U.S. patent application number 13/284255 was filed with the patent office on 2012-06-14 for detection and quantification of modified proteins.
This patent application is currently assigned to PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to Steven P. Gygi, Junmin Peng.
Application Number | 20120149883 13/284255 |
Document ID | / |
Family ID | 28041735 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120149883 |
Kind Code |
A1 |
Gygi; Steven P. ; et
al. |
June 14, 2012 |
Detection and quantification of modified proteins
Abstract
The invention provides a method detecting and quantifying
proteins by mass spectrophotometric analysis using peptide internal
standards and provides a highly sensitive way of detecting protein
modifications. In one aspect, the invention provides a method for
determining a site of ubiquitination in a polypeptide and for
evaluating ubiquitination targets in a population of polypeptides.
In this way, a proteome ubiquitination map can be obtained which
comprises information relating to the ubiquitination states of a
plurality of cellular polypeptides. Maps can be obtained for a
variety of different types of cells and cell states. For example,
ubiquitination targets in normal and diseased cells can be
evaluated. Preferably, the map is stored as data files in a
database. Individual ubiquitinated polypeptides identified can be
used to generate molecular probes diagnostic of a cell state and/or
can serve as targets for agents that modulate one or more cellular
processes.
Inventors: |
Gygi; Steven P.; (Foxboro,
MA) ; Peng; Junmin; (Memphis, TN) |
Assignee: |
PRESIDENT AND FELLOWS OF HARVARD
COLLEGE
Cambridge
MA
|
Family ID: |
28041735 |
Appl. No.: |
13/284255 |
Filed: |
October 28, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10506877 |
Jul 29, 2005 |
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PCT/US03/07527 |
Mar 11, 2003 |
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13284255 |
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60363179 |
Mar 11, 2002 |
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Current U.S.
Class: |
530/388.1 ;
530/389.1; 530/391.1 |
Current CPC
Class: |
C07K 7/08 20130101; G01N
33/6803 20130101; G01N 33/6842 20130101; C07K 1/22 20130101; C12Q
1/37 20130101; G01N 33/6848 20130101; G01N 33/6896 20130101; G01N
2440/36 20130101; Y10T 436/24 20150115; G01N 2560/00 20130101 |
Class at
Publication: |
530/388.1 ;
530/389.1; 530/391.1 |
International
Class: |
C07K 17/00 20060101
C07K017/00; C07K 16/00 20060101 C07K016/00 |
Goverment Interests
GOVERNMENT GRANTS
[0002] At least part of the work contained in this application was
performed under government grant HG00041 from the National
Institutes of Health, U.S. Department of Health and Human Services.
The government may have certain rights in this invention.
Claims
1-31. (canceled)
32. A kit comprising an antibody that specifically recognizes a
peptide product of a protease-digested ubiquitinated protein which
comprises a ubiquitin remnant.
33. The kit according to claim 32, wherein the peptide comprises a
lysine residue at position 6, 11, 27, 29, 33, 48, and 63 of the
ubiquitin polypeptide.
34. A kit comprising an antibody which specifically recognizes a
ubiquitin polypeptide ubiquitinated at one or more of the K.sup.6,
K.sup.11, K.sup.27, K.sup.29, K.sup.33, K.sup.48, and K.sup.63
sites.
35-44. (canceled)
45. The kit according to claim 32, wherein the antibody comprises a
monoclonal antibody or a portion thereof.
46. The kit according to claim 32, wherein the antibody comprises a
polyclonal antibody or a portion thereof.
47. The kit according to claim 32, wherein the antibody is
associated with a solid phase.
48. The kit according to claim 47, wherein the solid phase is at
least one selected from the group consisting of: a bead, a
microparticle, a sphere, a chip, and a support.
49. The kit according to claim 47, wherein the antibody is directly
linked to the solid phase.
50. The kit according to claim 47, wherein the antibody is
chemically conjugated to the solid phase.
51. The kit according to claim 47, wherein a binding partner links
the antibody to the solid phase.
52. The kit according to claim 34, wherein the antibody comprises a
monoclonal antibody or a portion thereof.
53. The kit according to claim 34, wherein the antibody comprises a
polyclonal antibody or a portion thereof.
54. The kit according to claim 34, wherein the antibody is
associated with a solid phase.
55. The kit according to claim 54, wherein the solid phase is at
least one selected from the group consisting of: a bead, a
microparticle, a sphere, a chip, and a support.
56. The kit according to claim 54, wherein the antibody is directly
linked to the solid phase.
57. The kit according to claim 54, wherein the antibody is
chemically conjugated to the solid phase.
58. The kit according to claim 54, wherein a binding partner links
the antibody to the solid phase.
59. A kit comprising an antibody that specifically recognizes a
peptide product of a protease-digested ubiquitinated protein,
wherein the peptide product comprises a ubiquitin remnant.
60. An antibody that specifically recognizes a peptide product of a
protease-digested ubiquitinated protein which comprises a ubiquitin
remnant.
61. The antibody according to claim 60, wherein the ubiquitin
remnant is attached to the peptide product.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. utility
application Ser. No. 10/506,877 filed Jul. 29, 2005 which claims
the benefit of the PCT/US03/07527 filed Mar. 11, 2003 which claims
the benefit of the U.S. provisional application Ser. No. 60/363,179
filed Mar. 11, 2002, each of which is hereby incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0003] This invention provides methods, reagents and kits for
obtaining absolute quantification of proteins and their
modifications directly from cell lysates. In particular, the
invention provides peptide internal standards for use in high
performance liquid chromatography (HPLC) with online detection by
multistage mass spectrometry (MS.sup.n). In one aspect, the
invention also provided compositions, kits and methods for
detective ubiquitination sites in proteins.
BACKGROUND OF THE INVENTION
[0004] There is a need to provide novel methods for the
quantification of proteins and modified proteins from cell lysates.
The current standard for protein detection (quantification) is
based on immunoreactive detection (Western analysis). However, this
technique requires the availability of an appropriately specific
antibody. In addition, many antibodies only recognize proteins in
an unfolded (denatured) form, cross-reactivity can be severely
limiting, and quantification is generally relative.
[0005] The development of methods and instrumentation for
automated, data-dependent electrospray ionization (ESI) tandem mass
spectrometry (MS/MS) in conjunction with microcapillary liquid
chromatography (LC) and database searching has significantly
increased the sensitivity and speed of the identification of
gel-separated proteins. Microcapillary LC-MS/MS has 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). However, while
these approaches dramatically accelerate protein identification,
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 microcapillary LC/MS/MS method
without their prior enrichment.
[0006] There is thus a need to provide methods for the accurate
comparison of protein expression levels between cells in two
different states, particularly for comparison of low abundance
proteins.
[0007] Another metholology has recently been described. ICAT.TM.
reagent technology makes use of a class of chemical reagents called
isotope coded affinity tags (ICAT). These reagents exist in
isotopically heavy and light forms which are chemically identical
with the exception of eight deuterium or hydrogen atoms,
respectively. Proteins from two cells lysates can be labeled
independently with one or the other ICAT reagent at cysteinyl
residues. After mixing and proteolysing the lysates, the
ICAT-labeled peptides are isolated by affinity to a biotin molecule
incorporated into each ICAT reagent. ICAT-labeled peptides are
analyzed by LC-MS/MS where they elute as heavy and light pairs of
peptides. Quantification is performed by determining the relative
expression ratio relating to the amount of each ICAT-labeled
peptide pair in the sample.
[0008] Identification of each ICAT-labeled peptide is performed by
a second stage of mass spectrometry (MS/MS) and sequence database
searching. The end result is relative protein expression ratios on
a large scale. The major drawback to this technique are 1)
quantification is only relative; 2) specialized chemistry is
required, and 3) database searches are hindered by the presence of
the large ICAT reagent molecule, and 4) relative amounts of
posttranslationally modified (e.g., phosphorylated) proteins are
transparent to analysis.
SUMMARY
[0009] The present invention provides reagents, kits, and methods
for accurate quantification of proteins and methods for using the
same. In particular, the method is useful for detecting and
quantitating modified proteins and identifying sites of protein
modification, such as sites of ubiquitination. The reagents, kits,
and methods of the invention are useful for rapid, high throughput
analysis of proteomes.
[0010] The invention also provides a method for generating a
peptide internal standard. The method comprises identifying a real
or predicted peptide digestion product of a target polypeptide,
determining the amino acid sequence of the peptide digestion
product and synthesizing a peptide having the amino acid sequence.
The peptide is labeled with a mass-altering label (e.g., by
incorporating labeled amino acid residues during the synthesis
process) and fragmented (e.g., by multi-stage mass spectrometry).
Preferably, the label is a stable isotope. A peptide signature
diagnostic of the peptide is determined, after one or more rounds
of fragmenting, and the signature is used to identify the presence
and/or quantity of a peptide of identical amino acid sequence in a
sample.
[0011] Preferably, a labeled peptide is provided which co-elutes
with an unlabeled peptide having the same amino acid sequence
(i.e., a target peptide) in a chromatographic separation procedure
(e.g., such as HPLC).
[0012] In one aspect, the mass-altering label is part of a peptide
comprising a modification, and the peptide is fragmented to
determine a peptide signature diagnostic of such a modified
peptide. The modified residue in the peptide internal standard
comprises a phosphorylated residue, a glycosylated residue, an
acetylated residue, a ubiquitinated residue, a ribosylated residue,
or a farnesylated residue, or another modification found in a
cellular protein. In one aspect, panels of peptide internal
standards are generated corresponding to (i.e., diagnostic of)
different modified forms of the same protein.
[0013] Peptide internal standards corresponding to different
peptide subsequences of a single target protein also can be
generated to provide for redundant controls in a quantitative
assay. In one aspect, different peptide internal standards
corresponding to the same target protein are generated and
differentially labeled (e.g., peptides are labeled at multiple
sites to vary the amount of heavy label associated with a given
peptide).
[0014] In another aspect, a panel of peptide internal standards
corresponding to different amino acid subsequences of a single
protein is used to scan for mutations in that protein. In a further
aspect, peptide internal standards corresponding to different
variant sequences of a single amino acid subsequence of a single
protein are provided. A match between a peptide internal standard
and a target peptide in a sample indicates the presence of a
variant sequence in the sample. In one aspect, the multiple peptide
internal standards corresponding to variant sequences are
differentially labeled.
[0015] In a further aspect, a panel of peptide internal standards
corresponding to amino acid subsequences of different proteins in a
molecular pathway is generated. Molecular pathways, include, but
are not limited to signal transduction pathways, cell cycle
pathways, metabolic pathways, blood clotting pathways, and the
like. In one aspect, the panel includes peptide standards which
correspond to different modified forms of one or more proteins in a
pathway and the panel is used to determine the presence and/or
quantity of the activated or inactivated form of a pathway
protein.
[0016] The invention also provides a method for determining the
presence and/or quantity of a target polypeptide in at least one
mixture of different polypeptides. The method comprises providing a
mixture of different polypeptides and spiking the mixture with a
known quantity of a peptide internal standard labeled with a
mass-altering label. Preferably, the labeled peptide internal
standard comprises a subsequence of the target polypeptide and
possesses a known peptide fragment signature diagnostic of the
presence of the peptide subsequence. The spiked mixture is treated
with a protease activity to generate a plurality of peptides
including the labeled peptide internal standard and peptides
corresponding to the target polypeptide. Preferably, a
chromatographic separation step is performed to isolate the labeled
peptide internal standard and any target peptide present in the
spiked mixture which comprises the same amino acid sequence as the
standard. Preferably, the internal standard and target peptide
co-elute with each other.
[0017] The labeled peptide internal standard and target peptide are
fragmented (e.g., using multistage mass spectrometry) and the ratio
of labeled fragments to unlabeled fragments; is determined. The
quantity of the target polypeptide can be calculated using both the
ratio and known quantity of the labeled internal standard. The
mixtures of different polypeptides can include, but are not limited
to, such complex mixtures as a crude fermenter solution, a
cell-free culture fluid, a cell or tissue extract, blood sample, a
plasma sample, a lymph sample, a cell or tissue lysate; a mixture
comprising at least about 100 different polypeptides; at least
about 1000 different polypeptides, at least about 100,000 different
polypeptides. or a mixture comprising substantially the entire
complement of proteins in a cell or tissue. In one preferred
aspect, the method is used to determine the presence of and/or
quantity of one or more target polypeptides directly from one or
more cell lysates, i.e., without separating proteins from other
cellular components or eliminating other cellular components.
[0018] In one aspect, the presence and/or quantity of target
polypeptide in a mixture are diagnostic of a cell state. In another
aspect, the cell state is representative of an abnormal
physiological response, for example, a physiological response which
is diagnostic of a disease. In a further aspect, the cell state is
a state of differentiation or represents a cell which has been
exposed to a condition or agent (e.g., a drug, a therapeutic agent,
a potential toxin). In one aspect, the method is used to diagnose
the presence or risk of a disease. In another aspect, the method is
used to identify a condition or agent which produces a selected
cell state (e.g., to identify an agent which returns one or more
diagnostic parameters of a cell state to normal).
[0019] In a further aspect, the method comprises determining the
presence and/or quantity of target peptides in at least two
mixtures. In another aspect, one mixture is from a cell having a
first cell state and the second mixture is from a cell having a
second cell state. In a further aspect, the first cell is a normal
cell and the second cell is from a patient with a disease. In still
a further aspect, the first cell is exposed to a condition and/or
treated with an agent and the second cell is not exposed and/or
treated. Preferably, first and second mixtures are evaluated in
parallel.
[0020] Alternatively, the two mixtures can be from identical
samples or cells. In one aspect, a labeled peptide internal
standard is provided in different known amounts in each mixture. In
another aspect, pairs of labeled peptide internal standards are
provided each comprising mass-altering labels which differ in mass,
e.g., by including different amounts of a heavy isotope in each
peptide.
[0021] The invention also provides a method of determining the
presence of and/or quantity of a modification in a target
polypeptide. Preferably, the label in the internal standard is part
of a peptide comprising a modified amino acid residue or to an
amino acid residue which is predicted to be modified in a target
polypeptide. In one aspect, the presence of the modification
reflects the activity of a target polypeptide and the assay is used
to detect the presence and/or quantity of an active polypeptide.
The method is advantageous in enabling detection of small
quantities of polypeptide (e.g., about 1 part per million (ppm) or
less than about 0.001% of total cellular protein).
[0022] The invention additionally provides a method for scanning
for mutations in a protein sequence using panels of peptide
internal standards corresponding to different variant forms of a
single sequence or multiple peptide internal standards representing
different amino acid subsequences of a protein. In the first
scenario, a match to a variant peptide internal standard in a
sample indicates the presence of the variant in the sample. In the
second scenario, a lack of match to a one peptide internal standard
and matches to one or more other peptide internal standards
indicates the presence of a mutation in the amino acid sequence
corresponding to the mismatched peptide.
[0023] In a further aspect, the invention provides a method for
profiling the activity of a molecular pathway using panels of
peptide internal standards corresponding to different pathway
proteins and/or to different modified forms of the proteins. The
presence and/or quantity of the proteins can be used to profile the
function of a pathway in a particular cell. In one aspect, the
pathway is one or more of a signal transduction pathway, a cell
cycle pathway, a metabolic pathway, a blood clotting pathway and
the like. The coordinate function of multiple pathways can be
evaluated using a plurality of panels of standards. Similarly, the
peptide internal standards can be used to assay for the presence of
multiple diseases or pathological conditions by providing a panel
of peptide internal standards which comprises peptide internal
standards diagnostic of different diseases.
[0024] The invention further provides reagents useful for
performing the method. In one aspect, a reagent according to the
invention comprises a peptide internal standard labeled with a
stable isotope. Preferably, the standard has a unique peptide
fragmentation signature diagnostic of the peptide. The peptide is a
subsequence of a known protein and can be used to identify the
presence of and/or quantify the protein in sample, such as a cell
lysate. In one aspect, the peptide internal standard comprises a
label associated with a modified amino acid residue, such as a
phosphorylated amino acid residue, a glycosylated amino acid
residue, an acetylated amino acid residue, a farnesylated residue,
a ribosylated residue, and the like. In another aspect, a pair of
reagents is provided, a peptide internal standard corresponding to
a modified peptide and a peptide internal standard corresponding to
a peptide identical in sequence but not modified.
[0025] In one aspect, panels of peptide internal standards
representing different variant forms of a single amino acid
subsequence of a polypeptide are provided.
[0026] In another aspect, panels of peptide internal standards
corresponding to different amino acid subsequences of single
polypeptide are provided.
[0027] In a further aspect, panels of peptide internal standards
are provided which correspond to different proteins in a molecular
pathway (e.g., a signal transduction pathway, a cell cycle pathway,
a metabolic pathway, a blood clotting pathway and the like). In
still a further aspect, peptide internal standards corresponding to
different modified forms of one or more proteins in a pathway are
provided.
[0028] In still a further aspect, panels of peptide internal
standards are provided which correspond to proteins diagnostic of
different diseases, allowing a mixture of peptide internal
standards to be used to test for the presence of multiple diseases
in a single assay.
[0029] The invention additionally provides kits comprising one or
more peptide internal standards labeled with a stable isotope. In
one aspect, a kit comprises peptide internal standards comprising
different peptide subsequences from a single known protein. In
another aspect, the kit comprises peptide internal standards
corresponding to different variant forms of the same amino acid
subsequence of a target polypeptide. In still another aspect, the
kit comprises peptide internal standards corresponding to different
known or predicted modified fauns of a polypeptide. In a further
aspect, the kit comprises peptide internal standards corresponding
to sets of related proteins, e.g., such as proteins involved in a
molecular pathway (a signal transduction pathway, a cell cycle,
etc) and/or to different modified forms of proteins in the pathway.
In still a further aspect, a kit comprises a labeled peptide
internal standard as described above and software for performing
multistage mass spectrometry. The kit may also include a means for
obtaining access to a database comprising data files which include
data relating to the mass spectra of fragmented peptide ions
generated from peptide internal standards. The means for obtaining
access can be provided in the form of a URL and/or identification
number for accessing a database or in the form of a computer
program product comprising the data files. In one aspect, the kit
comprises a computer program product which is capable of
instructing a processor to perform any of the methods described
above.
[0030] The invention additionally provides a method for determining
a site of ubiquitination. The method comprises obtaining a
plurality of ubiquitinated polypeptides, digesting the
ubiquitinated polypeptides with a protease, thereby generating a
plurality of test peptides, and determining the presence of an
isopeptide bond in a test peptide by mass spectrometry, wherein the
presence of the bond indicates a site of ubiquitination. The test
peptide being evaluated can be ionized and/or fragmented prior to
the determining step. Preferably, ionizing is performed by
electrospray.
[0031] In one aspect, the invention provides a method for
determining a site of ubiquitination comprising: obtaining a
plurality of ubiquitinated polypeptides, digesting the
ubiquitinated polypeptides with a protease, thereby generating a
plurality of test peptides, at least some of which comprise a
ubiquitin remnant, identifying a mass difference between a test
peptide and a reference peptide comprising a known identical amino
acid sequence as the test peptide, the mass difference
corresponding to the mass of the ubiquitin remnant, wherein
detection of the mass difference indicates a site of ubiquitination
in the test peptide.
[0032] In another aspect, the methods further comprise the step of
mapping a sequence of a test peptide comprising a ubiquitin remnant
to a polypeptide sequence comprising the same amino acid sequence
as the test peptide, thereby determining the site of ubiquitination
in the polypeptide sequence. In another aspect, the ubiquitin
remnant comprises Gly-Gly amino acid residues and has a mass of
about 114 daltons. The methods can be used to detect one or more
sites of ubiquitination in a polypeptide, as well as the amount of
ubiquitination at particular sites in a population of
polypeptides.
[0033] The methods also can include the step of determining the
presence, site, and/or amount of a protein modification other than
ubiquitination.
[0034] Ubiquitinated polypeptides can be obtained by contacting
cellular polypeptides with binding partners which bind to a
ubiquitin molecule thereby forming ubiquitinated
polypeptide:binding partner complexes. The complexes can be
isolated using standard affinity purification methods. In one
aspect, the ubiquitin molecule comprises an affinity tag such as
6.times.-histidine. The ubiquitinated polypeptides can be obtained
from a cell expressing tagged ubiquitin molecules. The cell can be
a mammalian cell, e.g., a mouse cell.
[0035] In another aspect, the methods further comprise the step of
separating the ubiquitinated peptides obtained. Preferably,
separating is performed by at least one round of liquid
chromatography, such as reversed-phase liquid chromatography or by
HPLC.
[0036] In a further aspect, ubiquitination sites are identified for
a plurality of polypeptides in a first cell and in a second cell
and the sites identified in the first cell are compared to those in
the second cell. In one aspect, the first cell is a normal cell
(e.g., from a healthy patient), while the second cell is from a
patient with a pathological condition (e.g., a neurodegenerative
disease, cancer, a disease of the immune system). Preferably, the
second cell is the target of the pathology (e.g., a tumor cell from
a cancer patient; a neural cell from a patient with a
neurodegenerative disease). In another aspect, the second cell
differs from the first cell in expressing one or more recombinant
DNA molecules, but is otherwise genetically identical to the first
cell. In a further aspect, the site of ubiquitination is correlated
with disease and detection of ubiquitination at the site is
associated with risk of the disease. In one aspect, the disease is
a neurodegenerative disease, such as Alzheimer's or Pick's disease.
In another aspect, the disease is cancer. In a further aspect, the
disease is an abnormal immune response or inflammatory disease.
[0037] The methods can be used to identify regulators of
ubiquitination pathways. In one aspect, the methods further
comprise contacting a first cell with a compound and comparing
ubiquitination sites identified in the first cell with
ubiquitination sites in a second cell not contacted with the
compound. The compound may be a therapeutic agent for treating a
disease associated with an improper state of ubiquitination (e.g.,
abnormal sites or amounts of ubiquitination). Suitable agents
include, but are not limited to, drugs, polypeptides, peptides,
antibodies, nucleic acids (genes, cDNA's, RNA's, antisense
molecules, ribozymes, aptamers and the like), toxins, and
combinations thereof.
[0038] Preferably, the methods further comprise generating a
database comprising data files storing information relating to
ubiquitination sites for a plurality of polypeptides for a
plurality of different cells. Preferably, the data files also
include information relating to amount of ubiquitination of a
polypeptide in at least one cell. Additionally, the database
comprises data relating to the source of the cell (e.g., such as a
patient).
[0039] The invention further provides a computer memory comprising
data files storing information relating to ubiquitination sites for
a plurality of polypeptides for a plurality of different cells.
BRIEF DESCRIPTION OF THE FIGURES
[0040] The objects and features of the invention can be better
understood with reference to the following detailed description and
accompanying drawings.
[0041] FIG. 1 is a schematic diagram illustrating a method for
generating a peptide internal standard (IS) for a protein or
modified protein to be detected and/or quantified. The
representative IS containing amino acid sequence LSFVFGGTDEK (SEQ
ID NO:58) was produced for the corresponding protein (RLSFVFGGTDEK,
SEQ ID NO:59).
[0042] FIG. 2 illustrates characterization of peptide internal
standards by mass-to-charge ratio and retention time in reverse
phase chromatography according to one aspect of the invention.
[0043] FIGS. 3A and B show characterization of a peptide signature
by multistage mass spectrometry. FIG. 3A shows a signature obtained
after a second stage of mass spectrometry. FIG. 3B shows a
signature obtained after performing a third stage of mass
spectrometry.
[0044] FIGS. 4A and B illustrate steps in a method for absolute
quantitation of proteins in a complex mixture of proteins. FIG. 4A
shows sample processing steps in which a cell lysate is spiked with
a known amount of a labeled peptide internal standard according to
the invention. FIG. 4B shows mass spectra of a labeled peptide
internal standard and the corresponding unlabeled peptide in the
sample. The ratio of labeled to unlabeled peptide provides a means
to quantify the amount of unlabeled peptide in the sample.
[0045] FIG. 5A shows a peptide internal standard suitable for use
in detecting and/or quantitating a protein comprising the amino
acid sequence GFTALK (SEQ ID NO:2). The upper panel of the Figure
shows the native tryptic peptide. The lower portion of the Figure
shows a peptide internal standard corresponding to this peptide
which comprises a stable isotope (.sup.13C). As can be seen from
the Figure, the stable isotope provides a characteristic mass
difference in the two peptides without altering the essential
chemical structure of the peptide. FIG. 5B shows a peptide internal
standard suitable for use in detecting a phosphorylated foam of a
protein comprising the amino acid sequence GFTALK (SEQ ID NO:2).
FIG. 5C shows a peptide internal standard suitable for use in
detecting a methylated form of the amino acid sequence GFTALK (SEQ
ID NO:2).
[0046] FIG. 6 shows diagnostic peptide fragmentation signatures
obtained for two peptides comprising the sequences ALELFR (SEQ ID
NO:3) and LFTGHPETLEK (SEQ ID NO:4), respectively, from the
myoglobin protein. Each peptide produces a characteristic signature
ion that can be used to detect and/or quantify myoglobin in a
sample of cellular proteins. Providing both peptide internal
standards together in an assay can provide an additional control
for quantification.
[0047] FIG. 7 shows a schematic of an on-line nanoscale
microcapillary LC/MS/MS system used in one aspect of the
invention.
[0048] FIG. 8 is a schematic showing the isolation and sequence
analysis of yeast ubiquitin-conjugates according to one aspect of
the invention.
[0049] FIGS. 9A-C illustrate a strategy for identifying a site of
ubiquitination by tandem mass spectrometry according to one aspect
of the invention. FIG. 9A is a schematic diagram of a signature
peptide generated after trypsin digestion of a ubiquitinated
polypeptide. FIG. 9B shows an exemplary sequence LIFAGKQLEDGR (SEQ
ID NO:7) of a signature peptide produced by trypsin proteolysis.
FIG. 9C shows the fragmentation pattern (MS/MS spectrum) acquired
for the peptide shown in FIG. 9B.
[0050] FIGS. 10A-C show proteins identified comprising multiple
ubiquitination sites using methods according to the invention. FIG.
10A shows a table listing of amino acid sequences of
poly-ubiquitinated polypeptides. The third column contains amino
acid sequences of poly-ubiquitinated polypeptides (line 1 SEQ ID
NO:8 through line 19 SEQ ID NO:26, numbered consecutively). FIG.
10B shows a table listing of ubiquitination sites identified in
ubiquitin. The second column contains ubiquitinated signature
peptides (line 1 SEQ ID NO: 27 to line 5 SEQ ID NO: 31, numbered
consecutively). FIG. 10C shows a list of phosphorylated peptides
from candidate ubiquitinated polypeptides. The second column
contains amino acid sequences of phosphorylated peptides (line 1
SEQ ID NO: 32 through line 26 SEQ ID NO:57, numbered
consecutively).
[0051] FIGS. 11A-C show comparisons of protein expression, protein
environment and function in the yeast proteome characterized using
methods according to the invention.
DETAILED DESCRIPTION
[0052] The invention provides reagents, kits and methods for
detecting and/or quantifying proteins in complex mixtures, such as
a cell lysate. In one preferred aspect, the proteins comprise one
or more modifications. The methods can be used in high through put
assays to profile cellular proteomes and to correlate protein
modification patterns with particular cell states.
[0053] In one aspect, the invention provides a method for
determining a site of ubiquitination in a polypeptide and for
evaluating ubiquitination targets in a population of polypeptides.
In this way, a proteome ubiquitination map can be obtained which
comprises information relating to the ubiquitination states of a
plurality of cellular polypeptides. Maps can be obtained for a
variety of different types of cells and cell states. For example,
ubiquitination targets in normal and diseased cells can be
evaluated. Preferably, the map is stored as data files in a
database. Individual ubiquitinated polypeptides identified can be
used to generate molecular probes diagnostic of a cell state and/or
can serve as targets for agents which modulate one or more cellular
processes.
DEFINITIONS
[0054] The following definitions are provided for specific terms
which are used in the following written description.
[0055] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof. The term "a
protein" includes a plurality of proteins.
[0056] "Protein", as used herein, means any protein, including, but
not limited to peptides, enzymes, glycoproteins, hormones,
receptors, antigens, antibodies, growth factors, etc., without
limitation. Presently preferred proteins include those comprised of
at least 25 amino acid residues, more preferably at least 35 amino
acid residues and still more preferably at least 50 amino acid
residues. The terms "polypeptide" and "protein" are generally used
interchangeably herein to refer to a polymer of amino acid
residues.
[0057] As used herein, "a polypeptide" refers to a plurality of
amino acids joined by peptide bonds. Amino acids can include D-,
L-amino acids, and combinations thereof, as well as modified forms
thereof. As used herein, a polypeptide is greater than about 20
amino acids. The term "polypeptide" generally is used
interchangeably with the term "protein"; however, the term
polypeptide also may be used to refer to a less than full-length
protein (e.g., a protein fragment) which is greater than 20 amino
acids.
[0058] As used herein, the term "peptide" refers to a compound of
two or more subunit amino acids, and typically less than 20 amino
acids. The subunits are linked by peptide bonds.
[0059] As used herein, a "target protein" or a "target polypeptide"
is a protein or polypeptide whose presence or amount is being
determined in a protein sample. The protein/polypeptide may be a
known protein (i.e., previously isolated and purified) or a
putative protein (i.e., predicted to exist on the basis of an open
reading frame in a nucleic acid sequence).
[0060] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[0061] As used herein, a "protease activity" is an activity which
cleaves amide bonds in a protein or polypeptide. The activity may
be implemented by an enzyme such as a protease or by a chemical
agent, such as CNBr.
[0062] As used herein, "a protease cleavage site" is an amide bond
which is broken by the action of a protease activity.
[0063] As used herein, a "labeled peptide internal standard" refers
to a synthetic peptide which corresponds in sequence to the amino
acid subsequence of a known protein or a putative protein predicted
to exist on the basis of an open reading frame in a nucleic acid
sequence and which is labeled by a mass-altering label such as a
stable isotope. The boundaries of a labeled peptide internal
standard are governed by protease cleavage sites in the protein
(e.g., sites of protease digestion or sites of cleavage by a
chemical agent such as CNBr). Protease cleavage sites may be
predicted cleavage sites (determined based on the primary amino
acid sequence of a protein and/or on the presence or absence of
predicted protein modifications, using a software modeling program)
or may be empirically determined (e.g., by digesting a protein and
sequencing peptide fragments of the protein). In one aspect, a
labeled peptide internal standard includes a modified amino acid
residue.
[0064] "Percent identity" and "similarity" between two sequences
can be determined using a mathematical algorithm (see, e.g.,
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Hcinje, G., Academic Press,
1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M Stockton Press, New York, 1991). For example, the percent
identity between two amino acid sequences can be determined using
the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453,
1970) which is part of the GAP program in the GCG software package
(available at http://www.gcg.com), by the local homology algorithm
of Smith & Waterman (Adv. Appl. Math. 2: 482, 1981), by the
search for similarity methods of Pearson & Lipman (Proc. Natl.
Acad. Sci. USA 85: 2444, 1988) and Altschul, et al. (Nucleic Acids
Res. 25(17): 3389-3402, 1997), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and BLAST in the Wisconsin
Genetics Software Package (available from, Genetics Computer Group,
575 Science Dr., Madison, Wis.), or by manual alignment and visual
inspection (see, e.g., Ausubel et al., supra). Gap parameters can
be modified to suit a user's needs. For example, when employing the
GCG software package, a NWSgapdna.CMP matrix and a gap weight of
40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6
can be used. Examplary gap weights using a Blossom 62 matrix or a
PAM250 matrix, are 16, 14, 12, 10, 8, 6, or 4, while exemplary
length weights are 1, 2, 3, 4, 5, or 6. The percent identity
between two amino acid or nucleotide sequences also can be
determined using the algorithm of E. Myers and W. Miller (CABIOS 4:
11-17, 1989) which has been incorporated into the ALIGN program
(version 2.0), using a PAM120 weight residue table, a gap length
penalty of 12 and a gap penalty of 4.
[0065] As used herein, "a peptide fragmentation signature" refers
to the distribution of mass-to-charge ratios of fragmented peptide
ions obtained from fragmenting a peptide, for example, by collision
induced disassociation, ECD, LID, PSD, IRNPD, SID, and other
fragmentation methods. A peptide fragmentation signature which is
"diagnostic" or a "diagnostic signature" of a target protein or
target polypeptide is one which is reproducibly observed when a
peptide digestion product of a target protein/polypeptide identical
in sequence to the peptide portion of a peptide internal standard,
is fragmented and which differs only from the fragmentation pattern
of the peptide internal standard by the mass of the mass-altering
label. Preferably, a diagnostic signature is unique to the target
protein (i.e., the specificity of the assay is at least about 95%,
at least about 99%, and preferably, approaches 100%).
[0066] A "relational" database as used herein means a database in
which different tables and categories of the database are related
to one another through at least one common attribute and is used
for organizing and retrieving data.
[0067] The term "external database" as used herein refers to
publicly available databases that are not a relational part of the
internal database, such as GenBank and Blocks.
[0068] As used herein, an "expression profile" refers to
measurement of a plurality of cellular constituents that indicate
aspects of the biological state of a cell. Such measurements may
include, e.g., abundances or proteins or modified forms
thereof.
[0069] As used herein, a "cell state profile" refers to values of
measurements of levels of one or more proteins in the cell.
Preferably, such values are obtained by determining the amount of
peptides in a sample having the same peptide fragmentation
signatures as that of peptide internal standards corresponding to
the one or more proteins. A "diagnostic profile" refers to values
that are diagnostic of a particular cell state, such that when
substantially the same values are observed in a cell, that cell may
be determined to have the cell state. For example, in one aspect, a
cell state profile comprises the value of a measurement of p53
expression in a cell. A diagnostic profile would be a value which
is significantly higher than the value determined for a normal cell
and such a profile would be diagnostic of a tumor cell. A "test
cell state profile" is a profile which is unknown or being
verified.
[0070] As used herein, a processor that "receives a diagnostic
profile" receives data relating to the values diagnostic of a
particular cell state. For example, the processor may receive the
values by accessing a database where such values are stored through
a server in communication with the processor.
[0071] As used herein, a "ubiquitin remnant" is that portion of a
ubiquitin protein which remains attached to the digestion product
of a polypeptide which has been exposed to a protease.
[0072] As used herein, "a binding partner" refers to a first
molecule which can form a stable, and specific, non-covalent
association with a second molecule to be bound, enabling isolation
of the second molecule from a population of molecules including the
second molecule. "Stable" refers to an association which is strong
enough to permit complexes to form which may be isolated.
[0073] As used herein, an "antibody" refers to monoclonal or
polyclonal, single chain, double chain, chimeric, humanized, or
recombinant antibody, or antigen binding portion thereof (e.g.,
F(ab')2 fragments and Fab' fragments).
[0074] As used herein, the term "biological sample" refers to any
material obtained from a living source, for example, an animal such
as a human or other mammal, a plant, a bacterium, a fungus, a
protist or a virus. The biological sample can be in any form,
including a solid material such as a tissue, cells, a cell pellet,
a cell extract, a biopsy, a biological fluid such as urine, blood,
saliva, spinal fluid, amniotic fluid, exudate from a region of
infection or inflammation, or a mouthwash containing buccal
cells.
[0075] As used herein, "computer readable media" or a "computer
memory" refers to any media that can be read and accessed directly
by a computer. Such media include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc storage medium, and
magnetic tape; optical storage media such as CD-ROM; electrical
storage media such as RAM and ROM; and hybrids of these categories
such as magnetic/optical storage media.
Labeled Peptide Internal Standards
[0076] The invention provides labeled peptide internal standards
for use in determining the presence of, and/or quantifying the
amount of, a target protein in a sample which comprises an amino
acid subsequence identical to the peptide portion of the internal
standard. Peptide internal standards are generated by examining the
primary amino acid sequence of a protein and synthesizing a peptide
comprising the same sequence as an amino acid subsequence of the
protein (see, e.g., FIG. 1). In one aspect, the peptide's
boundaries are determined by predicting the cleavage sites of a
protease. In another aspect, a protein is digested by the protease
and the actual sequence of one or more peptide fragments is
determined. Suitable proteases include, but are not limited to one
or more of: serine proteases (e.g., such as trypsin, hepsin, SCCE,
TADG12, TADG14); metallo proteases (e.g., such as PUMP-1);
chymotrypsin; cathepsin; pepsin; elastase; pronase; Arg-C; Asp-N;
Glu-C; Lys-C; carboxypeptidases A, B, and/or C; dispase;
thermolysis; cysteine proteases such as gingipains, and the like.
Proteases may be isolated from cells or obtained through
recombinant techniques. Chemical agents with a protease activity
also can be used (e.g., such as CNBr).
[0077] The target protein can be a known protein or a protein
predicted to exist on the basis of an open reading frame in a
nucleic acid sequence. Such open reading frames can be identified
from a database of sequences including, but not limited to, the
GenBank database, EMBL data library, the Protein Sequence Database
and PIR-International, SWISS-PROT, The ExPASy proteomics server of
the Swiss Institute of Bioinformatics (SIB) and databases described
in PCT/US01/25884. Predicted cleavage sites also can be identified
through modeling software, such as IVIS-Digest (available at
http://prospector.ucsf.edu/). Predicted sites of protein
modification also can be determined using software packages such as
Scansite, Findmod, NetOGlyc (for prediction of type-O-glycosylation
sequences), YinOYang (for prediction of O-beta-GlcNac attachment
sites), big-PI Predictor (for prediction of GPI modifications),
NetPhos (for prediction of Ser, Thr, and Tyr phosphorylation
sites), NMT (for prediction of N-terminal N-myristolation) and
Sulfinator (for prediction of tyrosine sulfation sites) which are
accessible through http://au.expasy.org/tools/#ptm, for
example.
[0078] A peptide sequence within a target protein is selected
according to one or more criteria to optimize the use of the
peptide as an internal standard. Preferably, the size of the
peptide is selected to minimize the chances that the peptide
sequence will be repeated elsewhere in other non-target proteins.
Preferably, therefore, a peptide is at least about 6 amino acids.
The size of the peptide is also optimized to maximize ionization
frequency. Thus, peptides longer than about 20 amino acids are not
preferred. In one aspect, an optimal peptide ranges from about 6
amino acids to about 20 amino acids, and preferably from about 7
amino acids to about 15 amino acids.
[0079] A peptide sequence is also selected which is not likely to
be chemically reactive during mass spectrometry. Thus, peptide
sequences which comprise cysteine, tryptophan or methionine
residues are avoided.
[0080] Peptides also are selected based on the presence of one or
more bonds that preferentially fragment. For example, because
peptides will preferentially fragment at proline residues, intense
fragment ions may be produced at proline. Therefore in one aspect
of the invention, a peptide is selected from a region of a protein
comprising a proline amino acid residue.
[0081] In another aspect, a peptide is selected from a region of a
protein which is not expected or not known to be modified, so that
the peptide internal standard can be used to determine the quantity
of all forms of the protein. However, in a further aspect, the
peptide internal standard does include an amino acid residue which
is expected to, or is known to be modified, to provide an internal
standard to quantify only the modified form the protein (see, e.g.,
FIGS. 5B and 5C). Peptide standards representing modified (e.g.,
FIGS. 5B and 5C) and unmodified forms of a protein (see, e.g., FIG.
5A) can be used together, to determine the extent of protein
modification in a particular sample of proteins, i.e., to determine
what fraction of the total amount of protein is represented by the
modified form.
[0082] The peptide is synthesized using one or more labeled amino
acids (i.e., the label is actually part of the peptide) or less
preferably, labels may be attached after synthesis. By providing
the label as part of the peptide (see, e.g., FIGS. 5A-5C), there
are minimal differences in the chemical structure of a peptide
internal standard and a native peptide obtained from the digestion
of a target protein with a protease activity. Further, because the
peptide is synthesized, it is unnecessary to separate and/or purify
the peptide from other cellular proteins.
[0083] Preferably, the label is a mass-altering label. The type of
label selected is generally based on the following considerations:
The mass of the label should preferably be unique to shift fragment
masses produced by MS analysis to regions of the spectrum with low
background. The ion mass signature component is the portion of the
labeling moiety which preferably exhibits a unique ion mass
signature in mass spectrometric analyses. The sum of the masses of
the constituent atoms of the label is preferably uniquely different
than the fragments of all the possible amino acids. As a result,
the labeled amino acids and peptides are readily distinguished from
unlabeled amino acids and peptides by their ion/mass pattern in the
resulting mass spectrum. In a preferred embodiment, the ion mass
signature component imparts a mass to a protein fragment produced
during mass spectrometric fragmentation that does not match the
residue mass for any of the 20 natural amino acids.
[0084] The label should be robust under the fragmentation
conditions of MS and not undergo unfavorable fragmentation.
Labeling chemistry should be efficient under a range of conditions,
particularly denaturing conditions and the labeled tag preferably
remains soluble in the MS buffer system of choice. Preferably, the
label does not suppress the ionization efficiency of the protein.
More preferably, the label does not alter the ionization efficiency
of the protein and is not otherwise chemically reactive.
Alternatively, or additionally, the label contains a mixture of two
or more isotopically distinct species to generate a unique mass
spectrometric pattern at each labeled fragment position.
[0085] In one preferred aspect, peptide internal standards comprise
mass-altering labels which are stable isotopes. In certain
preferred embodiments, the method utilizes isotopes of hydrogen,
nitrogen, oxygen, carbon, or sulfur. Suitable isotopes include, but
are not limited to, .sup.2H, .sup.13C, .sup.15N, .sup.17O,
.sup.18O, or .sup.34S. In another aspect, pairs of peptide internal
standards can be provided, comprising identical peptide portions
but distinguishable labels, e.g., peptides may be labeled at
multiple sites to provide different heavy forms of the peptide).
Multiple labeled amino acids may be incorporated in a peptide
during the synthesis process. In another aspect, the label is part
of a peptide comprising a modified amino acid residue, such as a
phosphorylated residue (see, e.g., FIG. 5B), a glycosylated
residue, an acetylated residue, a ribosylated residue, or a
farnesylated residue, a methlyated residue (see, e.g., FIG. 5C). In
this embodiment, pairs or larger sets of peptide internal standards
corresponding to modified and unmodified peptides also can be
produced. In one aspect, such a pair/set is differentially
labeled.
[0086] Peptide internal standards are characterized according to
their mass-to-charge ratio (m/z) and preferably, also according to
their retention time on a chromatographic column (e.g., such as an
HPLC column). Internal standards are selected which co-elute with
peptides of identical sequence but which are not labeled (see,
e.g., FIG. 2).
[0087] The peptide internal standard is then analyzed by
fragmenting the peptide. Fragmentation can be achieved by inducing
ion/molecule collisions by a process known as collision-induced
dissociation (CID) (also known as collision-activated dissociation
(CAD)). Collision-induced dissociation is accomplished by selecting
a peptide ion of interest with a mass analyzer and introducing that
ion into a collision cell. The selected ion then collides with a
collision gas (typically argon or helium) resulting in
fragmentation. Generally, any method that is capable of fragmenting
a peptide is encompassed within the scope of the present invention.
In addition to CID, other fragmentation methods include, but are
not limited to, surface induced dissociation (SID) (James and
Wilkins, Anal. Chem. 62: 1295-1299, 1990; and Williams, et al., J.
Amer. Soc. Mass Spectrom. 1: 413-416, 1990), blackbody infrared
radiative dissociation (BIRD); electron capture dissociation (ECD)
(Zubarev, et al., J. Am. Chem. Soc. 120: 3265-3266, 1998);
post-source decay (PSD), LID, and the like.
[0088] The fragments are then analyzed to obtain a fragment ion
spectrum. One suitable way to do this is by CID in multistage mass
spectrometry (MS.sup.n). Traditionally used to characterize the
structure of a peptide and/or to obtain sequence information, it is
a discovery of the present invention, that MS' provides enhanced
sensitivity in methods for quantitating absolute amounts of
proteins. Thus, in one aspect, peptide internal standards are
generated for low abundance proteins (e.g., below 2000
copies/cell).
[0089] Preferably, a peptide internal standard is analyzed by at
least two stages of mass spectrometry to determine the
fragmentation pattern of the peptide and to identify a peptide
fragmentation signature (see, e.g., FIG. 3A). More preferably, a
peptide signature is obtained in which peptide fragments have
significant differences in m/z ratios to enable peaks corresponding
to each fragment to be well separated. Still more preferably,
signatures are unique, i.e., diagnostic of a peptide being
identified and comprising minimal overlap with fragmentation
patterns of peptides with different amino acid sequences. If a
suitable fragment signature is not obtained at the first stage,
additional stages of mass spectrometry are performed until a unique
signature is obtained (see, e.g., FIG. 3B).
[0090] Fragment ions in the MS/MS and MS.sup.3 spectra are
generally highly specific and diagnostic for peptides of interest.
In contrast, to prior art methods, the identification of peptide
diagnostic signatures provides for a way to perform highly
selective analysis of a complex protein mixture, such as a cellular
lysate in which there may be greater than about 100, about 1000,
about 10,000, or even about 100,000 different kinds of proteins.
Thus, while conventional mass spectroscopy would not be able to
distinguish between peptides with different sequences but similar
m/z ratios (which would tend to co-elute with any labeled standard
being analyzed), the use of peptide fragmentation methods and
multistage mass spectrometry in conjunction with LC methods,
provide a way to detect and quantitate target proteins which are
only a small fraction of a complex mixture (e.g., present in less
than 2000 copies per cell or less than about 0.001% of total
cellular protein) through these diagnostic signatures.
[0091] Multiple peptide subsequences of a single protein may be
synthesized, labeled, and fragmented to identify optimal
fragmentation signatures. However, in one aspect at least two
different peptides are used as internal standards to
identify/quantify a single protein, providing an internal
redundancy to any quantitation system (see, e.g., as shown in FIG.
6). In another aspect, peptide internal standards are synthesized
which correspond to a single amino acid subsequence of a target
polypeptide but which vary in one or more amino acids. The peptide
internal standards may correspond to known variants or mutations in
the target polypeptide or can be randomly varied to identify all
possible mutations in an amino acid sequence.
[0092] In one preferred aspect, peptide internal standards
corresponding to proteins expressed from nucleic acids comprising
single nucleotide polymorphisms are synthesized to identify variant
proteins encoded by such nucleic acids. Thus, peptide internal
standards can be generated corresponding to SNP's which map to
coding regions of genes and can be used to identify and quantify
variant protein sequences on an individual or population level. SNP
sequences can be accessed through The Human SNP database available
at http://www-genome.wi.mit.edu/SNP/human/index.html.
[0093] Peptide internal standards may also be used to scan for
mutations in proteins including, but not limited to, BRCA1, BRCA2,
CFTR, p53, blood group antigens, HLA proteins, MHC proteins,
G-Protein Coupled Receptors, apolipoprotein E, kinases (e.g., such
as hCds1, MTKs, PTK, CDKs, STKs, CaMs, and the like) (see, e.g.,
U.S. Pat. No. 6,426,206), phosphatases, human drug metabolizing
proteins, viral proteins such as a viral envelope proteins (e.g.,
HIV envelope proteins), transporter proteins, and the like.
[0094] In a further aspect, peptides corresponding to different
modified forms of a protein are synthesized, providing internal
standards to detect and/or quantitate changes in protein
modifications in different cell states. In still a further aspect,
peptide internal standards are generated which correspond to
different proteins in a molecular pathway and/or modified forms of
such proteins (e.g., proteins in a signal transduction pathway,
cell cycle, metabolic pathway, blood clotting pathway, etc.)
providing panels of internal standards to evaluate the regulated
expression of proteins and/or the activity of proteins in a
particular pathway. Combinations of the above-described internal
standards can be used in a given assay.
Methods of Using Peptide Internal Standards
[0095] The labeled peptide internal standards according to the
invention can be used to facilitate quantitative determination 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 present in a single sample. Samples
that can be analyzed by method of the invention include, but are
not limited to, cell homogenates; cell fractions; biological
fluids, including, but not limited to urine, blood, and
cerebrospinal fluid; tissue homogenates; tears; feces; saliva;
lavage fluids such as lung or peritoneal lavages; and generally,
any mixture of biomolecules, e.g., such as mixtures including
proteins and one or more of lipids, carbohydrates, and nucleic
acids such as obtained partial or complete fractionation of cell or
tissue homogenates.
[0096] Preferably, a proteome is analyzed. By a proteome is
intended at least about 20% of total protein coming from a
biological sample source, usually at least about 40%, more usually
at least about 75%, and generally 90% or more, up to and including
all of the protein obtainable from the source. Thus, the proteome
may be present in an intact cell, a lysate, a microsomal fraction,
an organelle, a partially extracted lysate, biological fluid, and
the like. The proteome will be a mixture of proteins, generally
having at least about 20 different proteins, usually at least about
50 different proteins and in most cases, about 100 different
proteins, about 1000 different proteins, about 10,000 different
proteins, about 100,000 different proteins, or more In one aspect,
a proteome comprises substantially all of the proteins in a cell.
In one preferred aspect, as shown in FIG. 4A, a complex mixture of
cellular proteins is evaluated directly from a cell lysate, i.e.,
without any steps to separate and/or purify and/or eliminate
cellular components or cellular debris.
[0097] While the methods described herein are compatible with any
biochemical, immunological or cell biological fractionation methods
that reduce sample complexity and enrich for proteins of low
abundance, it is a particular advantage of the method that it can
be used to detect and quantitate peptides in complex mixtures of
polypeptides, such as cell lysates. Unlike methods in the prior
art, because the present invention detects diagnostic signatures
that are highly selective for individual peptides, the quantities
of such peptides can be discerned even in a mixture of peptides of
similar mass/charge ratios.
[0098] Generally, the sample will have at least about 0.01 mg of
protein, at least about 0.05 mg, and usually at least about 1 mg of
protein or 10 mg of protein or more, typically at a concentration
in the range of about 0.1-10 mg/ml. The sample may be adjusted to
the appropriate buffer concentration and pH, if desired.
[0099] In one aspect, as shown in FIG. 4A, a known amount of a
labeled peptide internal standard corresponding to a target protein
to be detected and/or quantitated, is added to a sample such as a
cell lysate. Preferably, about 10 femtomoles is spiked into the
sample. The sample is contacted with a protease activity (e.g., one
or more proteases or appropriate chemical agent(s) are added to the
sample) and the spiked sample is incubated for a suitable period of
time to allow peptide digestion. If the target protein is present
in the sample, the digestion step should liberate a target peptide
identical in sequence to the peptide portion of the internal
standard and the amount of target peptides so liberated from target
proteins in the sample should be proportional to the amount of
target protein in the sample.
[0100] Preferably, a separation procedure is performed to separate
a labeled peptide internal standard and corresponding target
peptide from other peptides in the sample. Representative examples
include high-pressure liquid chromatography (HPLC), Reverse
Phase-High Pressure Liquid Chromatography (RP-HPLC),
electrophoresis (e.g., capillary electrophoresis), anion or cation
exchange chromatography, and open column chromatography. Preferred
is microcapillary liquid chromatography. As discussed above,
internal standards are selected so that they co-elute with their
corresponding target peptides as pairs of peptides that differ only
in the mass contributed by the mass-altering label.
[0101] Each peptide then is examined by monitoring of a selected
reaction in the mass spectrometer. This involves using the prior
knowledge gained by the characterization of the peptide internal
standard and then requiring the mass spectrometer to continuously
monitor a specific ion in the MS/MS or MS.sup.n spectrum for both
the peptide of interest and the internal standard. After, elution,
the areas-under-the-curve (AUC) for both the peptide internal
standard and target peptide peaks are calculated (see, e.g., FIG.
4B). The ratio of the two areas provides the absolute
quantification that can be normalized for the number of cells used
in the analysis and the protein's molecular weight, to provide the
precise number of copies of the protein per cell.
[0102] In one aspect, the presence and/or quantity of target
polypeptide in a mixture is diagnostic of a cell state. In another
aspect, the cell state is representative of an abnormal
physiological response, for example, a physiological response which
is diagnostic of a disease. In a further aspect, the cell state is
a state of differentiation or represents a cell which has been
exposed to a condition or agent (e.g., a drug, a therapeutic agent,
a potential toxin). Preferably, protein quantities identified are
compared to a reference quantity obtained from a reference sample
(e.g., a sample from a normal patient, a sample not exposed to a
condition or agent, etc.).
[0103] In another aspect, the method comprises determining the
presence and/or quantity of target peptides in at least two
mixtures. In still another aspect, one mixture is from a cell
having a first cell state and the second mixture is from a cell
having a second cell state. In a further aspect, the first cell is
a normal cell and the second cell is from a patient with a disease.
Preferably, first and second mixtures are evaluated in
parallel.
[0104] Alternatively, the two mixtures can be from identical
samples or cells. In one aspect, the labeled peptide internal
standard is provided in different known amounts in each mixture. In
another aspect, pairs of labeled peptide internal standards are
provided each comprising mass-altering labels that differ in mass.
For example, differentially labeled peptides may be generated by
incorporating different amounts of a heavy label into each peptide
varying the number of sites within the peptides labeled by a heavy
isotope.
[0105] The invention also provides a method of determining the
presence of and/or quantity of a modification in a target
polypeptide. Preferably, the label in the internal standard is
attached to a peptide comprising a modified amino acid residue or
to an amino acid residue that is predicted to be modified in a
target polypeptide. In one aspect, multiple internal standards
representing different modified forms of a single protein and/or
peptides representing different modified regions of the protein are
added to a sample and corresponding target peptides (bearing the
same modifications) are detected and/or quantified. Preferably,
standards representing both modified and unmodified forms of a
protein are provided in order to compare the amount of modified
protein observed to the total amount of protein in a sample.
[0106] In another aspect, peptide internal standards comprising
different peptides from a single protein are added in known amounts
to a sample to provide additional controls or to scan for mutations
in different regions of a protein. In a further aspect, peptides
corresponding to a single amino acid subsequence in a protein but
representing different variant forms of the protein are added to a
sample as a means of detecting and/or quantifying a particular
variant form of the protein.
[0107] In still another aspect, peptide internal standards are
added to a sample that represents different proteins in a molecular
pathway (e.g., a signal transduction pathway, a cell cycle, a
metabolic pathway, a blood clotting pathway) and/or different
modified forms of such proteins. In this aspect, the function of
the pathway is evaluated by monitoring the presence, absence or
quantity of particular pathway proteins and/or their modified
forms. Multiple pathways may be evaluated at a time by combining
mixtures of different pathway peptide internal standards.
[0108] In a further aspect, peptide internal standards represent
proteins and/or modified forms thereof whose presence is diagnostic
of a particular tissue type (e.g., neural proteins, cardiac
proteins, skin proteins, lung proteins, liver proteins, pancreatic
proteins, kidney proteins, proteins characteristic of reproductive
organs, etc.). These can be used separately or in combination to
perform tissue-typing analysis.
[0109] Peptide internal standards may represent proteins or
modified forms thereof whose presence is characteristic of a
particular genotype (e.g., such as HLA proteins, blood group
proteins, proteins characteristic of a particular pedigree, etc.).
These can be used separately or in combination to perform forensic
analyses, for example.
[0110] In one aspect, peptide internal standards are used in
prenatal testing to detect the presence of a congenital disease or
to quantitate protein levels diagnostic of a chromosomal
abnormality.
[0111] Peptide internal standards may represent proteins or
modified forms thereof whose presence is characteristic of
particular diseases. Such peptides may correspond to target
proteins diagnostic of neurological disease (e.g.,
neurodegenerative diseases, including, but not limited to,
Alzheimer's disease; amyotrophic lateral sclerosis; dementia,
depression; Down's syndrome; Huntington's disease; peripheral
neuropathy; multiple sclerosis; neurofibromatosis; Parkinson's
disease; and schizophrenia). These standards can be used separately
or in combination to diagnose a neurological disease.
[0112] Preferably, sets of internal standards are used so that
diagnostic fragmentation signatures can be evaluated for a number
of different diseases in a single assay. Thus, a sample may be
obtained from a patient who presents with general symptoms
associated with a neurological disease, and a peptide internal
standard mixture comprising internal standards for proteins
diagnostic of different neurological diseases can be added to the
sample. The sample is contacted with a protease activity and
peptide fractions are obtained, e.g., such as by HPLC. Peptide ions
are subsequently fragmented as described above to detect any
diagnostic, fragmentation signatures present characteristic of a
particular disease. The uniqueness of the fragmentation signature
thus allows a specific diagnosis to be obtained while testing for a
plurality of different types of diseases. The peptide internal
standard mixture may include a peptide internal standard
corresponding to a control target protein, such as a constitutively
expressed protein of known abundance. A negative standard (e.g.,
such as a peptide internal standard corresponding to a plant
protein) may also be provided.
[0113] Similarly, peptide internal standards can be used to
diagnose an immune disease, including, but not limited to, acquired
immunodeficiency syndrome (AIDS); Addison's disease; adult
respiratory distress syndrome; allergies; ankylosing spondylitis;
amyloidosis; anemia; asthma; atherosclerosis; autoimmune hemolytic
anemia; autoimmune thyroiditis; bronchitis; cholecystitis; contact
dermatitis; Crohn's disease; atopic dermatitis; dermatomyositis;
diabetes mellitus; emphysema; episodic lymphopenia with
lymphocytotoxins; erythroblastosis fetalis; erythema nodosum;
atrophic gastritis; glomerulonephritis; Goodpasture's syndrome;
gout; Graves' disease; Hashimoto's thyroiditis; hypereosinophilia;
irritable bowel syndrome; myasthenia gravis; myocardial or
pericardial inflammation; osteoarthritis; osteoporosis;
pancreatitis; and polymyositis.
[0114] Similarly, peptide internal standards can be used to
characterize infectious diseases, respiratory diseases,
reproductive diseases, gastrointestinal diseases, dermatological
diseases, hematological diseases, cardiovascular diseases,
endocrine diseases, urological diseases, and the like.
[0115] Because peptide internal standards provide diagnostic
fragmentation signatures for detecting and/or quantitating proteins
or modified forms thereof, changes in the presence or amounts of
such fragmentation signatures in a sample of proteins from a cell
(e.g., such as a cell lystate), as discussed above, can be
diagnostic of a cell state. In one aspect, a single fragmentation
signature from a peptide internal standard is diagnostic. In other
aspects, sets of fragmentation signatures are diagnostic and
multiple peptide internal standards are spiked into a sample to
evaluate changes in cell state.
[0116] In one preferred embodiment, changes in cell state are
evaluated after exposure of the cell to a compound. Compounds are
selected which are capable of normalizing a cell state, e.g., by
selecting for compounds which alter fragmentation signatures from
those characteristic of abnormal physiological responses to those
representative of a normal cell.
[0117] For example, a three-way comparison of healthy, diseased,
and treated diseased individuals can identify which compounds are
able to restore a disease cell state to a one that more closely
resembles a normal cell state. This can be used to screen for drugs
or other therapeutic agents, to monitor the efficacy of treatment,
and to detect or predict the occurrence of side effects, whether in
a clinical trial or in routine treatment, and to identify protein
targets which are more important to the manifestation and treatment
of a disease.
[0118] Compounds which can be evaluated include, but are not
limited to: drugs; toxins; proteins; polypeptides; peptides; amino
acids; antigens; cells, cell nuclei, organelles, portions of cell
membranes; viruses; receptors; modulators of receptors (e.g.,
agonists, antagonists, and the like); enzymes; enzyme modulators
(e.g., such as inhibitors, cofactors, and the like); enzyme
substrates; hormones; nucleic acids (e.g., such as
oligonucleotides; polynucleotides; genes, cDNAs; RNA; antisense
molecules, ribozymes, aptamers), and combinations thereof.
Compounds also can be obtained from synthetic libraries from drug
companies and other commercially available sources known in the art
(e.g., including, but not limited, to the LeadQuest.RTM. library)
or can be generated through combinatorial synthesis using methods
well known in the art. In one aspect, a compound is identified as a
modulating agent if it alters the site of modification of a
polypeptide and/or if it alters the amount of modification by an
amount that is significantly different from the amount observed in
a control cell (e.g., not treated with compound) (setting p values
to <0.05). In another aspect, a compound is identified as a
modulating agent, if it alters the amount of the polypeptide
(whether modified or not).
[0119] Compounds identified as modulating agents are used in
methods of treatment of pathologies associated with abnormal
sites/levels of modification or abnormal levels or types of
protein. For administration to a patient, one or more such
compounds are generally formulated as a pharmaceutical composition.
Preferably, a pharmaceutical composition is a sterile aqueous or
non-aqueous solution, suspension or emulsion, which additionally
comprises a physiologically acceptable carrier (i.e., a non-toxic
material that does not interfere with the activity of the active
ingredient). More preferably, the composition also is non-pyrogenic
and free of viruses or other microorganisms. Any suitable carrier
known to those of ordinary skill in the art may be used
Representative carriers include, but are not limited to
physiological saline solutions, gelatin, water, alcohols, natural
or synthetic oils, saccharide solutions, glycols, injectable
organic esters such as ethyl oleate or a combination of such
materials. Optionally, a pharmaceutical composition may
additionally contain preservatives and/or other additives such as,
for example, antimicrobial agents, anti-oxidants, chelating agents
and/or inert gases, and/or other active ingredients.
[0120] Routes and frequency of administration, as well doses, will
vary from patient to patient. In general, the pharmaceutical
compositions is administered intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity or transdermally.
Between 1 and 6 doses is administered daily. A suitable dose is an
amount that is sufficient to show improvement in the symptoms of a
patient afflicted with a disease associated an aberrant
modification state or an abnormal level or type of a protein. Such
improvement may be detected by monitoring appropriate clinical or
biochemical endpoints as is known in the art. In general, the
amount of a modulating agent present in a dose, or produced in situ
by DNA present in a dose (e.g., where the modulating agent is a
polypeptide or peptide encoded by the DNA), ranges from about 1
.mu.g to about 100 mg per kg of host. Suitable dose sizes will vary
with the size of the patient, but will typically range from about
10 mL to about 500 mL for 10-60 kg animal. A patient can be a
mammal, such as a human, or a domestic animal.
Detection and Quantitation of Protein Modifications:
Identifying Protein Ubiquitination Sites
[0121] Protein ubiquitination is the most common of all
posttranslational modifications. Ubiquitin is a highly conserved 76
amino acid protein which is linked to a protein target after a
cascade of transfer reactions. Ubiquitin is activated through the
formation of a thioester bond between its C-terminal glycine and
the active site cysteine of the ubiquitin activating protein, E1
(Hershko, 1991, Trends Biochem. Sci. 16(7): 265-8). In subsequent
trans-thiolation reactions, Ubiquitin is transferred to a cysteine
residue on a ubiquitin conjugating enzyme, E2 (Hershko, et al.,
1983, J. Biol. Chem. 267: 8807-8812). In conjunction with E3, a
ubiquitin polypeptide ligase, E2 then transfers ubiquitin to a
specific polypeptide target (see, e.g., Scheffner, et al., 1995,
Nature 373(6509): 81-3), forming an isopeptide bond between the
C-terminal glycine of ubiquitin and the e-amino group of a lysine
present in the target.
[0122] The covalent attachment of ubiquitin to cellular
polypeptides, in most cases, marks them for degradation by a
multi-polypeptide complex called a proteosome. The
ubiquitin-proteosome system is the principal mechanism for the
turnover of short-lived polypeptides, including regulatory
polypeptides (Weissman, 2001, Nat. Rev. Mol. Cell. Biol. 2:
169-78). Some known targets of ubiquitination include: cyclins,
cyclin-dependent kinases (CDK's), Nak.beta., cystic fibrosis
transduction receptor, p53, ornithine decarboxylase (ODC),
7-membrane spanning receptors, Cdc25 (phosphotyrosme phosphatase),
Rb, Ga, c-Jun and c-Fos. Polypeptides sharing consensus sequences
such as PEST sequences, destruction boxes, and F-boxes generally
are also targets for ubiquitin-mediated degradation pathways (see,
e.g., Rogers, et al., 1986, Science 234: 364-368; Yamano, et al.,
1998, The EMBO Journal 17: 5670-5678; Bai, et al., 1996, Cell 86:
263-274).
[0123] Ubiquitin has been implicated in a number of cellular
processes including: signal transduction, cell-cycle progression,
receptor-mediated endocytosis, transcription, organelle biogenesis,
spermatogenesis, response to cell stress, DNA repair,
differentiation, programmed cell death, and immune responses (e.g.,
inflammation). Ubiquitin also has been implicated in the biogenesis
of ribosomes, nucleosomes, peroxisomes and myofibrils. Thus,
ubiquitin can function both as signal for polypeptide degradation
and as a chaperone for promoting the formation of organelles (see,
e.g., Fujimuro, et al., 1997, Eur. J. Biochem. 249: 427-433).
[0124] Deregulation of ubiquitination has been implicated in the
pathogenesis of many different diseases. For example, abnormal
accumulations of ubiquitinated species are found in patients with
neurodegenerative diseases such as Alzheimer's as well as in
patients with cell proliferative diseases, such as cancer (see,
e.g., Hershko and Ciechanover, 1998, Annu. Rev. Biochem. 67:
425-79; Layfield, et al., 2001, Neuropathol. Appl. Neurobiol.
27:171-9; Weissman, 1997, Immunology Today 18(4): 189).
[0125] While the importance of its biological role is well
appreciated, the ubiquitin pathway is inherently difficult to
study. Generally, studies of ubiquitination have focused on
particular polypeptides. For example, site-directed mutagenesis has
been used to evaluate critical amino acids which form the
"destruction boxes", or "D-boxes", of cyclin, sites which are
rapidly poly-ubiquitinated when cyclin is triggered for
destruction. See, e.g., Yamano, et al., 1998, The EMBO Journal 17:
5670-5678; Amon et al., 1994, Cell 77: 1037-1050; Glotzer, et al.,
1991, Nature 349: 132-138; King, et al., 1996, Mol. Biol. Cell 7:
1343. Corsi, et al., 1997, J. Biol. Chem. 272(5): 2977-2883,
describe a Western blotting approach to identify ubiquitination
sites in .alpha.-spectrin. In this technique, crude radiolabeled
.alpha.-spectrin fractions were ubiquitinated in vitro, digested
with proteases, and electrophoresed on gels. Ubiquitinated peptides
were identified by their differences in mass from peptides
generated by digestion of non-ubiquitinated .alpha.-spectrin.
[0126] Identification of Sites of Ubiquitination
[0127] In one aspect, the invention provides a method comprising
obtaining a test peptide and identifying a site of an isopeptide
bond within the peptide, e.g., such as is formed between the
terminal C-Gly group of a ubiquitin molecule and the
.epsilon.-amino group of a lysine residue within the peptide.
Preferably, the test peptide is obtained from a ubiquitinated
polypeptide which has been digested by a protease (e.g., such as
trypsin) to generate a plurality of digestion products, i.e., a
plurality of test peptides, one or more of which comprise(s) a
remnant of a ubiquitin molecule (e.g., a fragment of ubiquitin
refractory to the digestion process). For example, a digested
poly-ubiquitinated polypeptide will generate a plurality of test
peptides comprising isopeptide bonds, while a mono-ubiquitinated
polypeptide will generate only one test peptide which comprises an
isopeptide bond.
[0128] Digested peptides are purified to isolate individual test
peptides for analysis. Preferably, the presence of an isopeptide
bond in a test peptide is detected by comparing the mass of the
test peptide with the mass of a reference peptide in a panel of
non-ubiquitinated peptides of known sequence. A reference peptide
is "matched" to a test peptide when it is smaller than the test
peptide by the amount of mass characteristic of the ubiquitin
remnant. For example, for a trypsin-digested ubiquitinated
polypeptide, a test peptide comprising a ubiquitination site will
comprise a ubiquitin remnant comprising a Gly-Gly residue, and a
mass difference of approximately 114 daltons.
[0129] A match to a reference peptide indicates that the test
peptide has the same sequence as the reference peptide. The peptide
can then be mapped to the polypeptide sequence from which it is
derived, either directly, or after determining the masses/sequences
of other test peptides which have resulted from the digestion of
the ubiquitinated polypeptide. In this way the site of
ubiquitination on the polypeptide can be determined.
[0130] Isolating Ubiquitinated Polypeptides
[0131] Ubiquitinated polypeptides can be isolated by a variety of
methods. For example, cellular polypeptides can be contacted with
binding partners that bind to a ubiquitin molecule. A ubiquitinated
polypeptide:binding partner complex forms which can be isolated
through affinity purification of the binding partner. A binding
partner can be selected which binds directly to ubiquitin, or which
binds to a molecule linked to ubiquitin.
[0132] The binding partner can comprise an antibody which binds to
ubiquitin. Anti-ubiquitin antibodies are commercially available,
and include both polyclonal (e.g., available from Research
Diagnostics, Inc., Flanders, N.J.) and monoclonal antibodies (e.g.,
available from International Biosciences, Inc., Tokyo, Japan). In
one aspect, the antibody binds to a ubiquitinated polypeptide but
not to free ubiquitin. Such an antibody can be obtained from Signet
Antibodies, Inc., Dedham, Mass., for example. In another aspect,
the antibody binds to a poly-ubiquitinated polypeptide, but not to
free ubiquitin and not to a mono-ubiquitinated polypeptide.
Antibodies of this type are commercially available from Affiniti
Research, Ltd. (Mamhead Castle, United Kingdom), for example.
Additional antibodies can be generated using methods well known in
the art (see, e.g., Harlow and Lane, In Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.).
[0133] The antibody preferably is stably associated with a solid
phase (e.g., a bead, micro particle, sphere, chip, support, and the
like). Antibodies can be linked directly to a solid phase (e.g.,
chemically conjugated) or can be bound to the solid phase via other
binding, partners specific for the antibodies immobilized on the
solid phase. By contacting a population of polypeptides (e.g., from
a cell extract) to the support, ubiquitinated polypeptide: antibody
complexes can be isolated. Ubiquitinated polypeptides can be
separated from the antibodies using suitable washing conditions
known in the art.
[0134] Alternatively, or additionally, ubiquitinated polypeptides
can be isolated by linking ubiquitin to an affinity tag. As used
herein, an "affinity tag" refers to a molecule which facilitates
the purification of a polypeptide (e.g., ubiquitin) to which it is
attached. In one aspect, the affinity tag is a poly-histidine tract
(e.g., a tract of about 6-10 histidines) fused in frame to a
ubiquitin molecule. A histidine-tagged ubiquitin molecule can be
isolated by contacting a population of peptides, some of which are
ubiquitinated, to a solid phase comprising a binding molecule which
forms a stable association with histidine (e.g., such s a nickel
chelate). Bound molecules comprising ubiquitinated polypeptides are
separated from non-bound molecules and ubiquitinated polypeptides
are removed from the solid phase using suitable washing
conditions.
[0135] Ubiquitin conjugates comprising affinity tags can be
introduced into cells using methods known in the art; e.g.,
transfection, electroporation, microinjection, germline transfer,
and the like. In one aspect, a transgenic animal expressing
ubiquitin conjugates in one or more cells is used as a source of
ubiquitinated polypeptides. For example, a mouse expressing
histidine-tagged ubiquitin can be used (see, e.g., Tsirigotis, et
al., 2001, Biotechniques 31: 120-130).
[0136] The isolation step described above will not result in the
purification of polypeptide fragments or peptides obtained after
proteosomal processing, since digested ubiquitin molecules will not
be recognized by the binding partners described above. Thus,
cellular ubiquitinated polypeptides that are degraded extremely
rapidly, i.e., such that essentially little or no ubiquitinated
polypeptides accumulate, may not be detected by this method.
However, these polypeptides can be examined in a cellular
background deficient for the activity or expression of one or more
proteosomal polypeptides, i.e., a cell treated with one or more
proteasome inhibitors, enabling the degradation process to be
uncoupled from the ubiquitination process. Preferably, the
proteasome inhibitor is specific to the proteosome rather than
acting generally on cellular proteases. Suitable proteosome
inhibitors include, but are not limited to, epoxyomycin,
lactacystin,
4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl
sulfone, and the like. Novel inhibitors also may be identified
using methods known in the art, e.g., such as described in
PCT/US98/14638 9904033.
[0137] Obtaining Test Peptides
[0138] In one aspect, ubiquitinated polypeptides which are obtained
are digested with a protease to generate sets of test peptides,
each set corresponding the digestion products of a particular
ubiquitinated polypeptide. Suitable proteases are those which do
not cleave isopeptide bonds and include, but are not limited to,
one or more of serine proteases (e.g., such as trypsin, hepsin,
SCCE, TADG12, TADG14); metallo proteases (e.g., such as PUMP-1);
chymotrypsin; cathepsin; pepsin; elastase; pronase; Arg-C; Asp-N;
Glu-C; Lys-C; carboxypeptidases A, B, and/or C; dispase;
thermolysin; and the like. Generally, the type of protease is not
limiting; however, preferably, the protease is an extracellular
protease.
[0139] Creating this highly complex peptide mixture is
straightforward. In one aspect, a population of ubiquitinated
polypeptides (e.g., from a cellular extract) is solubilized in a
highly reducing and denaturing environment (e.g., 8M urea, 10 mM
dithiothreitol (DTT), 50 mM Tris-HCL, pH 8.3). Cysteinyl residues
can be alkylated, if desired, and the polypeptide mixture is
diluted (e.g., about 8-fold) in the presence of one or more
proteases for digestion (e.g., approximately overnight).
[0140] Preferably, digestion products are purified to obtain
individual test peptides which are substantially isolated from
other test peptides (e.g., test peptides which comprise less than
5% of other test peptides, or which comprise greater than about 95%
identical peptides). A number of separation strategies can be used,
such as gel-based strategies (e.g., 2D-electrophoresis) or liquid
chromatography. Liquid chromatography is preferred because it can
be integrated readily with subsequent peptide analysis steps,
maximizing the throughput of the analysis.
[0141] Liquid chromatography (LC) may be used to separate and/or
concentrate peptides based on one or more of their adsorption
characteristics, affinity characteristics, ionic properties, and/or
size. Suitable LC methods include, but are not limited to on-line
reversed phase liquid chromatography; nano-scale microcapillary
reverse-phase chromatography; high pressure liquid chromatography
(HPLC); capillary electrophoresis; micro-column liquid
chromatography; multidimensional electrophoresis; and the like
(see, e.g., Deterding, et al., 1991, J. Chromatogr. 554: 73-82; Guo
et al., 1988, Anal Biochem 168: 54; U.S. Pat. No. 5,496,460;
Matson, et al., 1984, Clin. Chem. 30/9: 1477-1488). One or more
separation systems can be combined.
[0142] Preferably, a separation system used in a method according
to the invention is one that can be coupled to a peptide analyzer
such as a mass spectrometer (MS). In one aspect, the separation
system comprises one or more of a pump or sample injector for
delivering a sample of ubiquitinated polypeptides; transfer tubing;
a pre-column flow splitter for controlling the rate of flow and/or
establishing a flow gradient; a capillary column for performing the
separation; and a delivery mechanism for delivering substantially
purified test peptides to the peptide analyzer (see, e.g., as shown
in FIG. 7). The delivery mechanism can comprise a liquid junction,
e.g., such as a gold wire at high voltage (1-2 kV), which can be
used to promote electrospray. Preferably, a processor is used to
control flow of fluids through both the separation system and the
peptide analyzer, to coordinate the separation process with the
analysis process. For example, elution of a test peptide from the
separation column can be synchronized with ionization by a mass
spectrometer.
[0143] Both column sizes and flow rates in the separation system
can be optimized to suit a particular separation. In one aspect,
the separation system comprises a capillary column comprising
fused-silica tubing and is packed with C18 silica beads. In another
aspect, the capillary comprises an about 75 .mu.m internal diameter
with about 5 .mu.m of C18 beads and a bed length of about 12
cm.
[0144] Flow rates through different portions of the separation
device can vary. In one aspect, a pump provides sample to a
capillary column at a flow rate of about 100 .mu.l/minute, while
the flow rate through the column itself is maintained at
approximately 300 nl/min using a column size of approximately 75
.mu.m in internal diameter. A flow restrictor can be used to permit
a gradient of flow rates to be formed quickly.
[0145] It should be obvious to those of skill in the art that the
column dimensions and flow rates described above are exemplary and
are not intended to be limiting. Chromatographic parameters can be
optimized using methods routine in the art, e.g., through empirical
testing and/or computer simulations. For example, a simulation
program for optimizing HPLC parameters is described by Dolan, et
al., 1987, Chrornatographia 24: 261-276. Further, a processor may
be provided which can monitor and optimize conditions in the
separation system. In one aspect, the system processor comprises an
expert system which is responsive to signals generated by sensors
coupled to various columns and pumps of the systems. The expert
system can be used to modulate flow rates, pH, and/or ionic
conditions in the separation system in response to feedback from
the sensors. Such an expert system is described in U.S. Pat. No.
5,039,409, for example.
[0146] A sample can be loaded and eluted in the separation system
in different ways depending on the peptide concentration and
volume. For example, a sample can be loaded through an injection
loop on a valve (e.g., such as a six-port valve) inserted between a
T-splitter and a separation column (e.g., a microcapillary).
Loading also can be performed off-line via a pressure cell. While
this latter approach maximizes sensitivity, more sample handling is
required. To increase throughput, sample can be loaded into a
pre-column-trap for concentration and rapid desalting and then
eluted onto the separation column (e.g., such as a reverse phase
separation column). Preferably, flow rates in the pre-column trap
are on the order of about .mu.l/minute.
[0147] In a preferred aspect of the invention, to increase
sensitivity in a separation system having sub-microliter flow
rates, a vented microcapillary column (V-column) is used to vary
the rate of flow of sample through the capillary. The first few
centimeters of the capillary column preferably are loaded with
sample at high flow rates exiting through the vent. After closing
the vent (e.g., switching the position of a valve, such as a
six-port valve), bound peptides are eluted at much lower flow rates
that are compatible with microcapillary separations, such as HPLC
(e.g., rates of approximately 300 nl/min)
[0148] To maximize separation efficiency, multi-dimensional
chromatography can be employed. For example, peptides can be
separated in a first dimension by strong cation-exchange (SCX)
chromatography. SCX chromatography has the advantage of removing
proteases and binding peptides in the presence of accessory
molecules that carry no positive charge at pH 3.0, the pH at which
peptide elution typically occurs. Thus, peptide binding and elution
can occur in the presence of molecules typically used in cellular
extraction processes, such as SDS, detergent, urea, DTT, and the
like.
[0149] At pH 3.0, amine functional groups of peptides almost
exclusively contribute to the solution charge state. The nominal
charge of any peptide can be determined by adding up the number of
lysine, arginine, and histidine residues, with one additional
charge contributed by the N-terminus of the peptide. Tryptic
peptides generally have solution charge states of 2+ because they
terminate in lysine or arginine and have a free N-terminus. A
solution charge state of 3+ is seen for tryptic peptides containing
one histidine residue. Tryptic peptides carrying a single, charge
in solution at pH 3.0 are highly specialized, representing either
the C-terminal peptide from a polypeptide, an N-terminal peptide
that is blocked (e.g., acetylated), or a phosphorylated peptide.
Peptides which elute with solution charge states of 4+ or more also
represent specialized peptides, e.g., such as disulfide-linked
tryptic peptides, missed cleavages, etc. SCX can be used to
distinguish among these various charged states.
[0150] Other separation methods can be used to complement SCX to
achieve additional dimensions of separation. Preferably, such
separation methods include, but are not limited to, one or more of:
affinity chromatography, liquid chromatography, a gel-based
separation method, capillary electrophoresis, reversed phase
chromatography, and the like. Preferably, the separation system
interfaced with the peptide analyzer is one whose buffering system
is compatible with the peptide analyzer being used. For example,
when peptides are being evaluated by mass spectrometry, preferably,
a separation system which relies on volatile buffers and which does
not utilize solutions comprising salts and/or detergents is used.
Therefore, in one preferred aspect, the separation system
interfaced with the mass spectrometer is a reversed phase liquid
chromatography device.
[0151] Determining the Mass of Test Peptides
[0152] In one aspect, substantially purified test peptides obtained
after one or more separation steps are analyzed by a peptide
analyzer which evaluates the mass of the peptide or a fragment
thereof. Suitable analyzers include, but are not limited to, a mass
spectrometer, mass spectrograph, single-focusing mass spectrometer,
static field mass spectrometer, dynamic field mass spectrometer,
electrostatic analyzer, magnetic analyzer, quadropole analyzer,
time of flight analyzer (e.g., a MALDI Quadropole time-of-flight
mass spectrometer), Wien analyzer, mass resonant analyzer,
double-focusing analyzer, ion cyclotron resonance analyzer, ion
trap analyzer, tandem mass spectrometer, liquid secondary
ionization MS, and combinations thereof in any order (e.g., as in a
multi-analyzer system). Such analyzers are known in the art and are
described in, for example, Mass Spectrometry for the Biological
Sciences, Burlingame and Carr eds., Human Press, Totowa, N.J.).
[0153] In general, any analyzer can be used which can separate
matter according to its anatomic and molecular mass. Preferably,
the peptide analyzer is a tandem MS system (an MS/MS system) since
the speed of an MS/MS system enables rapid analysis of low
femtomole levels of peptide and can be used to maximize
throughput.
[0154] In a preferred aspect, the peptide analyzer comprises an
ionizing source for generating ions of a test peptide and a
detector for detecting the ions generated. The peptide analyzer
further comprises a data system for analyzing mass data relating to
the ions and for deriving mass data relating to the test
peptide.
[0155] A sample comprising a test peptide can be delivered to the
peptide analyzer using a delivery mechanism as described above.
Interfaces between a sample source (e.g., an HPLC column) and ion
source can be direct or indirect. For example, there may be an
interface that provides for continuous introduction of the sample
to the ion source. Alternatively, sample can be intermittently
introduced to the ion source (e.g., in response to feedback from
the system processor during the separation process, or while the
separation system is off-line).
[0156] In one aspect, the ion source is an electrospray which is
used to provide droplets to the peptide analyzer, each droplet
comprising a substantially purified test peptide obtained from
previous separation step(s) (e.g., such as HPLC or reversed phase
liquid chromatography). During electrospray, a high voltage is
applied to a liquid stream causing large droplets to be subdivided
into smaller and smaller droplets' until a peptide enters the gas
phase as an ion. Ionization generally is accomplished when the test
peptide loses or gains a proton at one or more basis sites on the
peptide (e.g., at the amino terminus, and at lysine and arginine
residues). Ionization in electrospray is constant; MALDI can be
used to achieve pulsed ionization. Other methods of ionization,
include, but are not limited to, plasma desorption ionization,
thermospray ionization, and fast atom bombardment ionization as are
known in the art.
[0157] When MALDI is used, peptides can be delivered to a solid
support, e.g., such as a sample plate inserted into the mass
spectrometer. The support may comprise a light-absorbent matrix
(see, e.g., as described in U.S. Pat. No. 5,288,644). In one
aspect, a substantially purified ubiquitinated polypeptide is
provided on a sample plate and protease digestion occurs on the
sample plate prior to ionization (see, e.g., U.S. Pat. No.
5,827,65). For example, substantially purified ubiquitinated
peptides also can be obtained from protease digests as described
above and separation by a liquid chromatography method. Preferably,
the peptide analyzer further comprises an ion transfer section
through which ions are delivered from the ion source to the
detector. The ion transfer section comprises an electric and/or
magnetic field generator (e.g., an electrode ring) that modulates
the acceleration of ions generated by the ionizing source. The
electric/magnetic field generator directs ions through the ion
transfer section of the peptide analyzer to the ion detector.
[0158] Preferably, the peptide analyzer further comprises an ion
trap positioned between the ion transfer section of the analyzer
and the detector, for performing one or more operations such as ion
storage, ion selection and ion collision. The ion trap can be used
to fragment ions produced by the ion source (e.g., causing ions to
undergo collisional activated dissociation in the presence of a
neutral gas ions, such as helium ions). The ion trap also can be
used to store ions in stable orbits and to sequentially eject ions
based on their mass-to-charge values (m/z) to the detector. An
additional separation section can be provided between the ion trap
and detector to separate fragments generated in the ion trap (e.g.,
as in tandem MS). The detector detects the signal strength of each
ion (e.g., intensity), which is a reflection of the amount of
protonation of the ion.
[0159] The peptide analyzer additionally comprises a data system
for recording and processing information collected by the detector.
The data system can respond to instructions from processor in
communication with the separation system and also can provide data
to the processor. Preferably, the data system includes one or more
of: a computer, an analog to digital conversion module; and control
devices for data acquisition, recording, storage and manipulation.
More preferably, the device further comprises a mechanism for data
reduction, i.e., to transform the initial digital or analog
representation of output from the analyzer into a form that is
suitable for interpretation, such as a graphical display (e.g., a
display of a graph, table of masses, report of abundances of ions,
etc.).
[0160] The data system can perform various operations such as
signal conditioning (e.g., providing instructions to the peptide
analyzer to vary voltage, current, and other operating parameters
of the peptide analyzer), signal processing, and the like. Data
acquisition can be obtained in real time, e.g., at the same time
mass data is being generated. However, data acquisition also can be
performed after an experiment, e.g., when the mass spectrometer is
off line.
[0161] The data system can be used to derive a spectrum graph in
which relative intensity (i.e., reflecting the amount of
protonation of the ion) is plotted against the mass to charge ratio
(m/z ratio) of the ion or ion fragment. An average of peaks in a
spectrum can be used to obtain the mass of the ion (e.g., peptide)
(see, e.g., McLafferty and Turecek, 1993, Interpretation of Mass
Spectra, University Science Books, CA).
[0162] Mass spectra can be searched against a database of reference
peptides of known mass and sequence to identify a reference peptide
which matches a test peptide (e.g., comprises a mass which is
smaller by the amount of mass attributable to a ubiquitin remnant).
The database of reference peptides can be generated experimentally,
e.g., digesting non-ubiquitinated peptides and analyzing these in
the peptide analyzer. The database also can be generated after a
virtual digestion process, in which the predicted mass of peptides
is generated using a suite of programs such as PROWL (e.g.,
available from ProteoMetrics, LLC, New York; N.Y.). A number of
database search programs exist which can be used to correlate mass
spectra of test peptides with amino acid sequences from polypeptide
and nucleotide databases, including, but not limited to: the
SEQUEST program (Eng, et al., J. Am. Soc. Mass Spectrom. 5: 976-89;
U.S. Pat. No. 5,538,897; Yates, Jr., III, et al., 1996, J. Anal.
Chem. 68(17): 534-540A), available from Finnegan Corp., San Jose,
Calif.
[0163] Data obtained from fragmented peptides can be mapped to a
larger peptide or polypeptide sequence by comparing overlapping
fragments. Preferably, a ubiquitinated peptide is mapped to the
larger polypeptide from which it is derived to identify the
ubiquitination site on the polypeptide. Sequence data relating to
the larger polypeptide can be obtained from databases known in the
art, such as the nonredundant protein database compiled at the
Frederick Biomedical Supercomputing Center at Frederick, Md.
[0164] In one aspect, the amount and location of ubiquitination is
compared to the presence, absence and/or quantity of other types of
polypeptide modifications. For example, the presence, absence,
and/or quantity of phosphorylation, sulfation, glycosylation,
and/or acetylation can be determined using methods routine in the
art (see, e.g., Rossomando, et al., 1992, Proc. Natl. Acad. Sci.
USA 89: 5779-578; Knight et al., 1993, Biochemistry 32: 2031-2035;
U.S. Pat. No. 6,271,037). The amount and locations of one or
modifications can be correlated with the amount and locations of
ubiquitination sites. Preferably, such a determination is made for
multiple cell states.
[0165] Knowledge of ubiquitination sites can be used to identify
compounds that modulate particular ubiquitinated polypeptides
(either preventing or enhancing ubiquitination, as appropriate, to
normalize the ubiquitination state of the polypeptide). Thus, in
one aspect, the method described above may further comprise
contacting a first cell with a compound and comparing
ubiquitination sites/amounts identified in the first cell with
ubiquitination sites/amounts in a second cell not contacted with
the compound. Suitable cells that may be tested include, but are
not limited to: neurons, cancer cells, immune cells (e.g., T
cells), stem cells (embryonic and adult), undifferentiated cells,
pluripotent cells, and the like. In one preferred aspect, patterns
of ubiquitination are observed in cultured cells, such as P19
cells, pluripotent embryonic carcinoma cells capable of
differentiating into cardiac cells and skeletal myocytes upon
exposure to DMSO (see, Montross, et al., J. Cell Sci. 113 (Pt. 10):
1759-70).
[0166] Compounds which can be evaluated include, but are not
limited to: drugs; toxins; proteins; polypeptides; peptides; amino
acids; antigens; cells, cell nuclei, organelles, portions of cell
membranes; viruses; receptors; modulators of receptors (e.g.,
agonists, antagonists, and the like); enzymes; enzyme modulators
(e.g., such as inhibitors, cofactors, and the like); enzyme
substrates; hormones; nucleic acids (e.g., such as
oligonucleotides; polynucleotides; genes, cDNAs; RNA; antisense
molecules, ribozymes, aptamers), and combinations thereof.
Compounds also can be obtained from synthetic libraries from drug
companies and other commercially available sources known in the art
(e.g., including, but not limited, to the LeadQuest.RTM. library)
or can be generated through combinatorial synthesis using methods
well known in the art. A compound is identified as a modulating
agent if it alters the site of ubiquitination of a polypeptide
and/or if it alters the amount of ubiquitination by an amount that
is significantly different from the amount observed in a control
cell (e.g., not treated with compound) (setting p values to
<0.05).
[0167] Compounds identified as modulating agents are used in
methods of treatment of pathologies associated with abnormal
sites/levels of ubiquitination. For administration to a patient,
one or more such compounds are generally formulated as a
pharmaceutical composition. Preferably, a pharmaceutical
composition is a sterile aqueous or non-aqueous solution,
suspension or emulsion, which additionally comprises a
physiologically acceptable carrier (i.e., a non-toxic material that
does not interfere with the activity of the active ingredient).
More preferably, the composition also is non-pyrogenic and free of
viruses or other microorganisms. Any suitable carrier known to
those of ordinary skill in the art may be used. Representative
carriers include, but are not limited to: physiological saline
solutions, gelatin, water, alcohols, natural or synthetic oils,
saccharide solutions, glycols, injectable organic esters such as
ethyl oleate or a combination of such materials. Optionally, a
pharmaceutical composition may additionally contain preservatives
and/or other additives such as, for example, antimicrobial agents,
anti-oxidants, chelating agents and/or inert gases, and/or other
active ingredients.
[0168] Routes and frequency of administration, as well doses, will
vary from patient to patient. In general, the pharmaceutical
compositions is administered intravenously, intraperitoneally,
intramuscularly, subcutaneously, intracavity or transdermally.
Between 1 and 6 doses is administered daily. A suitable dose is an
amount that is sufficient to show improvement in the symptoms of a
patient afflicted with a disease associated an aberrant
ubiquitination state. Such improvement may be detected by
monitoring appropriate clinical or biochemical endpoints as is
known in the art. In general, the amount of modulating agent
present in a dose, or produced in situ by DNA present in a dose
(e.g., where the modulating agent is a polypeptide or peptide
encoded by the DNA), ranges from about 1 .mu.g to about 100 mg per
kg of host. Suitable dose sizes will vary with the size of the
patient, but will typically range from about 10 mL to about 500 mL
for 10-60 kg animal. A patient can be a mammal, such as a human, or
a domestic animal.
[0169] In another aspect, the ubiquitination states (e.g., sites
and amount of ubiquitination) of first and second cells are
evaluated. Preferably, the second cell differs from the first cell
in expressing one or more recombinant DNA molecules, but is
otherwise genetically identical to the first cell. Alternatively,
or additionally, the second cell can comprise mutations or variant
allelic forms of one or more genes. In one aspect, DNA molecules
encoding regulators of the ubiquitin pathway can be introduced into
the second cell (e.g., E1, E2, E3, deubiquitinating proteins,
fragments thereof, mutant forms thereof, variants, and modified
forms thereof, or compounds identified as above) and alterations in
the ubiquitination state in the second cell can be determined DNA
molecules can be introduced into the cell using methods routine in
the art, including, but not limited to: transfection,
transformation, electroporation, electrofusion, microinjection, and
germline transfer.
Computer Systems and Databases
[0170] The invention also provides methods for generating a
database comprising data files for storing information relating to
diagnostic peptide fragmentation signatures. Preferably, data in
the data files include one or more peptide fragmentation signatures
characteristic or diagnostic of a cell state (e.g., such as a state
which is characteristic of a disease, a normal physiological
response, a developmental process, exposure to a therapeutic agent,
exposure to a toxic agent or a potentially toxic agent, and/or
exposure to a condition). Data in the data files also preferably
includes values corresponding to level of proteins corresponding to
the peptide fragmentation signatures found in a particular cell
state.
[0171] In one aspect, for a cell state determined by the
differential expression of at least one protein, a data file
corresponding to the cell state will minimally comprise data
relating to the mass spectra observed after peptide fragmentation
of a peptide internal standard diagnostic of the protein.
Preferably, the data file will include a value corresponding to the
level of the protein in a cell having the cell state. For example,
a tumor cell state is associated with the overexpression of p53
(see, e.g., Kern, et al., 2001, Int. J. Oncol. 21(2): 243-9). The
data file will comprise mass spectral data observed after
fragmentation of a labeled peptide internal standard corresponding
to a subsequence of p53. Preferably, the data file also comprises a
value relating to the level of p53 in a tumor cell. The value may
be expressed as a relative value (e.g., a ratio of the level of p53
in the tumor cell to the level of p53 in a normal cell) or as an
absolute value (e.g., expressed in nM or as a % of total cellular
proteins).
[0172] Preferably, the data files also include information relating
to the presence or amount of a modified form of a target a
polypeptide in at least one cell and to mass spectral data
diagnostic of the modified form (i.e., peak data for a fragmented
peptide internal standard which corresponds to the modified form).
More preferably, the data files also comprise spectral data
diagnostic of the unmodified form as well as data corresponding to
the level of the unmodified form.
[0173] Thus, in one aspect, data relating to ubiquitination sites
and amounts of ubiquitination are stored in a database to create a
proteome map of ubiquitinated proteins. Preferably, the database
comprises a collection of data files relating to all ubiquitinated
polypeptides in a particular cell type. The database preferably
further comprises data relating to the origin of the cell, e.g.,
such as data relating to a patient from whom a cell was obtained.
More preferably, the database comprises data relating to cells
obtained from a plurality of patients. In one aspect, the database
comprises data relating to the ubiquitination of a plurality of
different cell types (e.g., cells from patients with a pathology,
normal patients, cells at various stages of differentiation, and
the like). In another aspect, data relating to ubiquitination
patterns in cells obtained from patients comprising a neurological
disease are stored in the database. For example, information
relating to ubiquitination in cell samples from patients having any
of Alzheimer's disease; amyotrophic lateral sclerosis; dementia,
depression; Down's syndrome; Huntington's disease; peripheral
neuropathy; multiple sclerosis; neurofibromatosis; Parkinson's
disease; and schizophrenia, can be included in the database.
[0174] In a further aspect, data relating to ubiquitination
patterns in cells from patients with cancer are stored in the
database, including, but not limited to patients with:
adenocarcinoma; leukemia; lymphoma; melanoma; myeloma; sarcoma;
teratocarcinoma; and, in particular, cancers of the adrenal gland;
bladder; bone; bone marrow; brain; breast; cervix; gall bladder;
ganglia; gastrointestinal tract; heart, kidney; liver; lung;
muscle; ovary; pancreas; parathyroid; prostate; salivary glands;
skin; spleen; testis; thymus; thyroid; and uterus.
[0175] Additionally, data of ubiquitination patterns in cells from
patients with an immune disorder may be included in the database.
Such a disorder can include: acquired immunodeficiency syndrome
(AIDS); Addison's disease; adult respiratory distress syndrome;
allergies; ankylosing spondylitis; amyloidosis; anemia; asthma;
atherosclerosis; autoimmune hemolytic anemia; autoimmune
thyroiditis; bronchitis; cholecystitis; contact dermatitis; Crohn's
disease; atopic dermatitis; dermatomyositis; diabetes mellitus;
emphysema; episodic lymphopenia with lymphocytotoxins;
erythroblastosis fetalis; erythema nodosum; atrophic gastritis;
glomerulonephritis; Goodpasture's syndrome; gout; Graves' disease;
Hashimoto's thyroiditis; hypereosinophilia; irritable bowel
syndrome; myasthenia gravis; myocardial or pericardial
inflammation; osteoarthritis; osteoporosis; pancreatitis;
polymyositis; psoriasis; Reiter's syndrome; rheumatoid arthritis;
scleroderma; Sjogren's syndrome; systemic anaphylaxis; systemic
lupus erythematosus; systemic sclerosis; thrombocytopenic purpura;
ulcerative colitis; uveitis; Werner syndrome; and viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections.
[0176] Data regarding ubiquitination in apoptotic cells and in
pathologies associated with the misregulation of apoptosis also can
be obtained using methods according to the invention.
[0177] In a further aspect, data regarding ubiquitination in
cardiac cells and cells from patients exhibiting a cardiac disease
or at risk for a cardiac disease are obtained. In one aspect, the
disease is an infarction or a condition relating to ischemia. In
another aspect, the disease is cardiomyopathy.
[0178] In still a further aspect, data is obtained from cells
obtained from a patient comprising a chromosomal deletion or
mutation of nucleic acids encoding one or more polypeptides
involved in the ubiquitination process. In one aspect, the patient
comprises a 22q11.2 deletion.
[0179] It should be obvious to those of skill in the art, that the
invention may be used to characterize a large number of pathologies
associated with ubiquitin degradation and that the above examples
are not intended to be limiting.
[0180] Differences in ubiquitination patterns (sites and/or
quantity of ubiquitination) in cells with different cell states can
be used to identify diagnostic markers for a cell state. Thus, in
one aspect, ubiquitination at a particular polypeptide site is
associated with disease or risk of developing a disease (e.g., a
statistically significant chance of having or developing the
disease). Correlations between a particular state of ubiquitination
and a disease can be identified using the database described above
and suitable statistical programs, expert systems, and/or data
mining systems, as are known in the art, for identifying
relationships between records in data files (e.g., such as records
relating to ubiquitination patterns and records relating to
patients from whom cells were derived). In one aspect, the
ubiquitination state of a cell is determined and used to determine
the presence or risk of a pathology, such as a neurological
disease, cancer, or an immune disease (i.e., any of the diseases
described above). Molecular probes can be developed based on this
information (e.g., antibodies which recognize a polypeptide
ubiquitinated at the site but not a polypeptide which is not
ubiquitinated at the site) and can be used in screening assays to
identify patients have a disease or who are at risk of developing a
disease.
[0181] In one aspect, the database also comprises data relating to
the source of a cell whose cell state is being evaluated. For
example, the database comprises data relating to identifying
characteristics of a patient from whom the cell is derived.
[0182] The invention further provides a computer memory comprising
data files for storing information relating to the diagnostic
fragmentation signatures of peptide internal standards. In one
preferred aspect, the database comprises peptide diagnostic
signatures, e.g., mass spectral data obtained after fragmentation
of one or more peptide internal standards, which can be used to
identify a cell having a particular cell state. More preferably,
the database includes data relating to a plurality of cell state
profiles, i.e., data relating to levels of target proteins
identified by the peptide internal standards in a plurality of
cells having different cell states. For example, profiles of
disease states may be included in the database and these profiles
will include measurements of levels of one or more proteins, or
modified forms thereof, characteristic of the disease state.
Profiles of cells exposed to different compounds include
measurements of levels of proteins or modified forms thereof
characteristic of the response(s) of the cells to the compounds. In
one aspect, the measurements are obtained by performing any of the
methods described above.
[0183] Preferably, the database is in electronic form and the cell
state profiles, which are also in electronic form, provide
measurements of levels of a plurality of proteins in a cell or
cells of one or more subjects. In one aspect, the database
comprises measurements of more than about 5, more than about 10,
more than about 30, more than about 50, more than about 100, more
than about 500, more than about 1000, more than about 10,000, or
more than about 100,000 proteins in a cell, i.e., the database
comprises data relating to the proteome of a cell. The measurements
represent levels of modified and/or unmodified forms of the
proteins. In one aspect, the measurements also include data
regarding the site of protein modifications in one or more proteins
in a cell.
[0184] In one preferred aspect, cell state profiles comprise
quantitative data relating to target proteins and/or modified forms
thereof obtained by using one or more of the methods described
above.
[0185] A variety of data storage structures are available for
creating a computer readable medium or memory comprising data files
of the database. The choice of the data storage structure will
generally be based on the means chosen to access the stored
information. For example, the data can be stored in a word
processing text file, formatted in commercially-available software
such as WordPerfect and Microsoft Word, or represented in the form
of an ASCII file, stored in a database application, such as DB2,
Sybase, Oracle, or the like. The skilled artisan can readily adapt
any number of data processor structuring formats (e.g., text files,
pdf files, or database structures) in order to obtain computer
readable medium or a memory having recorded thereon data relating
to diagnostic fragmentation signatures, e.g., such as mass spectral
data obtained after fragmentation of the peptide internal
standards, and protein levels and/or data relating to the presence
and quantity of modified proteins (e.g., such as ubiquitinated
proteins) in a sample.
[0186] Correlations between a particular diagnostic signature
observed and a cell state (e.g., a disease, genotype, tissue type,
etc.) may be known or may be identified using the database
described above and suitable statistical programs, expert systems,
and/or data mining systems, as are known in the art. In one aspect,
the diagnostic signature is provided by a diagnostic pattern of
protein modification, such as protein ubiquitination.
[0187] In another aspect, the invention provides a computer system
comprising: a database having data files containing information
identifying diagnostic fragmentation signatures (e.g., mass
spectral peaks) as corresponding to particular peptide internal
standards which in turn are identified as corresponding to
particular target proteins. Preferably, the data files also
comprise information for relating the diagnostic fragmentation
signatures so identified to one or more cell states, e.g., where
the target protein corresponding to the peptide internal standard
is diagnostic of a cell state, the peptide internal standard and
fragmentation signature are also identified within the data file as
being diagnostic of a cell state. In one preferred aspect, the
system further comprises a user interface allowing a user to
selectively view information relating to a diagnostic fragmentation
signature and to obtain information about a cell state. The
interface may comprise links allowing a user to access different
portions of the database by selecting the links (e.g. by moving a
cursor to the link and clicking a mouse or by using a keystroke on
a keypad). The interface may additionally display fields for
entering information relating to a sample being evaluated.
[0188] Still more preferably, the system is capable of comparing
diagnostic fragmentation signatures of known peptide internal
standards to mass spectral data obtained for peptides in a sample
spiked with one or more internal standards in order to determine
and/or quantify levels of target proteins corresponding to the
standards in the sample. When a match is identified, the system may
also provide information regarding the cell state for which the
peptide internal standard is diagnostic (i.e., the system will
identify the source of the cell, the compound to which a cell has
been exposed, and/or a disease which the cell is responding to). In
some aspects, sets of peptide internal standards are evaluated, as
only the set will be diagnostic.
[0189] The system may also be used to collect and categorize
peptide fragmentation signatures for different types of cell states
to identify sets of peptide internal standards characteristic of
particular cell states. In this aspect, preferably, the system
comprises a relational database. More preferably, the system
further comprises an expert system for identifying sets of peptide
internal standards that are diagnostic of different cell states. In
one aspect, the system is capable of clustering related
information. Suitable clustering programs are known in the art and
are described in, for example, U.S. Pat. No. 6,303,297.
[0190] The system preferably comprises a means for linking a
database comprising data files of diagnostic fragmentation
signatures to other databases, e.g., such as genomic databases,
pharmacological databases, patient databases, proteomic databases,
and the like.
[0191] Preferably, the system comprises in combination, a data
entry means, a display means (e.g., graphic user interface); a
programmable central processing unit; and a data storage means
comprising the data files and information described above,
electronically stored in a relational database.
[0192] Preferably, the central processing unit comprises an
operating system for managing a computer and its network
interconnections. This operating system can be, for example, of the
Microsoft Windows' family, such as Windows 95, Windows 98, or
Windows NT, or any new Windows programmed developed. A software
component representing common languages may be provided. Preferred
languages include C/C++, and JAVA.RTM.. In one aspect, methods of
this invention are programmed in software packages which allow
symbolic entry of equations, high-level specification of
processing, and statistical evaluations.
Reagents and Kits
[0193] Reagents and Kits Comprising Peptide Internal Standards
[0194] The invention further provides reagents useful for
performing the method. In one aspect, a reagent according to the
invention comprises a peptide internal standard labeled with a
stable isotope. Preferably, the standard has a unique peptide
fragmentation signature diagnostic of the peptide. The peptide is a
subsequence of a known protein and can be used to identify the
presence of and/or quantify the protein in sample, such as a cell
lysate.
[0195] The invention additionally provides kits comprising one or
more peptide internal standards labeled with a stable isotope or
reagents suitable for performing such labeling. In certain
preferred embodiments, the method utilizes isotopes of hydrogen,
nitrogen, oxygen, carbon, or sulfur. Suitable isotopes include, but
are not limited to .sup.2H, .sup.13C, .sup.15N, .sup.17O, .sup.18O,
or .sup.34S. In another aspect peptide internal, pairs of pep
standards are provided, comprising identical peptide portions but
distinguishable labels, e.g., peptides may be labeled at multiple
sites to provide different heavy forms of the peptide. Pairs of
peptide internal standards corresponding to modified and unmodified
peptides also can be provided.
[0196] In one aspect, a kit comprises peptide internal standards
comprising different peptide subsequences from a single known
protein. In another aspect, the kit comprises peptide internal
standards corresponding to different known or predicted modified
forms of a polypeptide. In a further aspect, the kit comprises
peptide internal standards corresponding to sets of related
proteins, e.g., such as proteins involved in a molecular pathway (a
signal transduction pathway, a cell cycle, etc), or which are
diagnostic of particular disease states, developmental stages,
tissue types, genotypes, etc. Peptide internal standards
corresponding to a set may be provided in separate containers or as
a mixture or "cocktail" of peptide internal standards.
[0197] In one aspect, a plurality of peptide internal standards
representing a MAPK signal transduction pathway is provided.
Preferably, the kit comprises at least two, at least about 5, at
least about 10 or more, of peptide internal standards corresponding
to any of MAPK, GRB2, mSOS, ras, raf, MEK, p85, KHS1, GCK1, HPK1,
MEKK 1-5, ELK1, c-JUN, ATF-2, 3APK, MLK1-4, PAK, MKK, p38, a SAPK
subunit, hsp27, and one or more inflammatory cytokines.
[0198] In another aspect, a set of peptide internal standards is
provided which comprises at least about two, at least about 5 or
more, of peptide internal standards which correspond to proteins
selected from the group including, but not limited to, PLC
isoenzymes, phosphatidylinositol 3-kinase (PI-3 kinase), an
actin-binding protein, a phospholipase D isoform, (PLD), and
receptor and nonreceptor PTKs.
[0199] In another aspect, a set of peptide internal standards is
provided which comprises at least about 2, at least about 5, or
more, of peptide internal standards which correspond to proteins
involved in a JAK signaling pathway, e.g., such as one or more of
JAK 1-3, a STAT protein, IL-2, TYK2, CD4, IL-4, CD45, a type I
interferon (IFN) receptor complex protein, an IFN subunit, and the
like.
[0200] In a further aspect, a set of peptide internal standards is
provided which comprises at least about 2, at least about 5, or
more of peptide internal standards which correspond to cytokines.
Preferably, such a set comprises standards selected from the group
including, but not limited to, pro- and anti-inflammatory cytokines
(which may each comprise their own set or which may be provided as
a mixed set of peptide internal standards).
[0201] In still another aspect, a set of peptide internal standards
is provided which comprises a peptide diagnostic of a cellular
differentiation antigen or CD. Such kits are useful for tissue
typing.
[0202] In one aspect, peptides corresponding to known variants or
mutations in a target polypeptide, or which are randomly varied to
identify all possible mutations in an amino acid sequence, are
provided in the kit. In a preferred aspect, peptide internal
standards corresponding to proteins expressed from nucleic acids
comprising single nucleotide polymorphisms are provided.
[0203] Peptide internal standards may include peptides
corresponding to variant proteins selected from the group
consisting of BRCA1; BRCA2; CFTR; p53; a JAK protein; a STAT
protein; blood group antigens; HLA proteins; MHC proteins;
G-Protein Coupled Receptors; apolipoprotein E; kinases (e.g., such
as hCds1, MTKs, PTK, CDKs, STKs, CaMs, and the like) (see, e.g.,
U.S. Pat. No. 6,426,206); phosphatases; human drug metabolizing
proteins; viral proteins, including but not limited to viral
envelope proteins (e.g., an HIV envelope protein); transporter
proteins; and the like.
[0204] In one aspect, the peptide internal standard comprises a
label associated with a modified amino acid residue, such as a
phosphorylated amino acid residue, a glycosylated amino acid
residue, an acetylated amino acid residue, a farnesylated residue,
a ribosylated residue, and the like. In another aspect, a pair of
reagents is provided, a peptide internal standard corresponding to
a modified peptide and a peptide internal standard corresponding to
a peptide, identical in sequence but not modified.
[0205] In another aspect, one or more control peptide internal
standards are provided. For example, a positive control may be a
peptide internal standard corresponding to a constitutively
expressed protein, while a negative peptide internal standard may
be provided corresponding to a protein known not to be expressed in
a particular cell or species being evaluated. For example, in a kit
comprising peptide internal standards for evaluating a cell state
in a human being, a plant peptide internal standard may be
provided.
[0206] In still another aspect, a kit comprises a labeled peptide
internal standard as described above and software for analyzing
mass spectra (e.g., such as SEQUEST).
[0207] Preferably, the kit also comprises a means for providing
access to a computer memory comprising data files storing
information relating to the diagnostic fragmentation signatures of
one or more peptide internal standards. Access may be in the form
of a computer readable program product comprising the memory, or in
the form of a URL and/or password for accessing an internet site
for connecting a user to such a memory. In another aspect, the kit
comprises diagnostic fragmentation signatures (e.g., such as mass
spectral data) in electronic or written faun, and/or comprises
data, in electronic or written form, relating to amounts of target
proteins characteristic of one or more different cell states and
corresponding to peptides which produce the fragmentation
signatures.
[0208] The kit may further comprise expression analysis software on
computer readable medium, which is capable of being encoded in a
memory of a computer having a processor and capable of causing the
processor to perform a method comprising: determining a test cell
state profile from peptide fragmentation patterns in a test sample
comprising a cell with an unknown cell state or a cell state being
verified; receiving a diagnostic profile characteristic of a known
cell state; and comparing the test cell state profile with the
diagnostic profile.
[0209] In one aspect, the test cell state profile comprises values
of levels of peptides in a test sample that correspond to one or
more peptide internal standards provided in the kit. The diagnostic
profile comprises measured levels of the one or more peptides in a
sample having the known cell state (e.g., a cell state
corresponding to a normal physiological response or to an abnormal
physiological response, such as a disease).
[0210] Preferably, the software enables a processor to receive a
plurality of diagnostic profiles and to select a diagnostic profile
that most closely resembles or "matches" the profile obtained for
the test cell state profile by matching values of levels of
proteins determined in the test sample to values in a diagnostic
profile, to identify substantially all of a diagnostic profile
which matches the test cell state profile.
[0211] Substantially all of a diagnostic profile is matched by a
test cell state profile when most of the cellular constituents
(e.g., proteins in the proteome) which are diagnostic of the cell
state, are found to have substantially the same value in the two
profiles within a margin provided by experimental error.
Preferably, at least about 75% of the diagnostic proteins can be
matched, at least about 80%, at least about 85%, at least about 90%
or at least about 95% can be matched. Preferably, where one, or
only a few proteins (e.g., less than 10) are used to establish s
diagnostic profile, preferably all of the proteins have
substantially the same value.
[0212] Kits For Detecting Protein Ubiquitination
[0213] The invention further provides a kit for detecting and/or
quantifying a protein modification, such as ubiquitination. In one
aspect, the kit comprises a ubiquitin binding molecule (e.g., an
antibody, an affinity molecule for recognizing, a tag coupled to a
ubiquitin molecule, and the like), and one or more components,
including, but not limited to: a protease (e.g., such as trypsin);
a ubiquitinated molecule comprising known ubiquitination sites;
acetonitrile; silica resin; heptafluorobutyric acid; urea (e.g., 8M
urea); an isotope-coded affinity tag (e.g., such as an ICAT label
or pair of ICAT labels) (see, Gygi and Rist., 1999, Nat.
Biotechnol. 17: 994-999; U.S. Provisional Application No.
60/305,808, filed Jul. 16, 2001) or an affinity tag coupleable to
an isotope; a mass modifying moiety; a sample plate for use with a
mass spectrometer; a light-absorbent matrix; an ion exchange resin;
software for analyzing mass spectra (e.g., such as SEQUEST); fused
silica capillary tubing; and access to a computer memory comprising
data files storing information relating to ubiquitination sites for
a plurality of polypeptides for a plurality of different cells.
Access may be in the form of a computer readable program product
comprising the memory, or in the form of a URL and/or password for
accessing an internet site for connecting a user to such a memory.
In one preferred aspect, an isotope-labeled peptide comprising
Gly-Gly residues and known peptide amino acid sequences is provided
as an internal standard. In still a further aspect, an
isotope-labeled Gly-Gly dipeptide is provided.
[0214] In one particularly preferred aspect, a kit is provided
which comprises an antibody that specifically recognizes a peptide
product of a protease-digested ubiquitinated protein which
comprises a ubiquitin remnant. Preferably, the antibody does not
recognize the same peptide when it does not comprise the ubiquitin
remnant. Methods of making antibodies which are specific for
modified forms of peptides are routine in the art.
[0215] More preferably, the kit comprises one or more antibodies
which specifically recognize peptides produced by protease
digestion of ubiquitinated forms of ubiquitin. In one aspect, at
least one antibody in the kit specifically recognizes a peptide
comprising any of the K.sup.48, K.sup.63, K.sup.11, K.sup.27,
K.sup.6, K.sup.29, and K.sup.33 sites of the ubiquitin polypeptide
modified by a ubiquitin remnant at that site. In another aspect, an
antibody is provided which specifically recognize a ubiquitin
polypeptide ubiquitinated at one or more of the K.sup.48, K.sup.63,
K.sup.11, K.sup.27, K.sup.6, K.sup.29, and K.sup.33 sites. Either
type of antibody can be used to evaluate the site specificity and
amount of ubiquitination at one or more sites on a ubiquitin
polypeptide, e.g., to diagnose a pathology or stage of
differentiation associated with a particular pattern of
ubiquitination. Preferably, these antibodies do not recognize forms
of ubiquitin not ubiquitinated at the site of interest (although
such antibodies also may be included in the kits of the invention
as controls).
[0216] Diagnosis may be performed by using the peptide-specific
antibodies (which may also be polypeptide-specific antibodies) or
the polypeptide-specific antibodies (which may also be
peptide-specific antibodies) or a combination thereof. In one
aspect, however, a sample is digested by a protease (e.g., such as
trypsin) and one or more of the antibodies specific for a peptide
comprising a ubiquitin remnant at a particular site is used to
determine whether the sample is reactive with the antibody, e.g.,
by performing a standard immunoassay. Thus, reagents useful for
conducting immunoassays also may be included in the kits. The
presence and level of reactivity of the antibodies can be used to
monitor the site specificity and amount of ubiquitination.
[0217] Panels of antibodies can be used simultaneously to perform
the analysis (e.g., by using antibodies comprising distinguishable
labels). Panels of antibodies also can be used in parallel or in
sequential assays. Therefore, in one preferred aspect, a kit
according to the invention comprises a panel of antibodies
comprising antibodies specific for ubiquitinated
peptides/polypeptides ubiquitinated at one or more of the K.sup.48,
K.sup.63, K.sup.11, K.sup.27, K.sup.6, K.sup.29, and K.sup.33
sites.
[0218] The presence, absence, level, and/or site-specificity of
other types of modifications, such as phosphorylation, also can be
determined along with the presence, absence, level and/or site
specificity of ubiquitination. For example, in addition to
identifying the presence and/or amount of ubiquitination at the
K.sup.48, K.sup.63, K.sup.11, K.sup.27, K.sup.6, K.sup.29, and
K.sup.33 sites of ubiquitin, the presence and/or absence of
phosphorylation at particular phosphorylation sites on the
ubiquitin polypeptide also can be determined. Phosphorylation can
be determined by using mass spectrometry or through the use of
antibodies specific to particular phosphorylated forms of ubiquitin
polypeptides or peptides. In one preferred aspect, the kit
according to the invention further comprises an antibody specific
for a phosphorylated form of a ubiquitin polypeptide or peptide and
which does not recognize the non phosphorylated form. More
preferably, the kit comprises an antibody which recognizes a
ubiquitin polypeptide or peptide phosphorylated at Ser.sup.57 and
which does not recognize polypeptides/peptides which are not
phosphorylated at this site.
EXAMPLE
[0219] The invention will now be further illustrated with reference
to the following example. It will be appreciated that what follows
is by way of example only and that modifications to detail may be
made while still falling within the scope of the invention.
Example 1
Preparation of Ubiquitin-Conjugates from S. cerevisiae
[0220] Isolation and identification of yeast ubiquitin-conjugates
was accomplished as illustrated in FIG. 8. 100 mg of whole yeast
lysates were harvested from cells growing through log phase (OD610
1-1.5) from two strains of yeast differing in the expression of
6.times.His-tagged ubiquitin. Strain SUB592 (JSY171), expressing
tagged ubiquitin, and control strain, SUB280 (Spence, et al., 2000,
Cell 102: 67-76), were grown to log phase and lysed in buffer A (10
mM Tris, pH 8.0, 0.2 M NaH.sub.2PO.sub.4, 8M Urea) using glass
beads. A Ni.sup.2+-NTA-agarose column (Qiagen, Chatsworth, Calif.)
was loaded with the clarified lysates, sequentially washed with 30
volumes (bed volume) of buffer A twice, 3 volumes of buffer B (10
mM Tris, pH 6.3, 0.1 M NaH.sub.2PO.sub.4, 8M Urea), and eluted with
3 volumes of buffer C (10 mM Tris, pH 4.5, 0.1 M NaH.sub.2PO.sub.4,
8M Urea). A portion (0.5%) of eluted polypeptides was examined by
SDS-PAGE and silver staining. The remaining polypeptides (99.5%)
were reduced, alkylated at cysteinyl residues, and proteolyzed with
trypsin to generate test peptides.
[0221] Two-Dimensional Liquid Chromatography With Tandem Mass
Spectrometry
[0222] Because the resulting peptide mixture was enormously
complex, it was separated by two dimensions of chromatography to
allow thousands of peptides to be sequenced. The tryptic peptides
were separated in the first dimension by strong cation exchange
(SCX) chromatography with fraction collection every minute,
followed by nano-scale microcapillary reverse-phase (RP)
chromatography. Peptides of the control strain were eluted in a
10-minute gradient from 0% to 100% solvent B. Ubiquitin-conjugated
peptides were fractionated in a 70-minute gradient from 5% to 30%
solvent B. All collected fractions (80) were reduced in volume and
then analyzed individually using 75 .mu.m i.d..times.12 cm
self-packed fused silica C18 capillary columns.
[0223] Peptides were eluted for each analysis during a 90-minute
gradient in which the eluted peptide ions were detected, isolated,
and fragmented in a completely automated fashion on an LCQ-DECA ion
trap mass spectrometer (Thermo Finnigan, San Jose, Calif.). During
elution, peptides ions were constantly detected and selected for
sequencing in an automated fashion with one peptide being sequenced
on average every 2 seconds. More than 96,000 sequencing attempts
were acquired for ubiquitin-conjugates during the entire
experiment.
[0224] Data Processing
[0225] All MS/MS spectra were searched against the yeast ORF's
database supplemented with the sequence of the recombinant
6.times.His myc-ubiquitin using the SEQUEST algorithm (Eng, et al.
1994, supra). Modifications were permitted to allow for the
detection of the following (mass shift shown in Daltons); oxidized
methionines (+16), carboxymethylated cysteine (+57), ubiquitinated
lysine (+114), and phosphorylated serine, threonine, tyrosine
(+80). SEQUEST criteria were as described in Washburn, et al, 2001,
Nat. Biotechnol. 19: 242-7, and further included: (i) an Xcorr of
greater than 2.0, 2.2, and 3.75 for 1+, 2+, and 3+, charge state
peptides, respectively; ii) the requirement that a peptide must be
partially or fully tryptic; and (iii) the requirement that a
peptide must have a fCn score of >0.1. Peptides were also were
manually verified from each polypeptide identified by two or less
qualifying peptides.
[0226] Identification of 1,051 Ubiquitin Conjugated Candidates.
[0227] Database searching with SEQUEST identified 12,922 peptides
using the acceptance criteria described above. After removing
redundancy, 5,424 unique peptides were identified, corresponding to
1,237 polypeptides. These polypeptides were further filtered by:
(i) removing 48 polypeptides detected in the control fraction; (ii)
removing 34 polypeptides which contained 3 or more consecutive
histidine residues; (iii) removing 104 polypeptides considered to
be highly abundant (codon bias greater than 0.35 and identified by
less than three peptides); and (iv) accepting 70 polypeptides for
which the precise ubiquitination site was found. This filtering
resulted in final acceptance of 1,051 polypeptides as candidate
targets of ubiquitination.
[0228] In addition to unambiguously identifying more than 1,000
candidates for ubiquitin conjugation, the precise site of
ubiquitination was identified for a number of polypeptides. As
shown in FIGS. 9A-C, using mass differences characteristic of
ubiquitin remnants of the protease digestion process, a peptide (a
ubiquitin peptide, in this example) comprising a site of
ubiquitination could be identified. For example, where trypsin is
used as the protease, a 114 dalton mass change can be observed due
to a Gly-Gly residue linked to a lysine in the peptide through an
isopeptide bond. A missed cleavage site also is observed where
ubiquitination has occurred. As can be shown in FIGS. 10A-C, this
approach identified polypeptides comprising multiple ubiquitination
sites, including ubiquitin itself. Methods of utilizing the SEQUEST
algorithm to detect modified peptide are described in Jaffe, et
al., 1998, Biochemistry 37(46): 16211-24, for example.
[0229] The types and classes of ubiquitinated polypeptides
identified were compared to the entire yeast proteome. Codon bias
is a measure of the propensity of a gene to utilize only a subset
of the 61 potential codons to produce its amino acids (Bennetzen,
et al., 1982, J. Biol. Chem. 257: 3026-31, 1982) and has been shown
to be a good indicator of polypeptide expression levels under
specific growth conditions (Futcher, et al., 1999, Mol. Cell. Biol.
19: 7357-68). As a general rule, a codon bias value of less than
0.1 would reflect medium to highly abundant polypeptides. More than
one-half of the genes in yeast (57%) have codon bias values less
than 0.1 and are thus thought to be expressed at low abundance. A
majority of ubiquitinated polypeptides are highly enriched for low
abundance proteins, such as regulatory proteins.
[0230] The molecular environment of the polypeptides detected is
shown in FIG. 11B. Of the sites detected, more than one-third were
attributable to integral membrane polypeptides, supporting prior
studies that have indicating that downregulation of some membrane
polypeptides requires modification by ubiquitin for their
internalization and degradation in lysosmes/vacuoles (see, e.g.,
Hicke, 1999, Trends Cell Biol. 9: 107-112). The cellular function
of the polypeptides identified was compared against the yeast
proteome (see, e.g., FIG. 11C). Polypeptides from every category of
cellular polypeptide were detected.
[0231] Polypeptides involved in metabolism and transport were
detected with the highest frequencies.
[0232] Ubiquitination sites for 70 polypeptides were identified and
98 sites were found. Of these sites, 17 were attributable to the
ubiquitination of ubiquitin itself. The qualitative abundance of
ubiquitination at different sites on the ubiquitin molecule could
be assessed based on the number of fractions in which the peptide
occurred, the magnitude of the peptide ion as measured by mass
spectrometry, and the number of times the peptide was independently
identified by the database searching software. The relative
abundance of different ubiquitinated forms was determined to be
K.sup.48>K.sup.63>K.sup.11>>K.sup.27 and K.sup.6 (e.g.,
see FIG. 10B). The K.sup.11, K.sup.27, and K.sup.6 sites were newly
identified by the method according to the invention. The K.sup.63
site has been implicated in processes other than degradation (e.g.,
DNA repair, endocytosis, etc.) (see, e.g., Finley, 2001, Nature
412: 283, 285-6) and in polychain foimation in vivo (Pickart, 2000,
Trends Biochem. Sci. 25: 544-8; Mastrandrea, et al., 1999, J. Biol.
Chem. 274: 27299-306; Spence, et al., 1995, Mol. Cell. Biol. 15:
1265-73; Babinoshin and Haas, 1996, J. Biol. Chem. 271: 2823-31).
Ubiquitination at the K.sup.29 and K.sup.33 sites was not
observed.
[0233] The 98 ubiquitination sites identified were randomly
distributed throughout the entire sequence of polypeptides detected
with no apparent consensus sequence. For one polypeptide (ECM21p),
five sites of ubiquitination were detected, all in the middle third
of the polypeptide. However, homologous lysine residues within
polypeptide families were found to be modified by ubiquitination
(see, FIGS. 10A-C). For example, the sites detected for SNC1p and
SNC2p (K62) and HXT6p and HXT7p (K560) were identical.
[0234] Examining the 70 polypeptides for the presence of other
types of modifications revealed 29 phosphorylation sites from 26
phosphopeptides derived from 19 polypeptides (see, e.g., FIG. 10C).
For example, in addition to the 5 ubiquitination sites found for
ECM21p, 5 phosphorylation sites were found.
[0235] Among the phosphorylated polypeptides detected was ubiquitin
itself. A phorphorylated serine residue was identified at S.sup.57.
This serine was recently found to be nonessential for viability in
an alanine scanning mutation experiment. However, the crystalline
structure of tetraubiquitin suggests this residue is solvent and
could potentially interfere with proteosome recognition. Using the
methods described above, the presence of phosphorylated species of
ubiquitin can be examined in diseased and healthy cells to assess
the biological relevance of this modification. Additional
ubiquitin-like targets also can be studied, including, but not
limited to: Rub1/Nedd8, SUMO, and Apg12 (see, e.g., Hochstrasser,
2000, Science 289: 563-564).
[0236] Variations, modifications, and other implementations of what
is described herein will occur to those of ordinary skill in the
art without departing from the spirit and scope of the invention as
described and claimed herein and such variations, modifications,
and implementations are encompassed within the scope of the
invention.
[0237] All of the references, patents and patent applications
identified hereinabove are expressly incorporated herein by
reference.
Sequence CWU 1
1
5716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6X-His tag 1His His His His His His1 526PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Gly
Phe Thr Ala Leu Lys1 536PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Ala Leu Glu Leu Phe Arg1
5411PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Leu Phe Thr Gly His Pro Glu Thr Leu Glu Lys1 5
1055PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Ile Arg Asn Pro Asp1 5610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic His tag 6His
His His His His His His His His His1 5 10712PRTArtificial
SequenceDescription of Artificial Sequence Exemplary sequence of a
signature peptide produced by trypsin proteolysis 7Leu Ile Phe Ala
Gly Lys Gln Leu Glu Asp Gly Arg1 5 10860PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 8Met Pro Phe Ile Thr Ser Arg Pro Val
Ala Lys Asn Ser Ser His Ser1 5 10 15Leu Ser Glu Thr Asp Leu Asn Gln
Ser Lys Gly Gln Pro Phe Gln Pro 20 25 30Ser Pro Thr Lys Lys Leu Gly
Ser Met Gln Gln Arg Arg Arg Ser Ser 35 40 45Thr Ile Arg His Ala Leu
Ser Ser Leu Leu Gly Gly 50 55 60960PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 9Ala Asn Val His Ser Pro Ala Val Leu
Asn Asn Thr Thr Lys Gly Gly1 5 10 15Asn Asn Asn Gly Asn Ile Arg Ser
Ser Asn Thr Asp Ala Gln Leu Leu 20 25 30Gly Lys Lys Gln Asn Lys Gln
Pro Pro Pro Asn Ala Arg Arg His Ser 35 40 45Thr Thr Ala Ile Gln Gly
Ser Ile Ser Asp Ser Ala 50 55 601060PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 10Thr Thr Thr Pro Arg Ser Ser Thr
Ser Asp Thr Asn Arg Arg Thr Ser1 5 10 15Gly Arg Leu Ser Val Asp Gln
Glu Pro Arg Ile Ser Gly Gly Arg Tyr 20 25 30Ser Gln Ile Glu Glu Asp
Ser Thr Val Leu Asp Phe Asp Asp Asp His 35 40 45Asn Ser Ser Ala Val
Val Ser Ser Asp Leu Ser Ser 50 55 601160PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 11Thr Ser Leu Thr Arg Leu Ala Asn
Ser Lys Lys Phe Asn Glu Gln Phe1 5 10 15Leu Ile Glu Tyr Leu Thr Ala
Arg Gly Leu Leu Gly Pro Lys Thr Val 20 25 30Leu Ser Asn Glu Tyr Leu
Lys Ile Ser Ile Ser Thr Ser Gly Glu Ser 35 40 45Val Phe Leu Pro Thr
Ile Ser Ser Asn Asp Asp Glu 50 55 601260PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 12Tyr Leu Ser Arg Leu Asn Gly Leu
Asn Asp Gly Thr Asp Asp Ala Glu1 5 10 15Ala Asp Phe Phe Met Asp Gly
Ile Asp Gln Gln Glu Gly Asn Thr Pro 20 25 30Ser Leu Ala Thr Thr Ala
Ala Ala Thr Glu Ser Gly Gly Ser Ile Asn 35 40 45Glu Asn Arg Asp Thr
Leu Leu Arg Glu Asn Asn Ser 50 55 601360PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 13Gly Asp His Pro Gly Ser Gly Ser
Glu Leu Asn Thr Arg Ser Val Glu1 5 10 15Ile Asp Ser Ser Met Val Ser
Tyr Ser Ile Ala Val Ile Val Ser Val 20 25 30Lys Lys Pro Thr Arg Phe
Thr Asp Met Gln Leu Glu Leu Cys Ser Arg 35 40 45Val Lys Val Phe Trp
Asn Thr Gly Val Pro Pro Thr 50 55 601460PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 14Lys Thr Phe Asn Glu Glu Phe Tyr
Asn Ala Ala Ser Met Lys Trp Asn1 5 10 15Leu Asn Asp Glu Asn Phe Asp
Leu Phe Val Pro Leu Ser Ile Ser Pro 20 25 30Asp Ile Asp Gln Met Glu
Asn Asn Ser Asn Asp Arg Gln Met Arg Leu 35 40 45Phe Lys Asn Ile Pro
Thr Glu Glu Arg Leu Tyr Leu 50 55 601560PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 15Asp Lys Thr Lys Thr Lys Ala Ser
Leu Leu Asn Ala Ile Asp Val Asn1 5 10 15Lys Thr His Leu Tyr Gln Pro
Gly Asp Tyr Val Phe Leu Val Pro Val 20 25 30Val Phe Ser Asn His Ile
Pro Glu Thr Ile Tyr Leu Pro Ser Ala Arg 35 40 45Val Ser Tyr Arg Leu
Arg Leu Ala Thr Lys Ala Ile 50 55 601660PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 16Asn Arg Lys Gly Phe Tyr Arg Gln
Asp Ser Asn Ser Pro Gln Pro Ile1 5 10 15Val Ser Pro Asp Ser Ser Ser
Ser Leu Ser Ser Thr Thr Ser Ser Leu 20 25 30Lys Leu Thr Glu Thr Glu
Ser Ala Gln Ala His Arg Arg Ile Ser Asn 35 40 45Thr Leu Phe Ser Lys
Val Lys Asn His Leu His Met 50 55 601760PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 17Ser Ser His Gln Leu Lys Asn Glu
Glu Ser Gly Glu Glu Asp Ile Phe1 5 10 15Ala Glu Tyr Pro Ile Lys Val
Ile Arg Thr Pro Pro Pro Val Ala Val 20 25 30Ser Thr Ala Asn Lys Pro
Ile Tyr Ile Asn Arg Val Trp Thr Asp Ser 35 40 45Leu Ser Tyr Glu Ile
Ser Phe Ala Gln Lys Tyr Val 50 55 601860PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 18Ser Leu Asn Ser Glu Val Pro Ile
Lys Ile Lys Leu Ala Pro Ile Cys1 5 10 15Lys Asn Val Cys Val Lys Arg
Ile His Val Ser Ile Thr Glu Arg Val 20 25 30Thr Phe Val Ser Lys Gly
Tyr Glu Tyr Glu Tyr Asp Gln Thr Asp Pro 35 40 45Val Ala Lys Asp Pro
Tyr Asn Pro Tyr Tyr Leu Asp 50 55 601960PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 19Phe Ala Ser Lys Arg Arg Lys Glu
Arg Ser Val Ser Leu Phe Glu Ile1 5 10 15Arg Thr Lys Glu Lys Gly Thr
Arg Ala Leu Arg Glu Glu Ile Val Glu 20 25 30Asn Ser Phe Asn Asp Asn
Leu Leu Ser Tyr Ser Pro Phe Asp Asp Asp 35 40 45Ser Asp Ser Lys Gly
Asn Pro Lys Glu Arg Leu Gly 50 55 602060PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 20Ile Thr Glu Pro Ile Ile Ile Glu
Thr Lys Leu Lys Phe Pro Lys Tyr1 5 10 15Glu Asp Leu Asp Lys Arg Thr
Ala Lys Ile Ile Pro Pro Tyr Gly Ile 20 25 30Asp Ala Tyr Thr Ser Ile
Pro Asn Pro Glu His Ala Val Ala Asn Gly 35 40 45Pro Ser His Arg Arg
Pro Ser Val Ile Gly Phe Leu 50 55 602160PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 21Ser Gly His Lys Gly Ser Lys Ser
His Glu Glu Asn Glu Lys Pro Val1 5 10 15Tyr Asp Pro Lys Phe His Gln
Thr Ile Ile Lys Ser Asn Ser Gly Leu 20 25 30Pro Val Lys Thr His Thr
Arg Leu Asn Thr Pro Lys Arg Gly Leu Tyr 35 40 45Leu Asp Ser Leu His
Phe Ser Asn Val Tyr Cys Arg 50 55 602260PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 22His Lys Leu Glu Ile Met Leu Arg
Ile Ser Lys Pro Asp Pro Glu Cys1 5 10 15Pro Ser Lys Leu Arg His Tyr
Glu Val Leu Ile Asp Thr Pro Ile Phe 20 25 30Leu Val Ser Glu Gln Cys
Asn Ser Gly Asn Met Glu Leu Pro Thr Tyr 35 40 45Asp Met Ala Thr Met
Glu Gly Lys Gly Asn Gln Val 50 55 602360PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 23Pro Leu Ser Met Asn Ser Asp Phe
Phe Gly Asn Thr Cys Pro Pro Pro1 5 10 15Pro Thr Phe Glu Glu Ala Ile
Ser Val Pro Ala Ser Pro Ile Val Ser 20 25 30Pro Met Gly Ser Pro Asn
Ile Met Ala Ser Tyr Asp Pro Asp Leu Leu 35 40 45Ser Ile Gln Gln Leu
Asn Leu Ser Arg Thr Thr Ser 50 55 602460PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 24Val Ser Gly Pro Ser Gly Tyr Ser
Asp Asp Ala Gly Val Pro Asn Val1 5 10 15Asn Arg Asn Ser Ile Ser Asn
Ala Asn Ala Met Asn Gly Ser Ile Ser 20 25 30Asn Ser Ala Phe Val Ser
Gly Asn Ser Gly Gln Gly Val Ala Arg Ala 35 40 45Arg Ala Thr Ser Val
Asn Asp Arg Ser Arg Phe Asn 50 55 602560PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 25Asn Leu Asp Lys Leu Leu Ser Thr
Pro Ser Pro Val Asn Arg Ser His1 5 10 15Asn Ser Ser Pro Thr Asn Gly
Leu Ser Gln Ala Asn Gly Thr Val Arg 20 25 30Ile Pro Asn Ala Thr Thr
Glu Asn Ser Lys Asp Lys Gln Asn Glu Phe 35 40 45Phe Lys Lys Gly Tyr
Thr Leu Ala Asn Val Lys Asp 50 55 602637PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
poly-ubiquitinated polypeptide 26Asp Glu Glu Gln Glu Gly Ile Val
Ser Ser Ser Ser Ala Asp Ser Leu1 5 10 15Leu Ser His Gly Asn Glu Pro
Pro Arg Tyr Asp Glu Ile Val Pro Leu 20 25 30Met Ser Asp Glu Glu
352712PRTArtificial SequenceDescription of Artificial Sequence
Synthetic signature peptide 27Leu Ile Phe Ala Gly Lys Gln Leu Glu
Asp Gly Arg1 5 102818PRTArtificial SequenceDescription of
Artificial Sequence Synthetic signature peptide 28Thr Leu Ser Asp
Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val1 5 10 15Leu
Arg2921PRTArtificial SequenceDescription of Artificial Sequence
Synthetic signature peptide 29Thr Leu Thr Gly Lys Thr Ile Thr Leu
Glu Val Glu Ser Ser Asp Thr1 5 10 15Ile Asp Asn Val Lys
203018PRTArtificial SequenceDescription of Artificial Sequence
Synthetic signature peptide 30Thr Ile Thr Leu Glu Val Glu Ser Ser
Asp Thr Ile Asp Asn Val Lys1 5 10 15Ser Lys3119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic signature
peptide 31Leu Ile Ser Glu Glu Asp Leu Gly Met Gln Ile Phe Val Lys
Thr Leu1 5 10 15Thr Gly Lys3219PRTArtificial SequenceDescription of
Artificial Sequence Synthetic phosphopeptide sequence 32Ala Val Ser
Val Ser Asp Leu Ser Tyr Val Ala Asn Ser Gln Ser Ser1 5 10 15Pro Leu
Arg3318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic phosphopeptide sequence 33Gly Ser Gly Gly Thr Ser Glu Leu
Gly Gly Ser Glu Ser Thr Pro Leu1 5 10 15Leu Arg3417PRTArtificial
SequenceDescription of Artificial Sequence Synthetic phosphopeptide
sequence 34Asp Glu Asn Asp Gly Tyr Ala Ser Asp Glu Val Gly Gly Thr
Leu Ser1 5 10 15Arg3512PRTArtificial SequenceDescription of
Artificial Sequence Synthetic phosphopeptide sequence 35Asp Asp Glu
Tyr Asp Asp Leu Asn Thr Ile Asp Lys1 5 103615PRTArtificial
SequenceDescription of Artificial Sequence Synthetic phosphopeptide
sequence 36Asn Pro Ser Thr Leu Leu Pro Thr Ser Ser Met Phe Trp Asn
Lys1 5 10 153716PRTArtificial SequenceDescription of Artificial
Sequence Synthetic phosphopeptide sequence 37Asn Glu Glu Ser Gly
Glu Glu Asp Ile Phe Ala Glu Tyr Pro Ile Lys1 5 10
153823PRTArtificial SequenceDescription of Artificial Sequence
Synthetic phosphopeptide sequence 38His Ala Leu Ser Ser Leu Leu Gly
Gly Ala Asn Val His Ser Pro Ala1 5 10 15Val Leu Asn Asn Thr Thr Lys
203912PRTArtificial SequenceDescription of Artificial Sequence
Synthetic phosphopeptide sequence 39Arg Pro Ser Val Ile Gly Phe Leu
Ser Gly His Lys1 5 104018PRTArtificial SequenceDescription of
Artificial Sequence Synthetic phosphopeptide sequence 40Ser His Asn
Ser Ser Pro Thr Asn Gly Leu Ser Gln Ala Asn Gly Thr1 5 10 15Val
Arg4114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic phosphopeptide sequence 41Glu Glu Ile Asp Ser Glu Phe Glu
Asp Glu Asp Phe Glu Lys1 5 104222PRTArtificial SequenceDescription
of Artificial Sequence Synthetic phosphopeptide sequence 42Ala Ser
Gly Glu Thr Ala Ile His Glu Pro Glu Pro Glu Ala Glu Gln1 5 10 15Ala
Val Glu Asp Thr Ala 204322PRTArtificial SequenceDescription of
Artificial Sequence Synthetic phosphopeptide sequence 43Leu Gln Val
Val Ser His Glu Thr Asp Ile Asn Glu Asp Glu Glu Glu1 5 10 15Ala His
Tyr Glu Asp Lys 204420PRTArtificial SequenceDescription of
Artificial Sequence Synthetic phosphopeptide sequence 44Lys Tyr Ser
Asp Asn Glu Asp Asp Glu Tyr Asp Asp Ala Asp Leu His1 5 10 15Gly Phe
Glu Lys 204518PRTArtificial SequenceDescription of Artificial
Sequence Synthetic phosphopeptide sequence 45Arg Gly Ser Val Tyr
His Val Pro Leu Asn Pro Val Gln Ala Thr Ala1 5 10 15Val
Arg4616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic phosphopeptide sequence 46Ile His Asp Thr Ser Asp Glu Asp
Met Ala Ile Asn Gly Leu Glu Arg1 5 10 154716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic phosphopeptide
sequence 47Asn Asn Asp Ile Glu Ser Ser Ser Pro Ser Gln Leu Gln His
Glu Ala1 5 10 154815PRTArtificial SequenceDescription of Artificial
Sequence Synthetic phosphopeptide sequence 48Ser Val Asn Tyr Asn
Glu Leu Ser Asp Asp Asp Thr Ala Val Lys1 5 10 15499PRTArtificial
SequenceDescription of Artificial Sequence Synthetic phosphopeptide
sequence 49Thr Leu Ser Asp Tyr Asn Ile Gln Lys1 55016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic phosphopeptide
sequence 50Ile Glu Glu Ile Asn Glu Asn Ser Pro Leu Leu Ser Ala Pro
Ser Lys1 5 10 155112PRTArtificial SequenceDescription of Artificial
Sequence Synthetic phosphopeptide sequence 51Thr Asn Ser Phe Asp
Met Pro Gln Leu Asn Thr Arg1 5 105216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic phosphopeptide
sequence 52Glu Thr Val Asp Asp Asp Ser Glu Thr Leu Asn Gln Leu Gln
Asp Arg1 5 10 155312PRTArtificial SequenceDescription of Artificial
Sequence Synthetic phosphopeptide sequence 53Leu Pro Ser Tyr Glu
Glu Ala Ala Gly Thr Pro Lys1 5 105416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic phosphopeptide
sequence 54Lys Asn Pro Asp Glu Asp Glu Phe Leu Ile Asn Ser Asp Asp
Glu Met1 5 10 155529PRTArtificial SequenceDescription of Artificial
Sequence Synthetic phosphopeptide sequence 55Ser Ser Gly Ile Asp
Glu Asp Glu Val Val Thr Pro Ala Glu Asp Ala1 5 10 15Lys Glu Glu Glu
Glu Glu His Pro Pro Leu Pro Ala Arg 20 255619PRTArtificial
SequenceDescription of Artificial
Sequence Synthetic phosphopeptide sequence 56Glu Gln His His Glu
Asp Ser Glu Glu Glu Asp Ser Trp Ser Gln Phe1 5 10 15Val Glu
Lys5721PRTArtificial SequenceDescription of Artificial Sequence
Synthetic phosphopeptide sequence 57His Val Ile Ala Asp Leu Glu Asp
His Glu Ser Ser Asp Glu Glu Gly1 5 10 15Thr Ala Leu Pro Lys 20
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References