux for
6 hours. After cooling in an ice bath, 5 mL 3M HCl was added with
vigorous stirring. The organic layer was separated, washed 3.times.
with a dilute brine solution, and dried (NaSO.sub.4) to yield a
yellowish oil. After washing and removal of solvent, the dichloride
was used without further purification.
[0167] Additional Steps
[0168] 1,13-dicyano-4,7,10-trioxatridecane) (17) To a stirred
solution of 0.78 g (15.5 mmol) sodium cyanide in 4 mL dimethyl
sulfoxide at 80.degree. C. was added 1 g (3.9 mmol) of 16. After 2
hours, the reaction was additioned with 10 mL of saturated sodium
chloride solution, 5 mL of water, and 50 mL ethyl acetate. The
organic layer was washed 3.times. with a brine solution as before,
after which the organic layer was dried (Na.sub.2SO.sub.4) and the
solvents removed. The final product was purified by silica
chromatography (methlyene chloride/ethyl acetate). ESI-MS:
predicted, 240.1; observed, 241.1 (M+H+).sup.+.
[0169] 1,15-diamino-5,8,11-trioxapentadecane (18) A stirred
solution of 50 mL dry THF containing 0.42 g (10.4 mmol) fresh LAH
was heated to gentle reflux under argon for 15 minutes. 0.5 g (2
mmol) 17 in 15 mL dry THF was added dropwise over 20 minutes,
maintaining a gentle reflux. The unreacted LAH was quenched with
ethanol, and the mixture was treated with dropwise addition of
saturated sodium sulfate under efficient stirring until a white
precipitate formed. The mixture was filtered, and the precipitate
was washed 6.times.30 mL with THF. The organic extracts were
combined and the solvent was removed by rotary evaporation to yield
an oil. ESI-MS: predicted, 248.1; observed, 249.1 (M+H+).sup.+.
[0170] Deuteration
[0171] Deuterium has been incorporated into the diamine linker by
reduction of 14 and 17 using lithium aluminum deuteride (98% D) to
achieve a d-8 deuterated diamine. No other aspects of the synthesis
were changed for this procedure. These diols are used in the
construction of the SFD conjugates as described later.
[0172] Clinical Substrate Synthesis for Sanfilippo Syndrome, type B
(N-.alpha.-D-glucosaminidase deficiency).
[0173] 13. p-Aminophenyl-.alpha.-D-N-acetylglucosamine (19) 20 mg
(0.07 mmol) p-Nitrophenyl-.alpha.-D-N-acetylglucosamine (Sigma) was
added to 5 mg washed palladium catalyst on activated carbon in 3 mL
methanol with stirring in a 5 mL septa-lined vial. The septum was
pierced by a 16-gauge needle and the vial headspace was flushed
with H.sub.2 gas. H.sub.2 gas was allowed to slowly bubble through
the solution for 2 hours, after which the catalyst was removed by
filtration over diatomaceous earth (Celite). The solvent was
removed by rotary evaporation to yield a semi-solid 18 mg
(90%).
[0174] 14. p-Acrylamidophenyl-.alpha.-D-N-acetylglucosamine (20) 10
mg (0.03 mmol) 19 was added to 15 mL methanol and 100 mcL
triethylamine with stirring. The solution was chilled in an ice
bath. 15 mg (0.17 mmol) acryloyl chloride was dissolved in 2 mL dry
methylene chloride and added dropwise to the stirred solution over
5 minutes. The reaction was allowed to return to room temperature,
followed by 2 hours of stirring. The solution was then treated with
successive anion and cation exchange resins (AG MP-1 and AG MP-50,
respectively, BioRad) until neutral pH was obtained with moist pH
paper. Solvent was removed by rotary evaporation to yield a solid
(11 mg, 95%). 1H-NMR. Yield 11 mg.
[0175] 15. 3,6-dioxa-1,9-nonanedinitrile (21) 2 g (0.032 mol)
ethylene glycol was added to 0.5 g dry potassium hydroxide in 30 mL
dry benzene, followed by 5 g (0.096 mmol) acrylonitrile with
stirring overnight at room temperature. The reaction was filtered
and the solvent was removed by rotary evaporation to yield an oil.
Final product was purified by silica chromatography
(chloroform/methanol) to yield a colorless oil 3.2 g (60%).
[0176] 16. 4,7-dioxa-1,10-decanediamine (22) Raney nickel (Aldrich)
was washed five times with anhydrous methanol by inversion and
decantation. 50 mg of the washed catalyst was placed in 20 mL
anhydrous methanol, followed by 1 g (6 mmol) 21 in a 50 mL
screw-cap vial fitted with a Teflon-lined rubber septum. The vial
headspace was evacuated with H.sub.2 gas via an 16-gauge needle
piercing the septum. The cap was screwed on tightly and the entire
assembly was charged to 40 psi H.sub.2 and placed in a hot water
bath (80.degree. C.) for 4 hours, after which the solid catalyst
was removed by filtration and the methanol evaporated. The final
product was purified by reverse-phase HPLC (Vydac C-18 prep-scale
column, 6 mL/min. Mobile phase: H.sub.2O (0.08% TFA)/ACN (0.08%
TFA)).
[0177] 17. Michael addition product of 20 and 22 (23) 5 mg (0.015
mmol) 20 was added to a stirred solution of 13 mg (0.06 mmol) 22 in
5 mL 0.2M sodium carbonate, pH 10.5 at 37.degree. C. The reaction
was allowed to proceed for 3 days, after which the solution was
neutralized with dilute trifluoroacetic acid and purified by
reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile
phase: H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 6 mg.
[0178] 18. SFB substrate conjugate of 4 and 23 (24) A 4 mg (0.013
mmol) quantity of 4 was dissolved in 1.5 mL anhydrous DMF with
stirring, under argon atmosphere. 10 mcL dry triethylamine was
added, followed by 4 mg (0.01 5 mmol) 1. The formation of active
ester was monitored by silica TLC (5:1 CHCl.sub.3/CH.sub.3OH, Rf
0.5, UV) by briefly drying the spotted TLC plate with a stream of
air. After 25 minutes, the mixture was added to 6 mg (0.012 mmol)
23 in 1 mL anhydrous DMF. After 2 hours, the solvent was removed by
vacuum centrifugation and the final product was purified by
reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile
phase: H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 4.2 mg.
[0179] 19. 1,9-tetradeutero-3,6-dioxa-1,9-nonanedinitrile (25) 0.5
g (8 mmol) ethylene glycol was added to 0.1 g dry potassium
hydroxide in 20 mL acetonitrile, followed by 1.4 g (24 mmol) d-3
acrylonitrile with stirring overnight at room temperature. The
reaction was filtered and the solvent was removed by rotary
evaporation to yield an oil. Final product was purified by silica
chromatography (chloroform/methanol) to yield a colorless oil 0.9 g
(65%).
[0180] 20. 1,9-tetradeutero-3,6-dioxa-1,9-nonanediamine (26) Raney
nickel (Aldrich) was washed five times with anhydrous methanol by
inversion and decantation. 20 mg of the washed catalyst was placed
in 30 mL anhydrous methanol, followed by 0.5 g (3 mmol) 25 in a 50
mL screw-cap vial fitted with a Teflon-lined rubber septum. The
vial headspace was evacuated with H.sub.2 gas via an 18-gauge
needle piercing the septum. The cap was screwed on tightly and the
entire assembly was charged to 40 psi H.sub.2 and placed in a hot
water bath (80.degree. C.) for 4 hours, after which the solid
catalyst was removed by filtration and the methanol evaporated. The
final product was purified by reverse-phase HPLC (Vydac C-18
prep-scale column, 6 mL/min. Mobile phase: H.sub.2O (0.08% TFA)/ACN
(0.08% TFA)). 21. Deuterated analog of 23 (27) 20 mg (0.07 mmol)
p-acrylamidophenyl .beta.-D-galactoside was added to a stirred
solution of 90 mg (0.4 mmol) 26 in 5 mL 0.2 M sodium carbonate, pH
10.5 at 37.degree. C. The reaction was allowed to proceed for 3
days, after which the solution was neutralized with dilute
trifluoroacetic acid and purified by reverse-phase HPLC (Vydac C-18
prep-scale column, 6 mL/min. Mobile phase: H.sub.2O (0.08% TFA)/ACN
(0.08% TFA)). Yield 2 mg.
[0181] 22. Deuterated analog of 24 (28) A 2 mg (6.3mcmol) quantity
of 4 was dissolved in 1.5 mL anhydrous DMF with stirring, under
argon atmosphere. 5 mcL triethylamine was added, followed by 2.1 mg
(7.6 mcmol) 1. The formation of active ester was monitored by
silica TLC (5:1 CHCl.sub.3/CH.sub.3OH, Rf 0.5, UV) by briefly
drying the spotted TLC plate with a stream of air. After 35
minutes, the mixture was added to 4 mg (7mcmol) 27 in 1 mL
anhydrous DMF. After 2 hours, the solvent was removed by vacuum
centrifugation and the final product was purified by reverse-phase
HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase:
H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 1.2 mg.
[0182] n23. SFB internal standard conjugate (29) 1.2 mg 28 was
added to 2 mL of 100 mM Tris/10 mM MgCl2, pH 7.3 bufferwith
stirring. 15 units recombinant .beta.-D-galactosidase (Sigma) was
added, and after 12 hours the mixture was purified by reverse-phase
HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase:
H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 0.7 mg.
[0183] Clinical Substrate Synthesis for Sanfilippo Syndrome, type D
(a sulfatase deficiency).
[0184] 24.
p-Acrylamidophenyl-.alpha.-D-N-acetylglucosamine-6-sulfate (30) 100
mg (0.28 mmol) 20 was added to 10 mL dry DMF under argon atmosphere
with stirring at room temperature. 89 mg (0.56 mmol) sulfur
trioxide-pyridine complex was dissolved in 2 mL dry DMF and was
added to the reaction in 0.7.times., 1.1.times., 1.3.times. and
1.9.times. equivalents (+700 mcL, +400 mcL, +200 mcL, and +600
mcL). The reaction progress was monitored by 1H-NMR shift of the
anomeric (C1) proton chemical shift from 5.29 to 5.24 ppm by
removal of 15 mcL of solution 1 hour after addition of each amount
of sulfating reagent. The removed mixture was dried by vacuum
centrifugation and redissolved in d-6 DMSO and analyzed. Upon the
appearance of more than two forms (starting material and C-6
sulfate) of the C1 anomeric proton, the reaction was removed to
-20.degree. C. and stored. The product was purified by vacuum
centrifugation to remove solvent, followed by reverse-phase HPLC
(Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H.sub.2O
(0.08% TFA)/ACN (0.08% TFA)). Yield 72%.
[0185] 25. Michael addition product of 18 and 30 (31) 25 mg (0.058
mmol)30 was added to a stirred solution of 83 mg (0.35 mmol) 18 in
5 mL 0.2M sodium carbonate, pH 10.5 at 37.degree. C. The reaction
was allowed to proceed for 3 days, after which the solution was
neutralized with dilute trifluoroacetic acid and purified by
reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile
phase: H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 10 mg.
[0186] 26. SFD substrate conjugate of 4 and 31 (32) A 5.7 mg (0.018
mmol) quantity of 4 was dissolved in 1.0 mL anhydrous DMF with
stirring, under argon atmosphere. 20 mcL dry triethylamine was
added, followed by 5.5 mg (0.020 mmol) 1. The formation of active
ester was monitored by silica TLC (5:1 CHCl.sub.3/CH.sub.3OH, Rf
0.5, UV) by briefly drying the spotted TLC plate with a stream of
air. After 25 minutes, the mixture was added to 10 mg (0.015 mmol)
31 in 1 mL anhydrous DMF. After2 hours, the solvent was removed by
vacuum centrifugation and the final product was purified by
reverse-phase HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile
phase: H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 5.4 mg.
[0187] 27. 1,2,14,15-octadeutero-1,15-diamino-5,8,11
-trioxapentadecane (33) as referenced in Polyether Diamine Linker
Synthesis, Second Generation.
[0188] 28. Deuterated analog of 31 (34) 25 mg (0.07 mmol) 20 was
added to a stirred solution of 100 mg (0.4 mmol) 11 in 5 mL 0.2M
sodium carbonate, pH 10.5 at 37.degree. C. The reaction was allowed
to proceed for 3 days, after which the solution was neutralized
with dilute trifluoroacetic acid and purified by reverse-phase HPLC
(Vydac C-18 prep-scale column, 6 mL/min. Mobile phase: H.sub.2O
(0.08% TFA)/ACN (0.08% TFA)). Yield 7 mg.
[0189] 29. SFD internal standard conjugate (35) A 4 mg (12.6 mcmol)
quantity of 4 was dissolved in 1 mL anhydrous DMF with stirring,
under argon atmosphere. 20 mcL triethylamine was added, followed by
4 mg (14 mcmol) 1. The formation of active ester was monitored by
silica TLC (5:1 CHCl.sub.3/CH.sub.3OH, Rf 0.5, UV) by briefly
drying the spotted TLC plate with a stream of air. After 20
minutes, the mixture was added to 7 mg (11 mcmol) 34 in 1 mL
anhydrous DMF. After 4 hours, the solvent was removed by vacuum
centrifugation and the final product was purified by reverse-phase
HPLC (Vydac C-18 prep-scale column, 6 mL/min. Mobile phase:
H.sub.2O (0.08% TFA)/ACN (0.08% TFA)). Yield 2.7 mg.
[0190] N-(d-Biotinyl-sarcosinyl)-12-aminododecanoic acid (36).
Compound 4 (32.2 mg, 0.102 mmole) was dried overnight in vacuo
(with P.sub.2O.sub.5). Dry DMF (2 mL) was added and the mixture was
stirred with warming to affect dissolution under nitrogen.
[0191] Triethylamine (34 mcL) was added followed by 1 (20.4 mcL,
0.115 mmole) added in two 10.2 mcL portions, 5 min apart. The
mixture was stirred for 1 hr at room temperature under nitrogen.
12-Aminododecanic acid (24.1 mg, 0.112 mmole, Sigma) was added in
one portion, and the mixture was stirred at room temperature for 2
hr under nitrogen. CHCl.sub.3 (80 mL) was added, and the organic
solution was washed with two 10 mL portions of 1 m HCl. CHCl.sub.3
was removed by rotary evaporation, and residual DMF was removed by
vacuum centrifugation. The compound was dissolved in methanol and
purified by HPLC (Vydac 218TP prep column). Solvent program is:
0-10 min, water with 0.06% TFA; 10-55 min, 0-100% methanol with
0.06% TFA, flow rate is 6 mL/min. Yield 31.7 mg. 1 H-NMR. ESI-MS,
calculated 513.4, observed 513.4 (M+H).sup.+
[0192] N-hydroxysuccinimidyl ester of 36 (37). Compound 36 (9.8 mg,
19 mcmole) is dissolved in 100 mcL of dry DMF under nitrogen.
N-hydroxysuccinimide (2.2 mg, 19 mcmole) was added followed by
dicyclohexylcarbodiimide (3.9 mg, 19 mcmole). The mixture was
stirred at room temperature for 60 h in the dark. Solvent was
removed by vacuum centrifugation, and the residue was submitted to
flash chromatography on silica gel using a gradient of
CHCl.sub.3/CH.sub.3OH (15/1) to CHCl.sub.3/CH.sub.3OH (12/1). Yield
9.8 mg. 1H-NMR. ESI-MS, calculated 610.8, observed 609.7
(M+H).sup.+.
[0193] N-(N-(d-Biotinyl-sarcosinyl)-12-aminododecanoyl)-pyschosine
(38). Compound 37 (6.2 mg, 10 mcmole) and pyschosine (4.7 mg, 10
mcmole, Sigma) were dissolved in 200 mcL of dry DMF under nitrogen.
Diisoproylethylamine (5 mcL) was added, and the mixture was stirred
under nitrogen for 2 days in the dark. The compound was injected
directly onto the HPLC column (Vydac 218TP semi-prep), and the
column was developed at 2 mL/min with 0-20 min, water with 0.06%
TFA, then 20-80 min, 0-100% methanol with 0.06% TFA. Yield 3.8 mg.
1H-NMR. ESI-MS, calculated 957.3, observed 956.8 (M+H).sup.+.
[0194] N-(N-(d-Biotinyl-sarcosinyl)-1
2-aminododecanoyl)-sphingosylphospho- rylcholine (39).
Sphingosylphosphorylcholine (4.0 mg, Sigma) was mixed with 1 mL dry
DMF and solvent was removed by vacuum centrifugation. This was
repeated two more times. The final dried residue weighed 2.5 mg
(5.4 mcmole). To this residue was added 3.3 mg of 37 (5.4 mcmole),
150 mcL of dry DMF, and 2.5 mcL of diisoproylethylamine. The
mixture was stirred under nitrogen in the dark for 3 days. The
compound was injected directly onto the HPLC column (Vydac 218TP
semi-prep), and the column was developed at 2 mL/min with 0-20 min,
water with 0.06% TFA, then 20-80 min, 0-100% methanol with 0.06%
TFA. Yield 3.8 mg. 1H-NMR. ESI-MS, calculated 960.3, observed 958.7
(M+H).sup.+.
[0195] Conjugate of d-biotin with 1,1
3-diamino-4,7,10-trioxatridecane (40). Compound 2 was reacted with
1,13-diamino-4,7,10-trioxatridecane (Fluka) essentially as
described for the synthesis of 3. The product was purified by HPLC
(Vydac 218TP, semi-prep) using 0-100% methanol with 0.06% TFA over
30 min at 1.5 mL/min.
[0196] lodoacetylated 40 (41). Compound 40 was treated with 5
equivalents of iodoacetic anhydride (Aldrich) in dry DMF with
stirring under nitrogen for 4 h at room temperature. The product
was purified on HPLC as for 40. The structure was confirmed by
ESI-MS.
[0197] Octadeuterated 41 (42). The title compound was prepared as
for the 40 using 9 instead of
1,13-diamino-4,7,10-trioxatridecane.
[0198] Octadeuterated 42 (43). The title compound was prepared from
42 as for 41. The structure was confirmed by ESI-MS.
[0199] Exemplary MS.sup.N Techniques and Instrumentation
[0200] An automated LC-MS/MS system for the identification of
proteins by their amino acid sequence has been developed. A
schematic representation is shown in FIG. 7 The system, which
consists of an autosample, a capillary HPLC system connected
on-line to an ESI triple quadrupole MS/MS instrument and a data
system is operated in the following way: Proteins (typically
separated by 1D or 2D gel electrophoresis) are cleaved with a
specific protease, usually trypsin. the resulting cleavage
fragments are placed in an autosampler. Every 37 minutes the
autosampler injects one sample into the HPLC system and the
peptides are separated by capillary reverse-phase chromatography.
As separated peptides elute from the chromatography column, they
are ionized by the ESI process, enter the MS and the mass to charge
ratio (m/z) is measured. Any peptide ion whose intensity exceeds a
predetermined intensity threshold is automatically selected by the
instrument and collided in the collision cell with inert gas. These
collisions result in peptide fragmentation, primarily at the bonds
of the peptide backbone (collision induced dissociation, CID). The
masses of the CID fragments are measured and recorded in the data
system. The CID spectrum of a peptide contains sufficient
information to identify the protein by searching sequence databases
with the uninterpreted MS/MS spectra. This is accomplished with the
Sequent program. The program identifies each peptide in a sequence
database which has the same mass as the peptide that was selected
in the MS for CID and predicts the MS/MS spectrum for each one of
the isobaric peptides. By matching the experimentally determined
CID spectrum with computer generated theoretical CID spectra, the
protein from which the observed peptide originated is identified.
The system is capable of analyzing protein samples in a fully
automated fashion at a pace of less than 40 min. per sample. Since
each peptide represents an independent protein identification and
usually multiple peptides are derived from one protein, protein
identification by this method is redundant and tolerant to proteins
co-migrating in a gel. The system is well suited for the detection
and characterization of modified residues within polypeptide
chains. The LC-MS/MS technique and automated analysis of the
generated CID spectra can be used for the methods of this
invention.
[0201] Identification of Proteins at Sub-femtomole Sensitivity by
Solid-phase Extraction Capillary Electrophoresis Tandem Mass
Spectrometry (SPE-CE-MSIMS)
[0202] Protein identification by this method is based on the same
principle as described above, except that peptide separation and
ionization are performed at significantly higher sensitivity. Fig.
8 shows a schematic representation of the key design elements. The
design of the system and its mode of operation have been published.
Peptides derived from protein digests are concentrated by SPE,
separated by CE and analyzed by ESI-MS/MS. The resulting
uninterpreted CID spectra are used to search sequence databases
with the Sequest software system. The SPE extraction device is a
small reversed-phase chromatography column of the dimensions
0.18.times.1 mm which is directly packed in a fused silica
separation capillary. Peptides contained in a sample solution are
adsorbed and concentrated on the SPE device, eluted in an estimated
100-300 nl of organic solvent and further concentrated by
electrophoretic stacking and/or isotachophoresis to an estimated
volume of 5-30 nl. The peptides are then separated by CE in a 20
.mu.m or 50 .mu.m i.d. capillary and directly ionized by ESI as
they leave the capillary. With this system, peptide masses can be
determined at a sensitivity of 660 attomoles (approx. 500 fg for a
20 residue peptide) at a concentration limit of 33 amol/.mu.l and
that proteins can be identified by the CID spectra of automatically
selected peptides at less than 10 fmol (0.5 ng for a protein of 50
kDa) of sample at a concentration limit of less than 300
amol/.mu.l. this technique is used for the analysis at very high
sensitivity of the peptide samples generated by the experiments. It
has also been demonstrated that the analysis time available for
automated CID experiments can be significantly extended by
data-dependent modulation of the CE voltage. If several peptide
ions are detected coincidentally in the MS, the CE voltage is
automatically dropped. This results in a reduction of the
electroosmotic flow out of the capillary and therefore in an
extension of the time period available for selecting peptide ions
for CID. The net effect of this peak parking technique is an
extension of the dynamic range of the technique because the
increased time available is used for CID of ions with a low ion
current Once all the peptide ions are analyzed, electrophoresis is
automatically reaccelerated by increasing the CE voltage to the
original value.
2TABLE 1 Relative, redundant quantitation of .alpha.-lactalbumin
abundance (after mixing with known amount of the same protein with
cysteines modified with isotopically heavy biotinylating reagent)
Pep- Ratio tide m/z Charge Peptide (heavy: # (light) state Sequence
light) 1 518.4 2+ (K) IWCK 2.70 2 568.4 2+ (K) ALCSEK (SEQ ID NO:2)
2.68 3 570.4 2+ (K) CEVFR (SEQ ID NO:3) 2.90 4 760.5 2+ (K)
LDQWLCEK (SEQ ID NO:4) 2.82 5 710.1 3+ (K) FLDDDLTDDIMCVK 2.88 (SEQ
ID NO:5) 6 954.2 3+ (K) DDQNPHSSNICNISCDK 2.90 (SEQ ID NO:6) 7
1286.9 4+ (K) NA.sup.a GYGGVSLPEWVCTTFHTSGYDT QAIVQNNDSTEYGLFQINNK
(SEQ ID NO:7) (SEQ ID NO. .sup.aIsotope ratio was not analyzed
because on a 4.sup.- peptide the isotope patterns were highly
overlapping due to differences of only 2 amu between heavy and
light ions.
[0203]
3TABLE 2 Sequence identification and quantitation of the components
of a protein mixture in a single analysis. Observed Expected ratio
ratio % Gene Name* Peptide sequence identified (d0/d8).sup.+ Mean
.+-. SD (d0/d8).sup.= error LCA_BOVIN ALC#SEK (SEQ ID NO: 8 0.94
0.96 .+-. 0.06 1.00 4.2 C#EVFR 1.03 FLDDLTDDTMC#VK (SEQ 0.92 ID NO:
9) OVAL_CHICK ADHPFLFC#IK (SEQ ID 1.88 1.92 .+-. 0.06 2.00 4.0 NO:
10) YPILPEYLQC#VK (SEQ ID 1.96 NO: 11) BGAL_ECOLI LTAAC#FDR (SEQ ID
1.00 0.98 .+-. 0.07 1.00 2.0 NO: 12) IGLNC#QLAQVAER (SEQ 0.91 ID
NO: 13) IIFDGVNSAFHLWC#NGR 1.04 (SEQ ID NO: 14) LACB_BOVIN
WENGEC#AQK (SEQ ID 3.64 3.55 .+-. 0.13 4.00 11.3 NO: 15)
LSFNPTQLEEQC#HI (SEQ 3.45 ID NO: 16) G3P_RABIT VPTPNVSVVDLTC#R 0.54
0.56 .+-. 0.02 0.50 12.0 (SEQ ID NO: 17) IVSNASC#TTNC#LAPLAK 0.57
(SEQ ID NO: 18) PHS2_RABIT IC#GGWQMEEADDWLR 0.32 0.32 .+-. 0.03
0.33 3.1 (SEQ ID NO: 19) TC#AYTNHTVLPEALER 0.35 (SEQ ID NO: 20)
WLVLC#NPGLAEIIAER 0.30 (SEQ ID NO: 21) *Gene names are according to
Swiss Prot nomenclature (www.expasy.ch). .sup.+Ratios were
calculated for each peptide as shown in FIG. 3. .sup.=Expected
ratios were calculated from the known amounts of proteins present
in each mixture. # ICAT-labeled cysteinyl residue.
[0204]
4TABLE 3 Protein profiles from yeast growing on galactose or
ethanol as a carbon source. Gene Observed ratio.sup..dagger.
Galactose- Glucose- name* Peptide sequence identified
(Eth:Gal).sup.= repressed.sup..sctn. repressed.sup..sctn. ACH1
KHNC#LHEPHMLK (SEQ ID NO:22) >100:1 .check mark. ADH1
YSGVC#HTDLHAWHGDWPLPVK 0.57:1 (SEQ ID NO:23) 0.48:1 C#C#SDVFNQVVK
(SEQ ID NO:24) ADH2 YSGVC#HTDLHAWHGDWPLPTK >200:1 .check mark.
.check mark. (SEQ ID NO:25) >200:1 C#SSDVFNHVVK (SEQ ID NO:26)
ALD4 TFEVINPSTEEEIC#HIYEGR >100:1 .check mark. .check mark. (SEQ
ID NO:27) BMH1 SEHQVELIC#SYR (SEQ ID NO:28) 0.95:1 CDC19
YRPNC#PIILVTR (SEQ ID NO:29) 0.49:1 NC#TPKPTSTTETVAASAVAAVFEQK
0.65:1 (SEQ ID NO:30) 0.67:1 AC#DDK FBA1 SIAPAYGIPVVLHSDHC#AK
0.60:1 (SEQ ID NO:31) 0.63:1 EQVGC#K (SEQ ID NO:32) GAL1
LTGAGWGGC#TVHLVPGGPNGNIEK 1:>200 .check mark. (SEQ ID NO:33)
GAL10 HHIPFYEVDLC#DR (SEQ ID NO:34) 1:>200 .check mark. DC#VTLK
(SEQ ID NO:35) 1:>200 GCY1 LWC#TQHHEPEVALDQSLK (SEQ ID NO:36)
0.34:1 .check mark. GLK1 IC#SVNLHGDHTFSMEQMK (SEQ ID NO:37) 0.65:1
GPD1 IC#SQLK (SEQ ID NO:38) 0.54:1 .check mark. ICL1 GGTQC#SIMR
(SEQ ID NO:39) >100:1 .check mark. IPP1
NC#FPHHGYIHNYGAFPQTWEDPNVSHPETK 0.76:1 (SEQ ID NO:40) LPD1
VC#HAHPTLSEAFK (SEQ ID NO:41) 1.30:1 .check mark. PEP4
KGWTGQYTLDC#NTR (SEQ ID NO:42) 2.60:1 .check mark. PSA1 SVVLC#NSTIK
(SEQ ID NO:43) 0.56:1 PGM2 C#TGGIILTASHNPGGPENDMGIK 0.58:1 .check
mark. (SEQ ID NO:44) 0.62:1 LSIC#GEESFGTGSNHVR (SEQ ID NO:45) PCK1
C#PLK 1.59:1 IPC#LADSHPK (SEQ ID NO:46) 1.47:1 C#INLSAEKEPEIFDAIK
(SEQ ID NO:47) 1.52:1 .check mark. C#AYPIDYIPSAK (SEQ ID NO:48)
1.41:1 IVEEPTSKDEIWWGPVNKPC#SER 1.85:1 (SEQ ID NO:49) QCR6
ALVHHYEEC#AER (SEQ ID NO:50) 1.30:1 .check mark.
RPL1A.sup..paragraph. SC#GVDAMSVDDLKK (SEQ ID NO:51) 0.82:1 SAH1
HPEMLEDC#FGLSEETTTGVHHLYR 0.62:1 (SEQ ID NO:52) 0.74:1 EC#INIKPQVDR
(SEQ ID NO:53) SOD1 GFHIHEFGDATNGC#VSAGPHFNPFK 0.46:1 .check mark.
(SEQ ID NO:54) TEF1 RGNVC#GDAK (SEQ ID NO:55) 0.81:1 C#GGIDK (SEQ
ID NO:56) 0.70:1 FVPSKPMC#VEAFSEYPPLGR 0.74:1 (SEQ ID NO:57) VMA2
IPIFSASGLPHNEIAAQIC#R 0.70:1 (SEQ ID NO:58) YHB1 HYSLC#SASTK (SEQ
ID NO:59) 0.69:1 *Gene names are according to the Yeast Proteome
Database (YPD) (19). #Cysteinyl residue is ICAT-labeled.
.sup..dagger.Protein expression ratios were calculated as described
in FIG. 3. .sup.=Carbon source for yeast growth was 2% ethanol
(Eth) or 2% galactose (GAL0. .sup..sctn.Gene is known to be
galactose- or glucose-repressed (19). .sup..paragraph.Eight other
ribosomal proteins were detected at similar gene expression
levels.
[0205]
5TABLE 4 Disease Enzyme Dysfunction Butyrylcholinesterase BCHE
Decreased or absent deficiency enzyme activity Essential
fructosuria Fructokinase Deficient enzyme activity hepatic
fructokinase deficiency Hereditary fructose Fructose 1,6- Deficient
enzyme activity intolerance bisphosphate aldolase B Hereditary
fructose Fructose 1,6- Deficient enzyme activity 1,6-diphosphatase
bisphosphatase deficiency Erythrocyte aldolase Fructose 1,6-
Deficient enzyme activity deficiency with bisphosphate
nonspherocytic hemolytic aldolase A anemia (aldolase A deficiency)
Glycogen storage disease Glucose Absent or deficient type Ia (von
Gierke 6-phosphatase enzyme activity disease) Glycogen storage
disease Glucose Deficient transport of type Ib 6-phosphate glucose
6-phosphate translocase across the membrane of endoplasmic
reticulum Glycogen storage disease Amylo-1,6- Absent or deficient
type III glucosidase enzyme activity (debrancher enzyme) Glycogen
storage disease .alpha.-1,4 glucan-6-.alpha.- Deficient enzyme
activity type IV (Andersen glucosyltransferase disease) Glycogen
storage disease Muscle glycogen Absent or deficient type V (McArdle
phosphorylase enzyme disease) Glycogen storage disease
Phosphorylase Deficient or absent X-linked phosphorylase b-kinase
enzyme activity function kinase deficiency Glycogen storage disease
Phosphorylase Deficient enzyme activity autosomal phosphorylase
b-kinase kinase deficiency Glycogen storage disease Liver Deficient
enzyme activity liver phosphorylase phosphorylase deficiency
Glycogen storage disease Muscle phospho- Deficient enzyme activity
type VII (Tarui disease) fructokinase 1 Liver glycogen synthase
Liver glycogen Unknown deficiency synthase Phosphoglycerate kinase
Phosphoglycerate Deficient enzyme deficiency kinase
Phosphoglycerate mutase Phosphoglycerate Deficient enzyme
deficiency mutase Muscle lactate Muscle-specific Absence of M
subunit of dehydrogenase subunit of LDH. Muscle LDH is a deficiency
lactate tetramer of the heart- dehydrogenase specific subunit (LDH)
Glucose phosphate Glucose phosphate Unknown isomerase deficiency
isomerase Transferase deficiency Galactose Deficient enzyme
activity galactosemia 1-phosphate uridyltransferase Galactokinase
deficiency Galactokinase Deficient enzyme activity galactosemia
Epimerase deficiency Uridine diphosphate Deficient enzyme action
galactosemia galactose-4- in blood cells only epimerase (benign)
or, more rarely, in all tissues (generalized) Phenylketonuria (PKU)
Phenylalanine Deficient or absent PAH due to PAH deficiency
hydroxylase (PAH) activity (<1% normal) Hyperphenylalaninemia
Dihydropteridine Deficient or absent due to DHPR-deficiency
reductase (DHPR) DHPR activity Hyperphenylalaninemia Guanosine
Deficient enzyme activity due to GTP-CH- triphosphate deficiency
cyclohydrolase (GTP-CH) Hyperphenylalaninemia 6-Pyruvoyl Deficient
enzyme activity due to 6-PTS-deficiency tetrahydropterin synthase
(6-PTS) Oculocutaneous Tyrosine Decreased activity tyrosinemia
(tyrosinemia aminotransferase type II; tyrosine amino-transferase
deficiency) 4-Hydroxyphenylpyruvic 4-Hydroxy- Decreased activity
acid dioxygenase phenylpyruvic (tyrosinemia type III) acid
dioxygenase Maleylacetoacetate Maleylacetonacetate Presumably
decreased isomerase deficiency isomerase enzyme activity
(tyrosinemia type Ib) (tentative) Hepatorenal tyrosinemia
Fumarylacetoacetate Deficient enzyme activity (tyrosinemia type I:
hydroxylase fumarylacetoacetate hydrolase deficiency) Carbamyl
phosphate Carbamyl phosphate Absent or deficient synthetase
deficiency synthetase I enzyme activity Ornithine trans- Ornithine
Absent or reduced carbamylase deficiency transcarbamylase enzyme
activity Argininosuccinic acid Argininosuccinic Deficient enzyme
activity synthetase deficiency acid synthetase Argininosuccinase
Argininosuccinate Deficient enzyme activity deficiency lyase
Arginase deficiency Liver arginase Deficient enzyme activity
Familial hyperlysinemia .alpha.-Aminoadipic Deficient enzyme
activity (variant: saccharopinuria) semialdehyde synthase Maple
syrup urine Branched-chain Deficient or absent disease (MSUD) or
.alpha.-keto acid (<2%) BCKAD complex branched chain
dehydrogenase activity in mitochondria; ketoacidemia immunologic
absence or reduced levels of enzyme subunits; impairment of E1
subunit assembly Cystathionine .beta.-synthase Cystathionine
Deficient enzyme activity deficiency .beta.-synthase
.alpha.-Cystathionase .alpha.-Cystathionase Deficient enzyme
activity deficiency Hepatic methionine Isoenzyme of Deficient
enzyme activity adenosyltransferase methionine deficiency
adenosyltransferase Sarcosinemia Sarcosine Deficient enzyme
activity dehydrogenase? Nonketotic Glycine cleavage Deficient
enzyme activity hyperglycinemia system Hyperuracil thyminuria
Dihydropyrimidine Deficient enzyme activity dehydrogenase
Dihydropyrimidinuria Dihydro- Unknown pyrimidinase Pyridoxine
dependency Brain glutamic Deficient coenzyme with seizures acid
decar- binding? (brain) boxylase-1 GABA aminotransferase
GABA-.alpha.-keto- Deficient enzyme activity deficiency glutarate
transaminase 4-Hydroxybutyric Succinic Deficient enzyme activity
aciduria semialdehyde dehydrogenase Serum carnosinase Serum
Deficient enzyme deficiency and carnosinase homocarnosinosis
Alkaptonuria Homogentisic Absent or deficient acid oxidase enzyme
activity Isovaleric acidemia Isovaleryl-CoA Deficient enzyme
dehydrogenase activity, deficient protein, abnormal peptide size
Isolated 3- 3-Methylcrotonyl- Deficient enzyme activity
methylcrotonyl-CoA CoA carboxylase carboxylase deficiency
3-Methylglutaconic 3-Methylglutaconyl- Deficient enzyme activity
aciduria Mild form: CoA hydratase 3-Hydroxy-3- 3-Hydroxy-3-
Deficient enzyme activity methylglutaryl-CoA methylglutaryl-CoA
lyase deficiency lyase Mevalonic aciduria Mevalonate kinase
Deficient enzyme activity Mitochondrial Mitochondrial Deficient
enzyme acetoacetyl-CoA acetoacetyl-CoA activity, decreased thiolase
deficiency thiolase (T2) protein, unstable protein Propionic
acidemia (2 Propionyl-CoA Deficient enzyme activity nonallelic
forms carboxylase (PCC) (nonallelic forms reflect designated pccA
and mutations in nonidentical pccBC) subunits of PCC) Methylmalonic
acidemia Methyldmalonyl- Absent MUT activity in (2 allelic variants
CoA mutase (MUT) mut.degree., deficient MUT designated mut.degree.
apoenzyme activity due to reduced and mut.sup.- affinity for
cofactor (adenosylcobalamin) in mut.sup.- Glutaric acidemia type I
Glutaryl-CoA Deficient enzyme activity dehydrogenase Cytochrome
oxidase Cytochrome oxidase Decreased activity of the deficiency
polypeptides cytochrome oxidase complex Pyruvate dehydrogenase
Pyruvate Decreased enzyme complex deficiency-E.sub.1 decarboxylase,
activity, decreased decarboxylase E.sub.1.alpha. protein component
Pyruvate dehydrogenease Dihydrolipoamide Decreased enzyme E.sub.2
transacylase transacylase activity; abnormal protein
electrophoretic mobility Combined .alpha.-ketoacid Lipoamide
Decreased enzyme dehydrogenase dehydrogenase activity
deficiency/lipoamide dehydrogenase deficiency Pyruvate carboxylase
Pyruvate Absent enzyme activity; deficiency carboxylase 7 cases
absent enzyme, protein, and mRNA Carnitine palmitoyl Carnitine
Deficient enzyme transferase I (CPT I) palmitoyl deficiency
transferase I Carnitine/acylcarnitine Carnitine/ Deficient
translocase translocase deficiency acylcarnitine translocase
Carnitine palmitoyl Carnitine Deficient enzyme transferase II
palmitoyl (CPT II) deficiency transferase II Very long-chain acyl-
Very long-chain Deficient enzyme CoA dehydrogenase acyl-CoA (VLCAD)
deficiency dehydrogenase Long-chain acyl-CoA Long-chain acyl-
Deficient enzyme dehydrogenase (LCAD) CoA dehydrogenase Long-chain
L-3- L-3-hydroxyacyl- Deficient enzyme hydroxyacyl-CoA CoA
dehydrogenase dehydrogenase (LCHAD) deficiency Trifunctional enzyme
Trifunctional Deficient enzyme (TFE) deficiency enzyme Dienolyl-Co
reductase 2,4-dienoyl-CoA Deficient enzyme deficiency reductase
Medium-chain acyl-CoA Medium-chain Deficient enzyme dehydrogenase
(MCAD acyl-CoA deficiency dehydrogenase Short-chain acyl-CoA
Short-chain Deficient enzyme dehydrogenase (SCAD) acyl-CoA
deficiency dehydrogenase Glutaric acidemia type II Electron
transfer In some cases, no flavoprotein (ETF); enzyme antigen; in
ETF:ubiquinone others, no enzyme oxidoreductase activity Glycerol
kinase Glycerol kinase The microdeletion deficiency (Gkd) involves
not only GK but also the other deleted loci: AHC, DMD, OTC, and
other linked loci Primary gout: superactive PP-ribose-P Enhanced
enzyme variant of synthetase activity phosphoribosylpyro- phosphate
(PP-ribose-P) synthetase Primary gout: partial Hypoxanthine Absent
or deficient deficiency of guanine enzyme activity hypoxanthine
guanine phosphoribosyl phosphoribosyl- transferase transferase
(HPRT) (HPRT) Lesch-Nyhan syndrome: Hypoxanthine Absent or
deficient deficiency of guanine enzyme activity hypoxanthine
guanine phosphoribosyl- phosphoribosyl- transferase transferase
(HPRT) (HPRT) 2,8-Dihydroxyadenine Adenine Type I: absent enzyme
lithiasis (adenine phosphoribosyl- activity; type II: reducted
phosphoribosyl- transferase affinity for PP-ribose-P transferase
deficiency) Adenosine deaminase Adenosine Absent or greatly
deficiency with severe deaminase diminished enzyme combined immuno-
activity deficiency disease Purine nucleoside Purine Absent or
greatly phosphorylase deficiency nucleoside diminished enzyme with
cellular phosphorylase activity immunodeficiency Myoadenylate
deaminase Myoadenylate No enzyme activity; on deficiency deaminase
immunoreactive protein (AMPDI) Xanthinuria Xanthine Type I: absent
xanthine dehydrogenase dehydrogenase activity; (xanthine oxidase)
type II: absent xanthine dehydrogenase and alde- hyde oxidase
activity Hereditary orotic aciduria UMP synthase Deficient enzyme
activity (unstable protein) Pyrimidine 5'- Pyrimidine 5'- Absent or
unstable nucleotidase deficiency nucleotidase enzyme
Dihydropyrimidine Dihydropyrimidine Absent or unstable
dehydrogenase dehydrogenase enzyme deficiency Dihydropryimidase
Dihydropyrimidase Absent or unstable deficiency enzyme Familial
lipoprotein Lipoprotein Nonfunctional protein in lipase deficiency
lipase some, nondectable enzyme activity and protein in others
Familial lecithin: Lecithin: Absent enzyme protein chloesterol
acyl- cholesterol or deficient enzyme transferase deficiency
acyltransferase activity .delta.-Aminolevulinic acid
.delta.-Aminolevulinic Minimal enzyme activity dehydratase
porphyria acid dehydratase Acute intermittent Porphobilinogen
Decreased enzyme porphyria deaminase activity (.about.50%)
Congenital erythropoietic Uroporphyrinogen Minimal enzyme activity
porphyria III cosynthase Porphyria cutanea tarda Uroporphyrinogen
Decreased enzyme (familial form) decarboxylase activity
(.about.50%) Hepatoerythropoietic Uroporphyrinogen Minimal enzyme
activity porphyria decarboxylase Hereditary Coproporphyrinogen
Decreased enzyme coproporphyria oxidase activity (.about.50%)
Variegate porphyria Protoporphyrinogen Decreased enzyme oxidase
activity (.about.50%) Erythropoietic Ferrochelatase Decrease enzyme
activity protophorphyria (.about.50%) Crigler-Najjar syndrome,
Bilirubin UDP- Absent enzyme activity type I glucuronosyl-
transferase Crigler-Najjar syndrome, Bilirubin UDP- Markedly
reduced type II glucuronosyl- enzyme activity transferase, Gilbert
syndrome Bilirubin UDP- Reduced enzyme activity glucuronosyl-
transferase activity Refsum disease Phytanic acid Deficient enzyme
activity .alpha.-hydroxylase Primary hyperoxaluria
Alanine-glyoxylate Loss of enzyme catalytic type I aminotransferase
activity and aberrant subcellular distribution Primary
hyperoxaluria Glyoxylate Loss of enzyme catalytic type 2
reductase/D- activity glycerate dehydrogenase G.sub.M2
gangliosidosis: .beta.-hexosaminidase Absent or defective
hexosaminadase hexosamininidase A (.alpha..beta.) .alpha.-subunit
deficiency activity (variant B, Tay-Sachs disease) Glycogen storage
disease .alpha.-glucosidase Absent or deficient type II enzyme
activity Mucopolysaccharidosis I .alpha.-L-iduronidase Absent
enzyme activity (Hurler, Scheie, and Hurler-Sheie syndromes, MPS,
MPS IS, MPS IH/S) Mucopolysaccharidosis II Iduronate Absent enzyme
activity (Hunter syndrome) sulfatase Mucopolysaccharidosis IIIA:
Heparan N- Absent enzyme activity III (Saniflippo syndrome)
sulfatase types A, B, C and D IIIB: .alpha.-N-acetyl-
glucosaminidase IIIC: Acetyl-CoA: .alpha.-glucosaminide
acetyltransferase IIID: N-acetyl glucosamine- 6-sulfatase
Mucopolysaccharidosis IVA: Galactose 6- Absent enzyme activity IV
(Morquio syndrome) sulfatase types A and B IVB:
.beta.-Galactosidase Mucopolysaccharidosis N-acetyl- Absent enzyme
activity VI (Maroteaux-Lamy galactosamine syndrome) 4-sulfatase
Mucopolysaccharidosis .beta.-glucuronidase Absent enzyme activity
VII (Sly syndrome) I-cell disease (ML-II) N-acetyl- Phosphorylation
of many glucosaminyl- lysosomal enzymes I-phospho- transferase
Schindler disease (.alpha.-N- .alpha.-N-acetyl- Deficient activity
acetylgalactosaminidase galactosaminidase of .alpha.-N-acetyl-
deficiency) galactosaminidase .alpha.-Mannosidosis
.alpha.-D-mannosidase Deficient or unstable enzyme activity
.beta.-Mannosidosis .beta.-D-mannosidase Deficient enzyme activity
Sialidosis .alpha.-neuraminidase Deficient enzyme activity
Aspartylglucosaminuria Aspartygluco- Deficient enzyme activity
saminidase Fucosidosis .alpha.-L-fucosidase Deficient enzyme
activity Wolman disease and Acid lipase Deficient enzyme activity
cholesteryl ester storage disease Ceramidase deficiency Ceramidase
Deficient enzyme activity (Farber lipogranulo- matosis)
Niemann-Pick disease Sphingomyelinase Deficient sphingo- (NPD)
types A and B myelinase activity (primary sphingomyelin storage)
Gaucher disease type I Glucocere- Decreased catalytic
(nonneuronopathic) brosidase activity and some instability of
enzyme protein Globoid-cell Galacto- Absent enzyme activity
leukodystrophy sylceramidase (Krabbe disease) Metachromatic
Arylsulfatase A Deficient enzyme activity leukodystrophy Fabry
disease .alpha.-Galactosidase A Nonfunctional or unstable enzyme
protein G.sub.M1 gangliosidosis Acid .beta.- Deficient enzyme
activity galactosidase (GLBI) G.sub.M2 gangliosidosis:
.beta.-hexosaminidase Absent or defective hexosaminidase .alpha.-
hexosaminidase A (.alpha..beta.) subunit deficiency activity
(variant B, Tay-Sachs disease) Steroid 21-hydroxylase Steroid 21-
Absent or truncated deficiency salt-losing hydroxylase
enzyme with no activity form Steroid 5.alpha.-reductase 2 Steroid
5.alpha.- Absent or unstable deficiency reductase 2 enzyme activity
Steroid sulfatase 3.beta.-hydroxysteroid Absent immunoreactive
deficiency (X-linked sulfatase and enzymatically active ichthyosis)
protein (both deletion and nondeletion patients) Methylenetetra-
Methylenetetra- Absent or deficient hydrofolate reductase
hydrofolate enzyme activity. deficiency reductase Thermolabile
variants have been described. Holocarboxylase Holocarboxylase
Deficient holocar- synthetase deficiency synthetase boxylase
synthetase activity Biotinidase deficiency Biotinidase Deficient
biotinidase activity Hereditary Cytochrome b.sub.5 Deficient enzyme
activity methemoglobinemia reductase in erythrocyte cytosol
secondary to cytochrome only (type I), in all b.sub.5 reductase
deficiency, tissues (type II), and in types I, II, and III all
hematopoetic cells (type III) Pyruvate kinase Pyruvate Deficient
enzyme activity deficiency hemolytic kinase anemia Hexokinase
deficiency Hexokinase Deficient enzyme activity hemolytic anemia
Glucosephosphate Glucose- Deficient enzyme activity isomerase
deficiency phosphate hemolytic anemia isomerase Aldolase deficiency
Aldolase Deficient enzyme activity hemolytic anemia (A type)
Triosephosphate Triosephosphate Enzyme activity deficient isomerase
deficiency isomerase in all tissues hemolytic anemia
Phosphoglycerate kinase Phosphoglycerate Deficient enzyme activity
deficiency hemolytic kinase in hemizygotes anemia 2,3-Diphospho-
2,3-Diphospho- Deficient enzyme activity glyceromutase and
glyceratemutase phosphatase deficiency and phosphatase (1 protein)
6-Phosphogluconate 6-Phosphogluconate Enzyme activity dehydrogenase
dehydrogenase deficiency deficiency Glutathione peroxidase
Glutathione Diminished enzyme deficiency peroxidase activity
Glutathione reductase Glutathione Deficient enzyme activity
deficiency reductase Glutathione synthetase Glutathione Deficient
enzyme activity deficiency hemolytic synthetase anemia
.gamma.-Glutamylcysteine .gamma.-Glutamyl- Deficient enzyme
activity synthetase deficiency cysteine hemolytic anemia synthetase
Adenosine deaminase Adenosine Overproduction of hyperactivity
hemolytic deaminase structurally normal anemia enzyme protein
mediated at mRNA translation level Pyrimidine nucleotidase
Pyrimidine Deficient enzyme activity deficiency hemolytic
nucleotidase anemia Myeloperoxidase Myeloperoxidase Absent or
deficient deficiency enzyme activity Carbonic anhydrase II Carbonic
Quantitative deficiency deficiency syndrome anhydrase II of
carbonic anhydrase II (osteopetrosis with renal tubular acidosis)
Albinism, Tyrosinase Absent, reduced, or oculocutaneous unusual
enzyme activity tyrosinase-negative type (OCAIA) Canavan disease
Aspartoacylase Deficient enzyme activity
[0206]
6TABLE 5 Molecular masses of protonated and sodiated
substrate-conjugates, products, and internal standards for CDGS
enzymes. m/z Internal Substrate Product standard Enzyme (M+
H).sup.+ (M + Na).sup.+ (M + H).sup.+ (M + Na).sup.+ (M + H).sup.+
(M + Na).sup.+ Type Ia, b 711 733 549 571 555 577 Manose- 725 747
563 585 570 592 transferase Type II 1156 1178 1343 1365 1348 1370
Type 1126 1148 2362.sup.a 2384.sup.a 2367.sup.a 2389.sup.a
.sup.aCalculated for the GlcNAc-T II product and internal standard
containin a GlcNAc-GlcNAc-Mannose-(Mannose-GlcNAc).sub.2
residue.
[0207] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
23
[0208] References
[0209] Ashikaga, K. et al. (1988) Bull. Chem. Soc. Jpn.
61:2443-2450.
[0210] Bayer, E. and Wilchek, M. (eds.) "Avidin=Biotin Technology,"
(1990) Methods Enzymol. 184:49-51.
[0211] Bleasby, A. J. et al. (1994), "OWL--a non-redundant
composite protein sequence database,"Nucl. Acids Res.
22:3574-3577.
[0212] Boucherie, H. et al. (1996), "Two-dimensional gel protein
database of Saccharomyces cerevisiae," Electrophoresis
17:1683-1699.
[0213] Brockhausen, I.; Hull, E.; Hindsgaul, O.; Schachter, H.;
Shah, R. N.; Michnick, S. W.;
[0214] Carver, J. P. (1989) Control of glycoprotein synthesis. J.
Biol. Chem. 264,11211-11221.
[0215] Chapman, A.; Fujimoto, K.; Kornefeld, S. (1980) The primary
glycosylation defect in class E Thy-1-negative mutant mouse
lymphoma cells is an inability to synthesize dolichol-P-mannose. J.
Biol. Chem. 255, 4441-4446.
[0216] Chen, Y.-T. and Burchell, A. (1995), The Metabolic and
Molecular Bases of Inherited Disease, Scriver, C. R. et al. (eds.)
McGraw-Hill, N.Y., pp.935-966.
[0217] Clauser, K. R. et al. (1995), "Rapid mass spectrometric
peptide sequencing and mass matching for characterization of human
melanoma proteins isolated by two-dimensional PAGE," Proc. Natl.
Acad. Sci. USA 92:5072-5076.
[0218] Cole, R. B. (1997) Electrospray Ionization Mass
Spectrometry: Fundamentals, Instrumentation and Practice, Wiley,
N.Y.
[0219] De Leenheer, A. P. and Thienpont, L. M. (1992), "Application
of isotope dilution-mass spectrometry in clinical chemistry,
pharmacokinetics, and toxicology," Mass Spectrom. Rev.
11:249-307.
[0220] DeRisi, J. L. et al. (1997), "Exploring the metabolic and
genetic control of gene expression on a genomic scale," Science
278:680-6
[0221] Dongr'e, A. R., Eng, J. K., and Yates, J. R., 3rd (1997),
"Emerging tandem-mass-spectrometry techniques for the rapid
identification of proteins," Trends Biotechnol. 15:418-425.
[0222] Ducret, A., VanOostveen, I., Eng, J. K., Yates, J. R., and
Aebersold, R. (1998), "High throughput protein characterization by
automated reverse-phase chromatography/electrospray tandem mass
spectrometry," Prot. Sci. 7:706-719.
[0223] Eng, J., McCormack, A., and Yates, J. I. (1994), "An
approach to correlate tandem mass spectral data of peptides with
amino acid sequences in a protein database," J. Am. Soc. Mass
Spectrom. 5:976-989.
[0224] Figeys, D. et al. (1998), "Electrophoresis combined with
mass spectrometry techniques: Powerful tools for the analysis of
proteins and proteomes," Electrophoresis 19:1811-1818.
[0225] Figeys, D., and Aebersold, R. (1998), "High sensitivity
analysis of proteins and peptides by capillary electrophoresis
tandem mass spectrometry: Recent developments in technology and
applications," Electrophoresis 19:885-892.
[0226] Figeys, D., Ducret, A., Yates, J. R., and Aebersold, R.
(1996), "Protein identification by solid phase
microextraction-capillary zone
electrophoresis-microelectrospray-tandem mass spectrometry," Nature
Biotech. 14:1579-1583.
[0227] Figeys, D., Ning, Y., and Aebersold, R. (1997), "A
microfabricated device for rapid protein identification by
microelectrospray ion trap mass spectrometry," Anal. Chem.
69:3153-3160.
[0228] Freeze, H. H. (1998) Disorders in protein glycosylation and
potential therapy. J. Pediatrics 133, 593-600.
[0229] Freeze, H. H. (1999) Human glycosylation disorders and sugar
supplement therapy. Biochem. Biophys. Res. Commun. 255,189-193.
[0230] Gamper, H. B., "Facile preparation of nuclease resistant
3'-modified oligodeoxy-nucleotides, " Nucl. Acids Res., 21:145-150
(January 1993)
[0231] Garrels, J. I., McLaughlin, C. S., Warner, J. R., Futcher,
B., Latter, G. I., Kobayashi, R., Schwender, B., Volpe, T.,
Anderson, D. S., Mesquita, F.-R., and Payne, W. E. (1997),
"Proteome studies of Saccharomyces cerevisiae: identification and
characterization of abundant proteins. Electrophoresis,"
18:1347-1360.
[0232] Gerber, S. A.; Scott, C. R.; Turecek, F.; Gelb, M. H. (1999)
Analysis of rates of multiple enzymes in cell lysates by
electrospray ionization mass spectrometry. J. Am. Chem. Soc.
121,1102-1103.
[0233] Glaser, L. (1966) Phosphomannomutase from yeast. In Meth.
Enzymol. Vol. Vil, Neufeld, E. F.; Ginsburg, V. Eds; Academic
Press: N.Y. 1966, pp.183-185.
[0234] Gygi, S. P. et al. (1999), "Correlation between portein and
mRNA abundance in yeast," Mol. Cell. Biol. 19:1720-1730.
[0235] Gygi, S. P. et al. (1999), "Protein analysis by mass
spectrometry and sequence database searching: tools for cancer
research in the post-genomic era," Electrophoresis 20:310-319.
[0236] Haynes, P. A., Fripp, N., and Aebersold, R. (1998),
"Identification of gel-separated proteins by liquid chromatography
electrospray tandem mass spectrometry: Comparison of methods and
their limitations," Electrophoresis 19:939-945.
[0237] Hodges, P. E. et al. (1999), "The Yeast Proteome Database
(YPD): a model for the organization and presentation of genome-wide
functional data," Nucl. Acids Res. 27:69-73.
[0238] Johnston, M. and Carlson, M. (1992), in The Molecular and
Cellular Biology of the Yeast Saccharomyces, Johnes, E. W. et al.
(eds.), Cold Spring Harbor Press, New York City, pp.193-281.
[0239] Kataky, R. et. al. J Chem Soc Perk T 2 (2) 321-327 FEB
1990.
[0240] Kaur, K. J.; Hingsgaul, 0. (1991) A simple synthesis of
octyl 3,6-di-O-(.alpha.-D-mannopyranosyl)-.beta.-D-manopyranoside
and its use as an acceptor for the assay of
N-acetylglucosaminetransferase I activity. Glycoconjugate J. 8,
90-94.
[0241] Kaur, K. J.; Alton, G.; Hindsgaul, 0. (1991) Use of
N-acetylglucosaminyltranserases I and 11 in the preparative
synthesis of oligosaccharides. Carbohydr. Res. 210,145-153.
[0242] Korner, C.; Knauer, R.; Holzbach, U.; Hanefeld, F.; Lehle,
L.; von Figura, K. (1998) Carbohydrate-deficient glycoprotein
syndrome type V: deficiency of
dolichyl-P-Glc:Man9GlcNAc2-PP-dolichyl glucosyltransferase. Proc
Nati Acad Sci U.S.A. 95,13200-13205.
[0243] Link, A. J., Hays, L. G., Carmack, E. B., and Yates, J. R.,
3rd (1997), "Identifying the major proteome components of
Haemophilus influenzae type-strain NCTC 8143," Electrophoresis
18:1314-1334.
[0244] Link, J. et al. (1999), "Direct analysis of large protein
complexes using mass spectrometry," Nat. Biotech. 17:676-682 (July
1999)
[0245] Mann, M., and Wilm, M. (1994), "Error-tolerant
identification of peptides in sequence databases by peptide
sequence tags," Anal. Chem. 66:4390-4399.
[0246] McMurry, J. E.; Kocovsky, P. (1984) A method for the
palladium-catalyzed allylic oxidation of olefins. Tetrahedron Lett.
25, 4187-4190.
[0247] Morris, A. A. M. and Turnbull, D. M. (1994) Curr. Opin.
Neurol. 7:535-541.
[0248] Neufeld, E. and Muenzer, J. (1995), "The
mucopolysaccharidoses" In The Metabolic and Molecular Bases of
Inherited Disease, Scriver, C. R. et al. (eds.) McGraw-Hill, New
York, pp. 2465-2494.
[0249] Oda, Y. et al. (1999), "Accurate quantitation of protein
expression and site-specific phosphorylation," Proc. NatI. Acad.
Sci. USA 96:6591-6596.
[0250] Okada, S. and O'Brien, J. S. (1968) Science 160:10002.
[0251] Opiteck, G. J. et al. (1997), "Comprehensive on-line
LC/LC/MS of proteins," Anal. Chem. 69:1518-1524.
[0252] Paulsen, H.; Meinjohanns, E. (1992) Synthesis of modified
oligosaccharides of N-glycoproteins intended for substrate
specificity studies of N-acetylglucosaminyltransferases II-V
Tetrahedron Lett. 33, 7327-7330.
[0253] Paulsen, H.; Meinjohanns, E.; Reck, F.; Brockhausen, l.
(1993) Synthese von modifizierten Oligosacchariden der
N-Glycoproteine zur Untersuchung der Spezifitat der
N-Acetylglucosaminyltransferase II. Liebigs Ann. Chem. 721-735.
[0254] Pennington, S. R., Wilkins, M. R., Hochstrasser, D. F., and
Dunn, M. J. (1997), "Proteome analysis: From protein
characterization to biological function," Trends Cell Bio.
7:168-173.
[0255] Preiss, J. (1966) GDP -mannose pyrophosphorylase from
Arthrobacter. In Meth. Enzymol. Vol. Vill, Neufeld, E. F.;
Ginsburg, V. Eds; Academic Press: New York 1966, pp. 271-275.
[0256] Qin, J. et al. (1997), "A strategy for rapid,
high-confidence protein identification," Anal. Chem.
69:3995-4001.
[0257] Ronin, C.; Caseti, C.; Bouchilloux, C. (1981) Transfer of
glucose in the biosynthesis of thyroid glycoproteins. l. Inhibition
of glucose transfer to oligosaccharide lipids by GDP-mannose.
Biochim. Biophys. Acta 674, 48-57.
[0258] Ronin, C.; Granier, C.; Caseti, C.; Bouchilloux, S.; Van
Rietschoten, J. (1981a) Synthetic substrates for thyroid
oligosaccharide transferase. Effects of peptide chain length and
modifications in the -Asn-Xaa-Thr- region. Eur. J. Biochem.
118,159-164.
[0259] Ronne, H. (1995), "Glucose repression in fungi," Trends
Genet. 11:12-17.
[0260] Rush, J. S.; Wachter, C. J. (1995) Transmembrane movement of
a water-soluble analogue of mannosylphosphoryldolichol is mediated
by an endoplasmic reticulum protein. J. Cell. Biol. 130,
529-536.
[0261] Schachter, H. (1986) Biosynthetic controls that determine
the branching and microheterogeneity of protein-bound
oligosaccharides. Biochem. Cell Biol. 64, 163-181.
[0262] Scriver, C. R. et al. (1995), The Metabolic and Molecular
Bases of Inherited Disease,
[0263] Scriver, C. R. et al. (eds.) McGraw-Hill, N.Y., pp.
1015-1076.
[0264] Sechi, S. and Chait, B. T. (1998), "Modification of cysteine
residues by alkylation. A tool in peptide mapping and protein
identification," Anal. Chem. 70:5150-5158.
[0265] Segal, S. and Berry, G. T. (1995), The Metabolic and
Molecular Bases of Inherited Disease, Scriver, C. R. et al. (eds.),
McGraw-Hill, N.Y., pp. 967-1000.
[0266] Romanowska, A. et al. (1994), "Michael Additions for
Synthesis of Neoglycoproteins," Methods Enzymol. Neoconjugates Part
A (Synthesis) 242:90-101.
[0267] Roth, F. P. et al. (1998), "Finding DNA regulatory motifs
within unaligned noncoding sequences clustered by whole-genome mRNA
quantitation," Nat. Biotechnol. 16:939-945.
[0268] Shalon, D., Smith, S. J., and Brown, P. O. (1996), "A DNA
microarray system for analyzing complex DNA samples using two-color
fluorescent probe hybridization," Genome Res. 6:639-645.
[0269] Shevchenko, A., Jensen, 0. N., Podtelejnikov, A. V.,
Sagliocco, F., Wilm, M., Vorm, O., Mortensen, P., Shevchenko, A.,
Boucherie, H., and Mann, M. (1996), "Linking genome and proteome by
mass spectrometry: large-scale identification of yeast proteins
from two dimensional gels," Proc. Natl. Acad. Sci. U.S.A.
93:14440-14445.
[0270] Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996),
"Mass spectrometric sequencing of proteins silver-stained
polyacrylamide gels," Anal. Chem. 68:850-858.
[0271] Tan, J.; Dunn, J.; Jaeken, J.; Schachter, H. (1996)
Mutations in the MGAT2 gene controlling complex glycan synthesis
cause carbohydrate deficient glycoprotein syndrome type II, an
autosomal recessive disease with defective brain development. Am.
J. Hum. Genet. 59, 810-817.
[0272] Velculescu, V. E., Zhang, L., Zhou, W., Vogelstein, J.,
Basrai, M. A., Bassett, D. E., Jr., Hieter, P., Vogelstein, B., and
Kinzler, K. W. (1997), "Characterization of the yeast
transcriptome," Cell 88:243-251.
[0273] Wilbur, D.S. et al. (1997), "Biotin reagents for antibody
pretargeting. Synthesis, radioiodenation and in vitro evaluation of
water soluble, biotinidase resistant biotin derivatives,"
Bioconjugate Chem. 8:572-584.
[0274] Yates, J. R. d., Eng, J. K., McCormack, A. L., and Schieltz,
D. (1995), "Method to correlate tandem mass spectra of modified
peptides to amino acid sequences in the protein database," Anal.
Chem. 67:1426-1436.
[0275] All references cited herein are incorporated by reference in
their entirty herein.
Sequence CWU 1
1
64 1 7 PRT Artificial Sequence Description of Artificial Sequence
Heptapeptide motif found in substrates for glycosylation 1 Tyr Gln
Ser Asn Ser Thr Met 1 5 2 7 PRT Artificial Sequence Description of
Artificial Sequence Test peptide 2 Lys Ala Leu Cys Ser Glu Lys 1 5
3 6 PRT Artificial Sequence Description of Artificial Sequence Test
peptide 3 Lys Cys Glu Val Phe Arg 1 5 4 9 PRT Artificial Sequence
Description of Artificial Sequence Test peptide 4 Lys Leu Asp Gln
Trp Leu Cys Glu Lys 1 5 5 15 PRT Artificial Sequence Description of
Artificial Sequence Test peptide 5 Lys Phe Leu Asp Asp Asp Leu Thr
Asp Asp Ile Met Cys Val Lys 1 5 10 15 6 18 PRT Artificial Sequence
Description of Artificial Sequence Test peptide 6 Lys Asp Asp Gln
Asn Pro His Ser Ser Asn Ile Cys Asn Ile Ser Cys 1 5 10 15 Asp Lys 7
43 PRT Artificial Sequence Description of Artificial Sequence Test
peptide 7 Lys Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp Val Cys Thr
Thr Phe 1 5 10 15 His Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Gln
Asn Asn Asp Ser 20 25 30 Thr Glu Tyr Gly Leu Phe Gln Ile Asn Asn
Lys 35 40 8 6 PRT bovine VARIANT (3) C at position 3 is ICAT-
labeled cysteinyl residue 8 Ala Leu Cys Ser Glu Lys 1 5 9 13 PRT
bovine VARIANT (11) C at position 11 is ICAT-labeled cysteinyl
residue. 9 Phe Leu Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys 1 5
10 10 10 PRT chicken VARIANT (8) C at position 8 is ICAT-labeled
cystenyl residue. 10 Ala Asp His Pro Phe Leu Phe Cys Ile Lys 1 5 10
11 12 PRT chicken VARIANT (10) C at position 10 is ICAT labeled
cysteinyl residue. 11 Tyr Pro Ile Leu Pro Glu Tyr Leu Gln Cys Val
Lys 1 5 10 12 8 PRT E coli VARIANT (5) C at position 5 is
ICAT-labeled cysteinyl residue. 12 Leu Thr Ala Ala Cys Phe Asp Arg
1 5 13 13 PRT E coli VARIANT (5) C at position 5 is ICAT-labeled
cysteinyl residue. 13 Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala
Glu Arg 1 5 10 14 17 PRT E coli VARIANT (14) C at position 14 is
ICAT-labeled cysteinyl residue. 14 Ile Ile Phe Asp Gly Val Asn Ser
Ala Phe His Leu Trp Cys Asn Gly 1 5 10 15 Arg 15 9 PRT bovine
VARIANT (6) C at position 6 is ICAT-labeled cysteinyl residue. 15
Trp Glu Asn Gly Glu Cys Ala Gln Lys 1 5 16 14 PRT bovine VARIANT
(12) C at position 12 is ICAT-labeled cysteinyl residue. 16 Leu Ser
Phe Asn Pro Thr Gln Leu Glu Glu Gln Cys His Ile 1 5 10 17 14 PRT
rabbit VARIANT (13) C at position 13 is ICAT-labeled cysteinyl
residue. 17 Val Pro Thr Pro Asn Val Ser Val Val Asp Leu Thr Cys Arg
1 5 10 18 17 PRT rabbit VARIANT (1)..(17) C at positions 7 and 11
are ICAT-labeled cysteinyl residues. 18 Ile Val Ser Asn Ala Ser Cys
Thr Thr Asn Cys Leu Ala Pro Leu Ala 1 5 10 15 Lys 19 15 PRT rabbit
VARIANT (2) C at position 2 is ICAT-labeled cysteinyl residue. 19
Ile Cys Gly Gly Trp Gln Met Glu Glu Ala Asp Asp Trp Leu Arg 1 5 10
15 20 16 PRT rabbit VARIANT (2) C at position 2 is ICAT-labeled
cysteinyl residue. 20 Thr Cys Ala Tyr Thr Asn His Thr Val Leu Pro
Glu Ala Leu Glu Arg 1 5 10 15 21 16 PRT rabbit VARIANT (5) C at
position 5 is ICAT-labeled cysteinyl residue. 21 Trp Leu Val Leu
Cys Asn Pro Gly Leu Ala Glu Ile Ile Ala Glu Arg 1 5 10 15 22 12 PRT
yeast VARIANT (4) C at position 4 is ICAT-labeled cysteinyl
residue. 22 Lys His Asn Cys Leu His Glu Pro His Met Leu Lys 1 5 10
23 21 PRT yeast VARIANT (5) C at position 5 is ICAT-labeled
cysteinyl residue. 23 Tyr Ser Gly Val Cys His Thr Asp Leu His Ala
Trp His Gly Asp Trp 1 5 10 15 Pro Leu Pro Val Lys 20 24 11 PRT
yeast VARIANT (1)..(2) C at positions 1 and 2 are ICAT-labeled
cysteinyl residues. 24 Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys
1 5 10 25 21 PRT yeast VARIANT (5) C at position 5 is ICAT-labeled
cysteinyl residue. 25 Tyr Ser Gly Val Cys His Thr Asp Leu His Ala
Trp His Gly Asp Trp 1 5 10 15 Pro Leu Pro Thr Lys 20 26 11 PRT
yeast VARIANT (1) C at position 1 is ICAT-labeled cysteinyl
residue. 26 Cys Ser Ser Asp Val Phe Asn His Val Val Lys 1 5 10 27
20 PRT yeast VARIANT (14) C at position 14 is ICAT-labeled
cysteinyl residue. 27 Thr Phe Glu Val Ile Asn Pro Ser Thr Glu Glu
Glu Ile Cys His Ile 1 5 10 15 Tyr Glu Gly Arg 20 28 12 PRT yeast
VARIANT (9) C at position 9 is ICAT-labeled cysteinyl residue. 28
Ser Glu His Gln Val Glu Leu Ile Cys Ser Tyr Arg 1 5 10 29 12 PRT
yeast VARIANT (5) C at position 9 is ICAT-labeled cysteinyl
residue. 29 Tyr Arg Pro Asn Cys Pro Ile Ile Leu Val Thr Arg 1 5 10
30 25 PRT yeast VARIANT (2) C at position 2 is ICAT-labeled
cysteinyl residue. 30 Asn Cys Thr Pro Lys Pro Thr Ser Thr Thr Glu
Thr Val Ala Ala Ser 1 5 10 15 Ala Val Ala Ala Val Phe Glu Gln Lys
20 25 31 19 PRT yeast VARIANT (17) C at position 17 is ICAT-labeled
cysteinyl residue. 31 Ser Ile Ala Pro Ala Tyr Gly Ile Pro Val Val
Leu His Ser Asp His 1 5 10 15 Cys Ala Lys 32 6 PRT yeast VARIANT
(5) C at position 5 is ICAT-labeled cysteinyl residue. 32 Glu Gln
Val Gly Cys Lys 1 5 33 24 PRT yeast VARIANT (9) C at position 9 is
ICAT-labeled cysteinyl residue. 33 Leu Thr Gly Ala Gly Trp Gly Gly
Cys Thr Val His Leu Val Pro Gly 1 5 10 15 Gly Pro Asn Gly Asn Ile
Glu Lys 20 34 13 PRT yeast VARIANT (11) C at position 11 is
ICAT-labeled cysteinyl residue. 34 His His Ile Pro Phe Tyr Glu Val
Asp Leu Cys Asp Arg 1 5 10 35 6 PRT yeast VARIANT (2) C at position
2 is ICAT-labeled cysteinyl residue. 35 Asp Cys Val Thr Leu Lys 1 5
36 18 PRT yeast VARIANT (3) C at position 3 is ICAT-labeled
cysteinyl residue. 36 Leu Trp Cys Thr Gln His His Glu Pro Glu Val
Ala Leu Asp Gln Ser 1 5 10 15 Leu Lys 37 18 PRT yeast VARIANT (2) C
at position 2 is ICAT labeled cysteinyl residue. 37 Ile Cys Ser Val
Asn Leu His Gly Asp His Thr Phe Ser Met Glu Gln 1 5 10 15 Met Lys
38 6 PRT yeast VARIANT (2) C at position 2 is ICAT-labeled
cysteinyl residue. 38 Ile Cys Ser Gln Leu Lys 1 5 39 9 PRT yeast
VARIANT (5) C at position 5 is ICAT-labeled cysteinyl residue. 39
Gly Gly Thr Gln Cys Ser Ile Met Arg 1 5 40 30 PRT yeast VARIANT (2)
C at position 2 is ICAT-labeled cysteinyl residue. 40 Asn Cys Phe
Pro His His Gly Tyr Ile His Asn Tyr Gly Ala Phe Pro 1 5 10 15 Gln
Thr Trp Glu Asp Pro Asn Val Ser His Pro Glu Thr Lys 20 25 30 41 13
PRT yeast VARIANT (2) C at position 2 is ICAT-labeled cysteinyl
residue. 41 Val Cys His Ala His Pro Thr Leu Ser Glu Ala Phe Lys 1 5
10 42 14 PRT yeast VARIANT (11) C at position 11 is ICAT-labeled
cysteinyl residue. 42 Lys Gly Trp Thr Gly Gln Tyr Thr Leu Asp Cys
Asn Thr Arg 1 5 10 43 10 PRT yeast VARIANT (5) C at position 5 is
ICAT-labeled cysteinyl residue. 43 Ser Val Val Leu Cys Asn Ser Thr
Ile Lys 1 5 10 44 23 PRT yeast VARIANT (1) C at position 1 is
ICAT-labeled cysteinyl residue. 44 Cys Thr Gly Gly Ile Ile Leu Thr
Ala Ser His Asn Pro Gly Gly Pro 1 5 10 15 Glu Asn Asp Met Gly Ile
Lys 20 45 17 PRT yeast VARIANT (4) C at position 4 is ICAT-labeled
cysteinyl residue. 45 Leu Ser Ile Cys Gly Glu Glu Ser Phe Gly Thr
Gly Ser Asn His Val 1 5 10 15 Arg 46 10 PRT yeast VARIANT (3) C at
position 3 is ICAT-labeled cysteinyl residue. 46 Ile Pro Cys Leu
Ala Asp Ser His Pro Lys 1 5 10 47 17 PRT yeast VARIANT (1) C at
position 1 is ICAT-labeled cysteinyl residue. 47 Cys Ile Asn Leu
Ser Ala Glu Lys Glu Pro Glu Ile Phe Asp Ala Ile 1 5 10 15 Lys 48 12
PRT Yeast VARIANT (1) C at position 1 is ICAT-labeled cysteinyl
residue. 48 Cys Ala Tyr Pro Ile Asp Tyr Ile Pro Ser Ala Lys 1 5 10
49 23 PRT yeast VARIANT (20) C at position 20 is ICAT-labeled
cysteinyl residue. 49 Ile Val Glu Glu Pro Thr Ser Lys Asp Glu Ile
Trp Trp Gly Pro Val 1 5 10 15 Asn Lys Pro Cys Ser Glu Arg 20 50 12
PRT yeast VARIANT (9) C at position 9 is ICAT-labeled cysteinyl
residue. 50 Ala Leu Val His His Tyr Glu Glu Cys Ala Glu Arg 1 5 10
51 14 PRT yeast VARIANT (2) C at position 2 is ICAT-labeled
cysteinyl residue. 51 Ser Cys Gly Val Asp Ala Met Ser Val Asp Asp
Leu Lys Lys 1 5 10 52 24 PRT yeast VARIANT (8) C at position 8 is
ICAT-labeled cysteinyl residue. 52 His Pro Glu Met Leu Glu Asp Cys
Phe Gly Leu Ser Glu Glu Thr Thr 1 5 10 15 Thr Gly Val His His Leu
Tyr Arg 20 53 11 PRT yeast VARIANT (2) C at position 2 is
ICAT-labeled cysteinyl residue. 53 Glu Cys Ile Asn Ile Lys Pro Gln
Val Asp Arg 1 5 10 54 25 PRT yeast VARIANT (14) C at position 14 is
ICAT-labeled cysteinyl residue. 54 Gly Phe His Ile His Glu Phe Gly
Asp Ala Thr Asn Gly Cys Val Ser 1 5 10 15 Ala Gly Pro His Phe Asn
Pro Phe Lys 20 25 55 9 PRT yeast VARIANT (5) C at position 5 is
ICAT-labeled cysteinyl residue. 55 Arg Gly Asn Val Cys Gly Asp Ala
Lys 1 5 56 6 PRT yeast VARIANT (1) C at position 1 is ICAT-labeled
cysteinyl residue. 56 Cys Gly Gly Ile Asp Lys 1 5 57 20 PRT yeast
VARIANT (8) C at position 8 is ICAT-labeled cysteinyl residue. 57
Phe Val Pro Ser Lys Pro Met Cys Val Glu Ala Phe Ser Glu Tyr Pro 1 5
10 15 Pro Leu Gly Arg 20 58 20 PRT yeast VARIANT (19) C at position
19 is ICAT-labeled cysteinyl residue. 58 Ile Pro Ile Phe Ser Ala
Ser Gly Leu Pro His Asn Glu Ile Ala Ala 1 5 10 15 Gln Ile Cys Arg
20 59 10 PRT yeast VARIANT (5) C at position 5 is ICAT-labeled
cysteinyl residue. 59 His Tyr Ser Leu Cys Ser Ala Ser Thr Lys 1 5
10 60 14 PRT rabbit VARIANT (13) C at position 13 is ICAT-labeled
cysteinyl residue. 60 Val Pro Thr Pro Asn Val Ser Val Val Asp Leu
Thr Cys Arg 1 5 10 61 18 PRT Streptomyces lividans 61 Leu Gly Lys
Pro Val Leu Thr Ala Asn Gln Val Thr Ile Trp Glu Gly 1 5 10 15 Leu
Arg 62 19 PRT Unknown Description of Unknown Organism Unidentifed
62 Ile Ala Asn Pro Asn Val Tyr Thr Glu Thr Leu Thr Ala Ala Thr Val
1 5 10 15 Cys Thr Ile 63 19 PRT Unknown Description of Unknown
Organism Unidentifed 63 Leu Ala Leu Leu Pro Ser Asp Ala Glu Gly Pro
His Gly Gln Phe Val 1 5 10 15 Thr Asp Lys 64 20 PRT Homo sapiens 64
Ala Leu Leu Val Leu Val Ala Pro Ala Met Ala Ala Gly Asn Gly Glu 1 5
10 15 Asp Leu Arg Asn 20
* * * * *