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REAGENTS FOR THE DETECTION OF PROTEIN PHOSPHORYLATION
IN EGFR-SIGNALING PATHWAYS
FIELD OF THE INVENTION
The invention relates generally to antibodies and peptide reagents
for the detection of protein phosphorylation, and to protein phosphoryl-
ation in cancer.
BACKGROUND OF THE INVENTION
The activation of proteins by post-translational modification is an
important cellular mechanism for regulating most aspects of biological
organization and control, including growth, development, homeostasis,
and cellular communication. Protein phosphorylation, for example, plays
a critical role in the etiology of many pathological conditions and diseases,
including cancer, developmental disorders, autoimmune diseases, and
diabetes. Yet, in spite of the importance of protein modification, it is not
yet well understood at the molecular level, due to the extraordinary
complexity of signaling pathways, and the slow development of
technology necessary to unravel it.
Protein phosphorylation on a proteome-wide scale is extremely
complex as a result of three factors: the large number of modifying
proteins, e.g. kinases, encoded in the genome, the much larger number of
sites on substrate proteins that are modified by these enzymes, and the
dynamic nature of protein expression during growth, development,
disease states, and aging. The human genome, for example, encodes
over 520 different protein kinases, making them the most abundant class
of enzymes known. See Hunter, Nature 411: 355-65 (2001). Most
kinases phosphorylate many different substrate proteins, at distinct
tyrosine, serine, and/or threonine residues. Indeed, it is estimated that
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one-third of all proteins encoded by the human genome are
phosphorylated, and many are phosphorylated at multiple sites by
different kinases. See Graves et al., Pharmacol. Ther. 82:111-21 (1999).
Many of these phosphorylation sites regulate critical biological
processes and may prove to be important diagnostic or therapeutic
targets for molecular medicine. For example, of the more than 100
dominant oncogenes identified to date, 46 are protein kinases. See
Hunter, supra. Understanding which proteins are modified by these
kinases will greatly expand our understanding of the molecular
mechanisms underlying oncogenic transformation. Therefore, the
identification of, and ability to detect, phosphorylation sites on a wide
variety of cellular proteins is crucially important to understanding the key
signaling proteins and pathways implicated in the progression of diseases
like cancer.
An important class of signaling proteins is the receptor tyrosine
kinase family (RTKs), which act as essential mediators of physiological
cell functions such as proliferation, differentiation, motility or survival.
On
the basis of their structural characteristics RTKs can be classified into 20
subfamilies, which share a homologous domain that specifies the catalytic
tyrosine kinase function (Zwick et aL, 1999 Trends in Pharmacological
Sciences 20: 408-412). The RTKs include the epidermal growth factor
receptor (EGFR) family, which consists of four closely related receptors:
EGFR (HER1), HER2 (ErbB2lneu), the kinase dead HER3, and HER4
(ErbB4) (Casalini et al. (2004) J. Cell. Physio1200: 343-350). Signaling
through EGFR is an important component of normal development, and
defective signaling through this receptor early in development can be
detrimental for embryogenesis and organogenesis (see Casalini et al.,
supra.)
Aberrant EGFR activity has been implicated in a variety of human
solid tumors including lung, bladder, breast, esophageal, head and neck,
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and gynecological tumors (Smith et aL 2004 Oncology Research 14, 175-
225). Over-expression of EGFR has also been shown in 53% of
malignant gliomas, and a mutated form of EGFR, the type III EGFR
deletion mutant (also known as EGFRvlll), is frequently found in human
glioblastomas, breast tumors, and meduloblastomas (see Smith et al.,
supra.). EGFR overexpression in non-small cell lung cancers (NSCLC) is
correlated with shorter patient survival times as compared to patients with
lower or normal levels of the receptor (Veale et al., 1993 Br. J. Cancer 68:
162-165).
EGFR, through its extracellular domain, binds to different ligands,
including epidermal growth factor (EGF), tumor growth factor-alpha
(TGFalpha), betacellulin, amphiregulin, epiregulin and heregulin (Riese et
a/. 1998 Bioessays 20: 41-48). Ligand binding induces homodimerization,
leading to ATP-mediated autophosphorylation or transphosphorylation (by
a partner kinase) of EGFR, which in turn activates its kinase function
(Russo et al., 1985 J. Biol. Chem. 260: 5205-5208). Within EGFR, the
sites reported to be most important in terms of receptor phosphorylation
and activation are Tyr 1148, Tyr1173, Tyr1068, and Tyr1086 (Downward
et al. (1984) Nature 311: 483-485; Margolis et al. (1989) J. Biol. Chem.
264: 10667-10671). Phosphorylation of these distinct tyrosine residues
creates binding sites for numerous proteins, typically containing Src
homology 2 (SH2)- and phosphotyrosine binding (PTB)- domains, many
of which are either tyrosine phosphorylated enzymes, such as Src or
Phospholipase C gamma, or adaptor molecules that link receptor
activation to downstream signaling pathways including MAPK-Erkl/2 and
PI3K-AKT (see Zwick et al.)
Despite the identification of some of the downstream targets and
effectors of EGFR, the molecular mechanisms contributing to EGFR-
mediated oncogenesis in a variety of human cancers remain incompletely
understood. At the same time, however, interest in EGFR as a therapeutic
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target has continued to increase, and targeted inhibitors of this RTK are
already on the market, or in clinical trials, for a variety of cancers
involving
activated EGFR. For example, Herceptin , an inhibitor of HER2/neu, is
currently an approved therapy for a certain subset of breast cancer.
IressaTM (ZD1839), a smali-molecule inhibitor of EGFR, has recently
entered clinical trials for the treatment of breast cancer, while another
small molecule inhibitor, TarcevaTM (OSI-774), is in clinical trials for the
treatment of non-small cell lung carcinoma (NSCLC). However, the
efficacy, mechanism of action, and clinical utility of these compounds in
mediating molecular effects downstream of EGFR remain to be seen.
Indeed, the limited success thus far observed with these highly specific
targeted inhibitors (each targeting only a single protein) evidences that
additional signaling molecules beyond just EGFR may be driving these
cancers.
For example, 30-50 percent of HER2-positive breast cancers do
not respond to the HER2-inhibtor, Herceptin (see Hortobagyi (2001)
Semin Oncol 6, Suppl 18: 43-7). These observations, along with recent
studies (with Gleevec and Rapamycin) establishing that combinations of
targeted therapeutics may be more effective than single agents (see Mohi
et al,, Proc Natl Acad Sci U.S.A. (2004),101(9): 3130-5), support the
widely-accepted belief that multiple signaling molecules are in fact driving
most cancers.
Accordingly, there is a continuing and pressing need to unravel the
molecular mechanisms of EGFR-driven oncogenesis by identifying the
downstream signaling proteins mediating cellular transformation in
diseases involving activated EGFR. Identifying particular phosphorylation
sites on such signaling proteins and providing new reagents, such as
phospho-specific antibodies and AQUA peptides, to detect and quantify
them remains particularly important to advancing our understanding of the
biology of these cancers.
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Presently, a handful of compounds targeting EGFR are in or
entering clinical trials for the treatment of various cancers, including
breast and lung. Although the activation and/or expression of EGFR itself
can be detected, it is clear that other downstream effectors of EGFR
signaling, having diagnostic, predictive, or therapeutic value, remain to be
elucidated. Identification of downstream signaling molecules and
phospho-sites involved in the progression of EGFR- driven cancers, and
development of new reagents to detect and quantify these sites and
proteins, may lead to improved diagnostic/prognostic markers, as well as
novel drug targets, for the detection and treatment of these diseases.
SUMMARY OF THE INVENTION
The invention discloses 168 novel phosphorylation sites identified
in signal transduction proteins and pathways downstream of, and
including, EGFR, and provides new reagents, including phosphorylation-
site specific antibodies and AQUA peptides, for the selective detection
and quantification of these phosphorylated sites/proteins. Also provided
are methods of using the reagents of the invention for the detection and
quantification of the disclosed phosphorylation sites.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1- Is a diagram broadly depicting the immunoaffinity
isolation and mass-spectrometric characterization methodology (IAP)
employed to identify the novel phosphorylation sites disclosed herein.
FIG. 2 - Is a table (corresponding to Table 1) enumerating the
EGFR signaling protein phosphorylation sites disclosed herein:
Column A = the name of the parent protein; Column B the SwissProt
accession number for the protein (human sequence); Column C = the
protein type/classification; Column D = the tyrosine residue (in the parent
protein amino acid sequence) at which phosphorylation occurs within the
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phosphorylation site; Column E = the phosphorylation site sequence
encompassing the phosphorylatable residue (residue at which
phosphorylation occurs (and corresponding to the respective entry in
Column D) appears in lowercase; and Column F= the cell type(s) in
which the phosphorylation site was discovered.
FIG. 3 - is an exemplary mass spectrograph depicting the detection
of the tyrosine 998 phosphorylation site in EGFR (see Row 123 in Figure
2/Table 1), as further described in Example 1(red and blue indicate ions
detected in MS/MS spectrum); pY and Y* indicate the phosphorylated
tyrosine (shown as lowercase "y" in Figure 2).
FIG. 4- is an exemplary mass spectrograph depicting the detection
of the tyrosine 653 phosphorylation site in insulin receptor substrate-2 (IRS-
2) (see Row 14 in Figure 2/Table 1), as further described in Example 1(red
and blue indicate ions detected in MS/MS spectrum; the purple M# indicates
an oxidized methionine residue detected); pY and Y* indicate the
phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
FIG. 5- is an exemplary mass spectrograph depicting the
detection of the tyrosine 1328 phosphorylation site in HER-3 (see Row
126 in Figure 2/Table 1), as further described in Example 1(red and blue
indicate ions detected in MS/MS spectrum); pY and Y* indicate the
phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
FIG. 6 - is an exemplary mass spectrograph depicting the
detection of the tyrosine 539 phosphorylation site in STAT-3 (see Row
154 in Figure 2/Table 1), as further described in Example 1(red and blue
indicate ions detected in MS/MS spectrum); pY and Y* indicate the
phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
FIG. 7 - is an exemplary mass spectrograph depicting the detection
of the tyrosine 1238 phosphorylation site in Ron kinase (see Row 142 in
Figure 2/ Table 1), as further described in Example 1(red and blue indicate
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ions detected in MS/MS spectrum); pY and Y* indicate the phosphorylated
tyrosine (shown as lowercase "y" in Figure 2).
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, 168 novel protein
phosphorylation sites in signaling proteins and pathways downstream of,
and including, the Epidermal Growth Factor Receptor kinase (EGFR)
have now been discovered. These newly described phosphorylation sites
were identified by employing the techniques described in "Immunoaffinity
Isolation of Modified Peptides From Complex Mixtures," U.S. Patent
Publication No. 20030044848, Rush et al., using cellular extracts from a
variety of tumor derived cell lines, e.g. A549 and HT-29, expressing
EGFR and stimulated with EGF, as further described below. The novel
phosphorylation sites, and their corresponding parent proteins, disclosed
herein are listed in Table 1. These phosphorylation sites correspond to
numerous different parent proteins (the full sequences of which (human)
are all publicly available in SwissProt database and their Accession
numbers listed in Column B of Table 1/Fig. 2), each of which fall into
discrete protein type groups, for example Adaptor/Scaffold proteins,
Cytoskeletal proteins, Receptor Tyrosine Kinases, and RNA Binding
proteins, etc. (see Column C of Table 1), the phosphorylation of which is
relevant to signal transduction activity downstream of EGFR, as disclosed
herein.
The discovery of the 168 novel protein phosphorylation sites
described herein enables the production, by standard methods, of new
reagents, such as phosphorylation site-specific antibodies and AQUA
peptides (heavy-isotope labeled peptides), capable of specifically
detecting and/or quantifying these phosphorylated sites/proteins. Such
reagents are highly useful, inter alia, for studying signal transduction
events underlying the progression of EGFR-mediated cancers.
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Accordingly, the invention provides novel reagents -- phospho-specific
antibodies and AQUA peptides -- for the specific detection and/or
quantification of an EGFR-related signaling protein/polypeptide only when
phosphorylated (or only when not phosphorylated) at a particular
phosphorylation site disclosed herein. The invention also provides
methods of detecting and/or quantifying one or more phosphorylated
EGFR-related signaling proteins using the phosphorylation-site specific
antibodies and AQUA peptides of the invention.
In part, the invention provides an isolated phosphorylation site-
specific antibody that specifically binds a given EGFR-related signaling
protein only when phosphorylated (or not phosphorylated, respectively) at
a particular tyrosine enumerated in Column D of Table 1/Figure 2
comprised within the phosphorylatable peptide site sequence enumerated
in corresponding Column E. In further part, the invention provides a
heavy-isotope labeled peptide (AQUA peptide) for the detection and
quantification of a given EGFR-related signaling protein, the labeled
peptide comprising a particular phosphorylatable peptide site/sequence
enumerated in Column E of Table 1/Figure 2 herein. For example,
among the reagents provided by the invention is an isolated
phosphorylation site-specific antibody that specifically binds the STAT3
transcription factor only when phosphorylated (or only when not
phosphorylated) at tyrosine 539 (see Row 154 (and Columns D and E) of
Table 1/Figure 2). By way of further example, among the group of
reagents provided by the invention is an AQUA peptide for the
quantification of phosphorylated STAT3 protein, the AQUA peptide
comprising phosphorylatable peptide sequence listed in Column E, Row
154, of Table 1/Figure 2 (which encompasses the phosphorylatable
tyrosine at position 539).
In one embodiment, the invention provides an isolated
phosphorylation site-specific antibody that specifically binds a human
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EGFR-related signaling protein selected from Column A of Table 1(Rows
2-169) only when phosphorylated at the tyrosine listed in corresponding
Column D of Table 1, comprised within the peptide sequence listed in
corresponding Column E of Table 1(SEQ ID NOs: 1-168), wherein said
antibody does not bind said signaling protein when not phosphorylated at
said tyrosine. In another embodiment, the invention provides an isolated
phosphorylation site-specific antibody that specifically binds an EGFR-
related signaling protein selected from Column A of Table 1 only when not
phosphorylated at the tyrosine listed in corresponding Column D of
Table 1, comprised within the peptide sequence listed in corresponding
Column E of Table 1(SEQ ID NOs: 1-168), wherein said antibody does
not bind said signaling protein when phosphorylated at said tyrosine.
Such reagents enable the specific detection of phosphorylation (or non-
phosphorylation) of a novel phosphorylatable site disclosed herein. The
invention further provides immortalized cell lines producing such
antibodies. In one preferred embodiment, the immortalized cell line is a
rabbit or mouse hybridoma.
In another embodiment, the invention provides a heavy-isotope
labeled peptide (AQUA peptide) for the quantification of an EGFR-related
signaling protein selected from Column A of Table 1, said labeled peptide
comprising the phosphorylatable peptide sequence listed in corresponding
Column E of Table 1(SEQ ID NOs: 1-168), which sequence comprises
the phosphorylatable tyrosine listed in corresponding Column D of
Table 1. In certain preferred embodiments, the phosphorylatable tyrosine
within the labeled peptide is phosphorylated, while in other preferred
embodiments, the phosphorylatable tyrosine within the labeled peptide is
not phosphorylated.
Reagents (antibodies and AQUA peptides) provided by the
invention may conveniently be grouped by the type of EGFR-related
signaling protein in which a given phosphorylation site (for which reagents
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are provided) occurs. The protein types for each respective protein (in
which a phosphorylation site has been discovered) are provided in
Column C of Table 1/Figure 2, and include: Actin Binding proteins,
Adaptor/Scaffold proteins, Adhesion proteins, Apoptosis proteins, Axon
Guidance proteins, Calcium-binding proteins, Cell Cycle Regulation
proteins, Cell Surface proteins, Cellular Metabolism enzymes,
Chaperones, Cytoskeletal proteins,'DNA Binding proteins, DNA
Replication proteins, GTPase Activating proteins, Guanine Nucleotide
Exchange Factors, Hydrolases, Immunoglobulin Superfamily proteins,
Lipid Kinases, Lipid Binding proteins, Motor proteins, Oxidoreductases,
Proteases, Protein Kinases, Protein Phosphatases, Receptor Protein
Phosphatases, Receptor Tyrosine Kinases, Receptor Tyrosine Kinase
ligands, Receptors, RNA Binding proteins, Transcription Factors,
Transcription Coactivator/Corepressor proteins, Transferases,
Transporter proteins, Tumor Suppressor proteins, Ubitquitin Conjugating
System proteins, and Vesicle proteins. Each of these distinct protein
groups is considered a preferred subset of EGFR-related signal
transduction protein phosphorylation sites disclosed herein, and reagents
for their detection/quantification may be considered a preferred subset of
reagents provided by the invention.
Particularly preferred subsets of the phosphorylation sites (and
their corresponding proteins) disclosed herein are those occurring on the
following protein types/groups listed in Column C of Table 1/Figure 2:
Actin Binding proteins, Adaptor/Scaffold proteins, Calcium-Binding
Proteins, Cell Cycle Regulation proteins, Cytoskeletal proteins, DNA
Binding and Replication Proteins, GTPase Activating proteins, Guanine
Nucleotide Exchange Factor proteins, Lipid Kinases, Receptor Tyrosine
Kinases, Receptor Tyrosine Kinase ligands, Protein Kinases, Receptor
and Protein Phosphatases, Transcription Factor proteins, Tumor
Suppressor proteins, and Vesicle proteins. Accordingly, among preferred
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subsets of reagents provided by the invention are isolated antibodies and
AQUA peptides useful for the detection and/or quantification of the
foregoing preferred protein/phosphorylation site subsets.
In one subset of preferred embodiments, there is provided:
(i) An antibody that specifically binds an Actin Binding protein selected
from Column A, Rows 2-8, of Table I only when phosphorylated at the
tyrosine listed in corresponding Column D, Rows 2-8, of Table 1,
comprised within the phosphorylatable peptide sequence listed in
corresponding Column E, Rows 2-8, of Table 1(SEQ ID NOs: 1-7),
wherein said antibody does not bind said protein when not phosphorylated
at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Actin Binding
protein when not phosphorylated at the disclosed site (and does not bind
the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of an Actin Binding protein selected from Column A, Rows
2-8, said labeled peptide comprising the phosphorylatable peptide
sequence listed in corresponding Column E, Rows 2-13, of Table 1(SEQ
ID NOs: 1-7), which sequence comprises the phosphorylatable tyrosine
listed in corresponding Column D, Rows 2-8, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Actin Binding
protein phosphorylation sites are particularly preferred: Catenin delta-1
(Y174, Y213, Y248, Y321, Y335) (see SEQ ID NOs: 2-6).
In a second subset of preferred embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a an Adaptor/Scaffold protein selected from Column A, Rows 9-38,
of Table 1 only when phosphorylated at the tyrosine listed in
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corresponding Column D, Rows 9-38, of Table 1, comprised within the
phosphorylatable peptide sequence listed in corresponding Column E,
Rows 9-38, of Table 1(SEQ ID NOs: 8-37), wherein said antibody does
not bind said protein when not phosphorylated at said tyrosine.
S (ii) An equivalent antibody to (i) above that only binds the
Adaptor/Scaffold protein when not phosphorylated at the disclosed site
(and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of a an Adaptor/Scaffold protein selected from Column A,
Rows 9-38, said labeled peptide comprising the phosphorylatable peptide
sequence listed in corresponding Column E, Rows 9-38, of Table 1(SEQ
ID NOs: 8-37), which sequence comprises the phosphorylatable tyrosine
listed in corresponding Column D, Rows 9-38, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Adaptor/Scaffold
protein phosphorylation sites are particularly preferred: Cbl (Y445), IRS-2
(Y653, Y675, Y823), STAM1 (Y198), and STAM2 (Y113) (see SEQ ID
NOs: 9, 13-15, and 25-26).
In another subset of preferred embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a Calcium-Binding or Cell Cycle Regulation protein selected from
Column A, Rows 60-62, of Table I only when phosphorylated at the
tyrosine listed in corresponding Column D, Rows 60-62, of Table 1,
comprised within the phosphorylatable peptide sequence listed in
corresponding Column E, Rows 60-62, of Table 1(SEQ ID NOs: 59-61),
wherein said antibody does not bind said protein when not phosphorylated
at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Calcium-
Binding or Cell Cycle Regulation protein when not phosphorylated at the
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disclosed site (and does not bind the protein when it is phosphorylated at
the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of a Calcium-Binding or Cell Cycle Regulation protein
selected from Column A, Rows 60-62, said labeled peptide comprising the
phosphorylatable peptide sequence listed in corresponding Column E,
Rows 60-62, of Table 1(SEQ ID NOs: 59-61), which sequence comprises
the phosphorylatable tyrosine listed in corresponding Column D, Rows
60-62, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Calcium-Binding or
Cell Cycle Regulation protein phosphorylation sites are particularly
preferred: Ov/Br Septin (Y260) (see SEQ ID NO: 61).
In another subset of preferred embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a Cytoskeletal protein selected from Column A, Rows 66-81, of
Table 1 only when phosphorylated at the tyrosine listed in corresponding
Column D, Rows 66-81, of Table 1, comprised within the phosphorylatable
peptide sequence listed in corresponding Column E, Rows 66-81, of
Table 1(SEQ ID NOs: 65-80), wherein said antibody does not bind said
protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Cytoskeletal
protein when not phosphorylated at the disclosed site (and does not bind
the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of a Cytoskeletal protein selected from Column A, Rows
66-81, said labeled peptide comprising the phosphorylatable peptide
sequence listed in corresponding Column E, Rows 66-81, of Table 1(SEQ
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ID NOs: 65-80), which sequence comprises the phosphorylatable tyrosine
listed in corresponding Column D, Rows 66-81, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Cytoskeletal
protein phosphorylation sites are particularly preferred: Talin-1 (Y26), (see
SEQ ID NO: 77).
In still another subset of preferred embodiments there is provided:
(i) An isolated phosphoryiation site-specific antibody that specifically
binds a DNA Binding or Replication protein selected from Column A, Rows
82-85, of Table 1 only when phosphorylated at the tyrosine listed in
corresponding Column D, Rows 82-85, of Table 1, comprised within the
phosphorylatable peptide sequence listed in corresponding Column E,
Rows 82-85, of Table 1(SEQ ID NOs: 81-84), wherein said antibody does
not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the DNA Binding
or Replication protein when not phosphorylated at the disclosed site (and
does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of DNA Binding or Replication protein selected from
Column A; Rows 82-85, said labeled peptide comprising the
phosphorylatable peptide sequence listed in corresponding Column E,
Rows 82-85, of Table 1(SEQ ID NOs: 81-84), which sequence comprises
the phosphorylatable tyrosine listed in corresponding Column D, Rows
82-85, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following DNA Binding or
Replication protein phosphorylation sites are particularly preferred: Smc5
(Y246) (see SEQ ID NO: 84).
In still another subset of preferred embodiments there is provided:
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(i) An isolated phosphorylation site-specific antibody that specifically
binds a GTPase Activating or Guanine Nucleotide Exchange Factor
protein selected from Column A, Rows 89-93, of Table 1 only when
phosphorylated at the tyrosine listed in corresponding Column D, Rows
89-93, of Table 1, comprised within the phosphorylatable peptide
sequence listed in corresponding Column E, Rows 89-93, of Table 1(SEQ
ID NOs: 88-92), wherein said antibody does not bind said protein when not
phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the GTPase
Activating or Guanine Nucleotide Exchange Factor protein when not
phosphorylated at the disclosed site (and does not bind the protein when it
is phosphoryiated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of a GTPase Activating or Guanine Nucleotide Exchange
Factor protein selected from Column A, Rows 89-93, said labeled peptide
comprising the phosphorylatable peptide sequence listed in corresponding
Column E, Rows 89-93, of Table 1(SEQ ID NOs: 88-92), which sequence
comprises the phosphorylatable tyrosine listed in corresponding Column
D, Rows 89-93, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following GTPase Activating
or Guanine Nucleotide Exchange Factor protein phosphorylation sites are
particularly preferred: RhoGAP p190B (Y1108), and ARHGEF5 (Y19) (see
SEQ ID NOs: 90-91).
In still another subset of preferred embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a Lipid Kinase selected from Column A, Rows 99-101, of Table I
only when phosphorylated at the tyrosine listed in corresponding
Column D, Rows 99-101, of Table 1, comprised within the
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phosphorylatable peptide sequence listed in corresponding Column E,
Rows 99-101 of Table 1(SEQ ID NOs: 98-100), wherein said antibody
does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Lipid Kinase
when not phosphorylated at the disclosed site (and does not bind the
protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of a Lipid Kinase selected from Column A, Rows 99-101,
said labeled peptide comprising the phosphorylatable peptide sequence
listed in corresponding Column E, Rows 99-101, of Table 1 (SEQ ID NOs:
98-100), which sequence comprises the phosphorylatable tyrosine listed in
corresponding Column D, Rows 99-101, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Lipid Kinase
phosphorylation sites are particularly preferred: P13K p85-beta (Y467,
Y605) (see SEQ ID NOs: 99-100).
In yet another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a Receptor Tyrosine Kinase ligand protein selected from Column A,
Rows 102-103, of Table I only when phosphorylated at the tyrosine listed
in corresponding Column D, Rows 102-103, of Table 1, comprised within
the phosphorylatable peptide sequence listed in corresponding Column E,
Rows 102-103, of Table 1(SEQ ID NOs: 101-102), wherein said 'antibody
does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Receptor
Tyrosine Kinase ligand protein when not phosphorylated at the disclosed
site (and does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of an EGFR-related signaling protein that is a Receptor
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Tyrosine Kinase ligand protein selected from Column A, Rows 102-103,
said labeled peptide comprising the phosphorylatable peptide sequence
listed in corresponding Column E, Rows 102-103, of Table 1(SEQ (D
NOs: 101-102), which sequence comprises the phosphorylatable tyrosine
listed in corresponding Column D, Rows 102-103, of Table 1.
In yet another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody specifically binds
a Protein Kinase (non-receptor) selected from Column A, Rows 112-118,
of Table 1 only when phosphorylated at the tyrosine listed in
corresponding Column D, Rows 112-118, of Table 1, comprised within the
phosphorylatable peptide sequence listed in corresponding Column E,
Rows 112-118, of Table 1(SEQ ID NOs: 111-117), wherein said antibody
does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Protein
Kinase when not phosphorylated at the disclosed site (and does not bind
the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of an EGFR-related signaling protein that is a Protein Kinase
(non-receptor) selected from Column A, Rows 112-118, said labeled
peptide comprising the phosphorylatable peptide sequence listed in
corresponding Column E, Rows 112-118, of Table 1(SEQ ID NOs: 111-
117), which sequence comprises the phosphorylatable tyrosine listed in
corresponding Column D, Rows 112-118, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Protein Kinase
(non-receptor) phosphorylation sites are particularly preferred: BRSK1
(Y121, Y123), MINK (Y906), FRK (Y46), and Fyn (Y212) (see SEQ ID
NOs: 111-112, and 115-117).
In yet another subset of preferred embodiments, there is provided:
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(i) An isolated phosphorylation site-specific antibody that specifically
binds a Protein Phosphatase selected from Column A, Rows 119-122, of
Table 1 only when phosphoryiated at the tyrosine listed in corresponding
Column D, Rows 119-122, of Table 1, comprised within the
phosphorylatable peptide sequence listed in corresponding Column E,
Rows 119-122, of Table 1(SEQ ID NOs: 118-121), wherein said antibody
does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Protein
Phosphatase when not phosphorylated at the disclosed site (and does not
bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of an, EGFR-related signaling protein that is a Protein
Phosphatase selected from Column A, Rows 119-122, said labeled
peptide comprising the phosphorylatable peptide sequence listed in
corresponding Column E, Rows 119-122, of Table 1(SEQ ID NOs: 118-
121), which sequence comprises the phosphorylatable tyrosine listed in
corresponding Column D, Rows 119-122, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Protein
Phosphatase phosphorylation sites are particularly preferred: PTP-kappa
(Y858) (see SEQ ID NO: 120).
In still another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a Receptor Tyrosine Kinase selected from Column A, Rows 123-
142, of Table 1 oniy when phosphorylated at the tyrosine listed in
corresponding Column D, Rows 123-142, of Table 1, comprised within the
phosphorylatable peptide sequence listed in corresponding Column E,
Rows 123-142, of Table 1(SEQ ID NOs: 122-141), wherein said antibody
does not bind said protein when not phosphorylated at said tyrosine.
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(ii) An equivalent antibody to (i) above that only binds the Receptor
Tyrosine Kinase when not phosphorylated at the disclosed site (and does
not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of an EGFR-related signaling protein that is a Receptor
Tyrosine Kinase selected from Column A, Rows 123-142, said labeled
peptide comprising the phosphorylatable peptide sequence listed in
corresponding Column E, Rows 123-142, of Table 1(SEQ ID NOs: 122-
141), which sequence comprises the phosphorylatable tyrosine listed in
corresponding Column D, Rows 123-142, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Receptor Tyrosine
Kinase phosphorylation sites are particularly preferred: EGFR (Y998),
HER2 (Y923), and HER3 (Y1307, Y1328) (see SEQ ID NOs: 122-125).
In still another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a Transcription Factor-Coactivator/Corepressor protein selected
from Column A, Rows 152-157, of Table 1 only when phosphorylated at
the tyrosine listed in corresponding Column D, Rows 152-157, of Table 1,
comprised within the phosphorylatable peptide sequence listed in
corresponding Column E, Rows 152-157, of Table 1(SEQ ID NOs: 151-
156), wherein said antibody does not bind said protein when not
phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Transcription
Factor-Coactivator/Corepressor protein when not phosphorylated at the
disclosed site (and does not bind the protein when it is phosphorylated at
the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of an EGFR-related signaling protein that is a Transcription
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Factor-Coactivator/Corepressor protein selected from Column A, Rows
152-157, said labeled peptide comprising the phosphorylatable peptide
sequence listed in corresponding Column E, Rows 152-157, of Table 1
(SEQ ID NOs: 151-156), which sequence comprises the phosphorylatable
tyrosine listed in corresponding Column D, Rows 152-157, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Transcription
Factor-Coactivator/Corepressor protein phosphorylation sites are
particularly preferred: STAT3 (Y539) (see SEQ ID NO: 153).
In still another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds a Vesicle protein selected from Column A, Rows 165-169, of Table I
only when phosphorylated at the tyrosine listed in corresponding Column
D, Rows 165-169, of Table 1, comprised within the phosphorylatable
peptide sequence listed in corresponding Column E, Rows 165-169, of
Table 1(SEQ ID NOs: 164-168), wherein said antibody does not bind said
protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds the Vesicle
protein when not phosphorylated at the disclosed site (and does not bind
the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of an EGFR-related signaling protein that is a Vesicle protein
selected from Column A, Rows 165-169, said labeled peptide comprising
the phosphorylatable peptide sequence listed in corresponding Column E,
Rows 165-169, of Table 1(SEQ ID NOs: 164-168), which sequence
comprises the phosphorylatable tyrosine listed in corresponding Column
D, Rows 165-169, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA
peptides for the detection/quantification of the following Vesicle protein
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phosphorylation sites are particularly preferred: Syntaxin 4(Y115) (see
SEQ ID NO: 168).
In yet a further subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically
binds the FAT tumor suppressor protein only when phosphorylated at
tyrosine 4244 (see Column D, Row 163 of Table 1), said tyrosine
comprised within the phosphorylatable peptide sequence listed in
corresponding Column E, Row 163 of Table 1(SEQ ID NO: 162), wherein
said antibody does not bind said protein when not phosphorylated at said
tyrosine.
(ii) An equivalent antibody to (i) above that only binds the FAT tumor
suppressor protein when not phosphorylated at the disclosed site (and
does not bind the protein when it is phosphorylated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the
quantification of the FAT tumor suppressor protein, said labeled peptide
comprising the phosphorylatable peptide sequence listed in Column E,
Row 163 of Table 1(SEQ ID NO: 162), which sequence comprises the
phosphorylatable tyrosine listed in corresponding Column D, Row 163 of
Table 1.
The invention also provides, in part, an immortalized cell line
producing an antibody of the invention, for example, a cell line producing
an antibody within any of the foregoing preferred subsets of antibodies. In
one preferred embodiment, the immortalized cell line is a rabbit hybridoma
or a mouse hybridoma.
In certain other preferred embodiments, a heavy-isotope labeled
peptide (AQUA peptide) of the invention (for example, an AQUA peptide
within an of the foregoing preferred subsets of AQUA peptides) comprises
a disclosed site sequence wherein the phosphorylatable tyrosine is
phosphorylated. In certain other preferred embodiments, a heavy-isotope
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labeled peptide of the invention comprises a disclosed site sequence
wherein the phosphorylatable tyrosine is not phosphorylated.
The foregoing subsets of preferred reagents of the invention
should not be construed as limiting the scope of the invention, which, as
noted above, includes reagents for the detection and/or quantification of
disclosed phosphorylation sites on any of the other protein type/group
subsets (each a preferred subset) listed in Column C of Table 1/Figure 2.
Also provided by the invention are' methods for detecting or
quantifying an EGFR-related signaling protein that is tyrosine-
phosphorylated, said method comprising the step of utilizing one or more
of the above-described reagents of the invention to detect or quantify one
or more EGFR-related signaling protein(s) selected from Column A of
Table 1 only when phosphorylated at the tyrosine listed in corresponding
Column D of Table 1. In certain preferred embodiments of the methods of
the invention, the reagents comprise a subset of preferred reagents as
described above.
The identification of the disclosed novel EGFR-related signaling
protein phosphorylation sites, and the standard production and use of the
reagents provided by the invention are described in further detail below
and in the Examples that follow.
AII cited references are hereby incorporated herein, in their
entirety, by reference. The Examples are provided to further illustrate the
invention, and do not in any way limit its scope, except as provided in the
claims appended hereto.
Table 1. Newly Discovered EGFR-related Phosphorylation Sites.
A B C D E G
Phospho-
1 Protein Accession Tyr
Name Number Protein Type Residue Phosphorylation Site Sequence SEQ ID NO:
Actin binding
2 afadin P55196 rotein Y1480 DLQyITVSKEELSSGDSLSPDPWKR SEQ ID NO: I
catenin, Actin binding
3 delta-1 060716 protein Y174 TVQPVAMGPDGLPVDASSVSNN IQTLGR SEQ ID NO: 2
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catenin, Actin binding
4 delta-I 060716 protein Y213 NFHYPPDGySR SEQ ID NO: 3
catenin, Actin binding
deita-1 060716 rotein Y248 YRPSMEGyR SEQ ID NO: 4
catenin, Actin binding
6 delta-I 060716 protein Y321 S EDMIGEEVPSDQYYWAPLAQHER SEQ ID NO: 5
catenin, Actin binding
7 delta-I 060716 protein Y335 SYEDMIGEEVPSDQYyWAPLAQHER SEQ ID NO: 6
Actin binding
8 Lasp-1 Q14847 protein Y122 TQDQISNIKyHEEFEK SEQ ID NO: 7
9 aveolin-1 Q03135 Adaptor/scaffold Y6 yVDSEGHLYTVPIR SEQ ID NO: 8
Cbl P22681 Adaptor/scaffold Y455 QGAEGAPSPNyDDDDDERADDTLFMMK SEQ ID NO: 9
11 DLG3 Q92796 Adaptor/scaffold Y673 RDNEVDGQDyHFVVSR SEQ ID NO: 10
AEPMPSASSAPPASSLySSPVNSSAPLAEDID
12 Hrs 014964 Adaptor/scaffold Y308 PELAR SEQ ID NO: 11
13 HSH2 Q96JZ2 Adaptor/scaffold Y265 GSQDHSGDPTSGDRGyTDPCVATSLK SEQ ID NO: 12
14 IRS-2 Q9Y4H2 Adaptor/scaffold Y653 SSSSNLGADDGyMPMTPGAALAGSGSGSCR SEQ ID
NO: 13
IRS-2 Q9Y4H2 Adaptor/scaffold Y675 SDDyMPMSPASVSAPK SEQ ID NO: 14
16 IRS-2 Q9Y4H2 Adaptor/scaffold Y823 SYKAPYTCGGDSDQyVLMSSPVGR SEQ ID NO: 15
liprin
17 beta 1 Q86W92-
iso2 2 Adaptor/scaffold Y336 KGKDGE EELLNSSSISSLLDAQGFSDLEK SEQ ID NO: 16
18 P130Cas P56945 Adaptor/scaffold Y267 GLLPSQYGQEVyDTPPMAVK SEQ ID NO: 17
19 P130Cas P56945 Adaptor/scaffold Y387 RPGPGTLyDVPR SEQ ID NO: 18
RPGRIP1 Q96KN7 Adaptor/scaffold Y864 FPVLVTSDLDHyLRR SEQ ID NO: 19
21 sciellin 095171 Adaptor/scaffold Y560 QAGPQDTVVyTR SEQ ID NO: 20
22 sciellin 095171 Adaptor/scaffold Y588 YIQTVySTSDR SEQ ID NO: 21
23 SH2D4A Q9H788 Adaptor/scaffold Y131 TKSQyHDLQAPDNQQTK SEQ ID NO: 22
24 Shb Q15464 Adaptor/scaffold Y355 AGKGESAGyMEPYEAQR SEQ ID NO: 23
Shb Q15464 Ada tor/scaffoid Y423 LPQDDDRPADEyDQPWEWNR SEQ ID NO: 24
26 STAM1 Q92783 Ada tor/scaffold Y198 QQSTTLSTLyPSTSSLLTNHQHEGR SEQ ID NO: 25
27 STAM2 075886 Adaptor/scaffold Y192 SLyPSSEIQLNNK SEQ ID NO: 26
28 s ntenin 000560 Adaptor/scaffold Y46 VIQAQTAFSANPANPAILSEASAPIPHDGNL PR SEQ
ID NO: 27
29 TEM6 Q81ZW7 Adaptor/scaffold Y333 WDSyENLSADGEVLHTQGPVDGSLYAK SEQ ID NO: 28
TEM6 Q81ZW7 Ada tor/scaffold Y354 WDSYENLSADGEVLHTQGPVDGSLyAK SEQ ID NO: 29
31 TEM6 Q8IZW7 Adaptor/scaffold Y584 KPSVSAQMQAYGQSSySTQTWVR SEQ ID NO: 30
32 TEM6 Q8IZW7 Adaptor/scaffold Y601 QQQMVVAHQySFAPDGEAR SEQ ID NO: 31
33 TEM6 Q8IZW7 Ada tor/scaffold Y780 KLSLGQyDNDAGGQLPFSK SEQ ID NO: 32
34 TEM6 Q8iZW7 Adaptor/scaffold Y802 AGVDyAPNLPPFPSPADVK SEQ ID NO: 33
ZO1 Q07157 Adaptor/scaffold Y1054 YESSSyTDQFSR SEQ ID NO: 34
36 ZOI Q07157 Adaptor/scaffold Y1343 SNHYDPEEDEEYyRK SEQ ID NO: 35
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37 Z02 Q9UDY2 Adaptor/scaffold Y911 MSYLTAMGAD LSCDSR SEQ ID NO: 36
Adaptor/scaffold;
38 Spry3 043610 Inhibitor protein Y27 STHASNDyVERPPAPCK SEQ ID NO: 37
39 DCBLD2 Q96PD2 Adhesion Y621 EVTTVLQADSAEyAQPLVGGIVGTLHQR SEQ ID NO: 38
40 DCBLD2 Q96PD2 Adhesion Y750 AGKPGLPAPDELV QVPQSTQEVSGAGR SEQ ID NO: 39
41 Erbin Q96RT1 Adhesion Y884 SHSITNMEIGGLKI DILSDNGPQQPSTTVK SEQ ID NO: 40
42 mucin 1 P15941 Adhesion Y1209 DTYHPMSE PTYHTHGR SEQ ID NO: 41
43 mucin 1 P15941 Adhesion Y1212 DTYHPMSEYPT HTHGR SEQ ID NO: 42
44 mucin 1 P15941 Adhesion Y1243 VSAGNGGSSLSyTNPAVAATSANL SEQ ID NO: 43
45 nectin I Q15223 Adhesion Y468 YDEDAKRPyFTVDEAEAR SEQ ID NO: 44
Plako-
46 philin 2 Q99959 Adhesion Y166 AHYTHSDyQYSQR SEQ ID NO: 45
Plako-
47 philin 2 Q99959 Adhesion Y168 AHYTHSDYQySQR SEQ ID NO: 46
Plako-
48 philin 2 Q99959 Adhesion Y845 AASVLLySLWAHTELHHAYKKAQFK SEQ ID NO: 47
Plako-
49 philin 3 Q9Y446 Adhesion Y176 AD DTLSLR SEQ ID NO: 48
Plako-
50 philin 3 Q9Y446 Adhesion Y195 LGPGGLDDRySLVSEQLEPAATSTYR SEQ ID NO: 49
Plako-
51 philin 4 Q99569 Adhesion Y1168 STTNyVDFYSTK SEQ ID NO: 50
Plako-
52 philin 4 Q99569 Adhesion Y157 SSTQMNSYSDSGyQEAGSFHNSQNVSK SEQ ID NO: 51
Plako-
53 philin 4 Q99569 Adhesion Y372 TVHDMEQFGQQQYDIyER SEQ ID NO: 52
Plako-
54 philin 4 Q99569 Adhesion Y470 NNyALNTTATYAEPYRPIQYR SEQ ID NO: 53
55 URP2 Q86UX7 Adhesion Yll TASGDyIDSSWELR SEQ ID NO: 54
56 zyxin Q15942 Adhesion Y316 LGHPEALSAGTGSPQPPSFTyAQQR SEQ ID NO: 55
Adhesion;
57 Desmo- Calcium-binding
glein 2 Q14126 protein Y1012 VIQPHGGGSNPLEGTQHLQDVPyVMVR SEQ ID NO: 56
58 Alix Q8WUM4 Apoptosis Y727 EPSAPSIPTPAyQSSPAGGHAPTPPTPAPR SEQ ID NO: 57
Axon guidance;
59 SLITRK6 Q9H5Y7 Cell surface Y814 ANLHAEPDyLEVLEQQT SEQ ID NO: 58
annexin Calcium-binding
60 A2 P07355 protein Y29 AyTNFDAERDALNIETAIK SEQ ID NO: 59
annexin Calcium-binding
61 A4 P09525 protein Y164 VLVSLSAGGRDEGNyLDDALVR SEQ ID NO: 60
Ov/Br Cell cycle
62 septin Q9Y5W4 regulation Y260 NEKAPVDFGyVGIDSILEQMR SEQ ID NO: 61
63 CDCPI Q96QU7 Cell surface Y806 LATEEPPPRSPPESESEPyTFSHPNNGDVSSK SEQ ID NO:
62
64 TSRC1 Q6UY14 Cell surface Y159 SRLRDPIKPGMFGyGR SEQ ID NO: 63
Chaperone;
65 Cytoskeletal
TBCB Q99426 protein Y239 YGAFVKPAVVTVGDFPEEDyGLDEI SEQ ID NO: 64
actinin, Cytoskeletal
66 alpha 1 P12814 protein Y246 AIMTYVSSFyHAFSGAQK SEQ ID NO: 65
Cytoskeletal
67 claudin 3 015551 protein Y214 STGPGASLGTGyDR SEQ ID NO: 66
Cytoskeletal
68 claudin 4 014493 protein Y208 SAAASN V SEQ ID NO: 67
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Cyto- Cytoskeletal
69 keratin 18 P05783 protein Y23 SLGSVQAPSyGARPVSSAASVYAGAGGSGSR SEQ ID NO: 68
Cyto- Cytoskeletal
70 keratin 19 P08727 protein Y391 SLLEGQEDH NNLSASK SEQ ID NO: 69
Cyto- Cytoskeletal
71 keratin 7 P08729 protein Y39 LSSARPGGLGSSSLyGLGASRPR SEQ ID NO: 70
Cyto- Cytoskeletal TTSGYAGGLSSAYGGLTSPGLSySLGSSFGS
72 keratin 8 P05787 protein Y436 GAGSSSFSR SEQ ID NO: 71
EHM2 Cytoskeletal
73 iso2 Q9H329-2 protein Y479 ASASGDDSHFDyVHDQNQK SEQ ID NO: 72
Cytoskeletal
74 ELMO2 Q96JJ3 protein Y49 EVCDGWSLPNPEYyTLR SEQ ID NO: 73
keratin,
75 hair, Cytoskeletal
basic I Q14533 protein Y282 AQ DDIVTR SEQ ID NO: 74
Cytoskeletal
76 plectin 1 Q15149 protein Y4611 GyYSPYSVSGSGSTAGSR SEQ ID NO: 75
SM22- Cytoskeletal
77 alpha P37802 protein Y192 GASQAGMTGyGMPR SEQ ID NO: 76
Cytoskeletal
78 talin I Q9Y490 protein Y26 TMQFEPSTMVyDACR SEQ ID NO: 77
Cytoskeletal
79 protein; Actin
cortactin Q14247 binding protein Y154 HASQKDySSGFGGK SEQ ID NO: 78
Cytoskeletal
80 Desmo- protein;
plakin 3 P14923 Adhesion Y19 VTEWQQT TYDSGIHSGANTCVPSVSSK SEQ ID NO: 79
Cytoskeletal
81 Desmo- protein;
plakin 3 P14923 Adhesion Y73 KTTTYTQGVPPSQGDLEyQMSTTAR SEQ ID NO: 80
DNA binding
82 ZFP42 Q8WXE2 protein Y146 ELPQKIVGENSLEYSEyMTGK SEQ ID NO: 81
DNA binding
83 ZNF185 015231 protein Y349 GILFVKEyVNASEVSSGKPVSAR SEQ ID NO: 82
84 Nuf2 Q9BZD4 DNA replication Y433 TALEKyHDGIEKAAEDSYAKIDEKTAELK SEQ ID NO:
83
85 Smc5 Q96SB9 DNA re lication Y246 yKQDVERFYERK SEQ ID NO: 84
Enzyme, cellular
86 G6PD P11413 metabolism Y111 NSYVAGQyDDAASYQR SEQ ID NO: 85
Enzyme, cellular
87 G6PD P11413 metabolism Y502 RVGFQyEGTYK SEQ ID NO: 86
Enzyme, cellular
88 G6PD P11413 metabolism Y506 VGFQYEGTyK SEQ ID NO: 87
GTPase
89 activating
MIG-6 Q9UJM3 rotein, Rac/Rho Y394 KVSSTHyYLLPERPPYLDKYEK SEQ ID NO: 88
GTPase
90 activating
MIG-6 Q9UJM3 protein, Rac/Rho Y395 VSSTHYyLLPERPPYLDKYEK SEQ ID NO: 89
GTPase
91 RhoGAP activating
190B Q13017 protein, Rac/Rho Y1108 GYSDEIyVVPDDSQNR SEQ ID NO: 90
Guanine nucleo-
92 ARHGE tide exchange
F5 Q12774 factor, Rac/Rho Y19 LINSSQLLyQEYSDVVLNK SEQ ID NO: 91
Guanine nucleo-
93 tide exchange
BCAR3 075815 factor, Ras Y266 CLEEHyGTSPGQAR SEQ ID NO: 92
94 BST1 Q10588 Hydrolase Y134 FMPLSDVLyGRVADFLSWCR SEQ ID NO: 93
Na,K- Hydrolase, non-
95 ATPase 1 P05023 esterase Y260 GIVVyTGDRTVMGR SEQ ID NO: 94
Immunoglobulin GPASDYGPEPTPPGPAAPAGTDTTSQLSyEN
96 NEPH1 Q7Z696 su erfamil Y520 YEK SEQ ID NO: 95
lmmunoglobulin GPASDYGPEPTPPGPAAPAGTDTTSQLSYEN
97 NEPHI Q7Z696 superfamily Y523 yEK SEQ ID NO: 96
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Immunoglobulin
98 SLAMF7 Q9NY08 superfamily Y284 ETPNICPHSGENTEyDTIPHTNR SEQ ID NO: 97
P13K
99 C2beta 000750 Kinase, lipid Y228 LLGSVDyDGINDAITR SEQ ID NO: 98
P13K p85-
100 beta 000459 Kinase, lipid Y467 SREYDQLYEEyTR SEQ ID NO: 99
P13K p85-
101 beta 000459 Kinase, lipid Y605 NETEDQyALMEDEDDLPHHEER SEQ ID NO: 100
Ligand, receptor
102 e hrin-B1 P98172 tyrosine kinase Y313 TTENN CPHYEK SEQ ID NO: 101
Ligand, receptor
103 e hrin-B1 P98172 tyrosine kinase Y317 TTENNYCPHyEK SEQ ID NO: 102
Lipid binding
104 PLEKHA5 Q9HAUO rotein Y366 LNSLPSEYESGSACPAQTVH RPINLSSSENK SEQ ID NO: 103
Lipid binding
105 PLEKHA5 Q9HAUO protein Y436 GVISyQTLPR SEQ ID NO: 104
Lipid binding
106 PLEKHA6 Q9Y2H5 protein Y493 SEDI ADPAAYVMR SEQ ID NO: 105
107 Myosin VI Q9UM54 Motor protein Y1114 SVTD DFAPFLNNSPQQNPAAQIPAR SEQ ID NO:
106
108 Myosin VI Q9UM54 Motor protein Y1159 IPFIRPADQyKDPQSK SEQ ID NO: 107
109 ARL-1 060218 Oxidoreductase Y315 ACNVLQSSHLEDYPFDAEy SEQ ID NO: 108
meltrin Protease (non-
110 gamma Q13443 proteasomal) Y815 VSSQGNLIPARPAPAPPLySSLT SEQ ID NO: 109
Protease (non-
111 USP34 Q70CQ2 proteasomal) Y1288 LL ALEIIEALGKPNR SEQ ID NO: 110
Protein kinase,
112 Ser/Thr (non-
BRSK1 Q8TDC3 rece tor Y121 K LYLVLEHVSGGELFDYLVKK SEQ ID NO: 111
Protein kinase,
113 Ser/Thr (non-
BRSKI Q8TDC3 rece tor Y123 KYLyLVLEHVSGGELFDYLVKK SEQ ID NO: 112
Protein kinase,
114 p38- Ser/Thr (non-
delta 015264 rece tor Y182 HADAEMTGyVVTR SEQ ID NO: 113
Protein kinase,
115 Ser/Thr (non-
STLK3 Q9UEW8 receptor) Y65 DAyELQEVIGSGATAVVQAALCKPRQER SEQ ID NO: 114
Protein kinase,
116 Ser/Thr (non-
MINK Q8N4C8 receptor) Y906 NLLHADSNG TNLPDVVQPSHSPTENSK SEQ ID NO: 115
Protein kinase,
117 tyrosine (non-
FRK P42685 receptor) Y46 HGHyFVALFDYQAR SEQ ID NO: 116
Protein kinase,
118 tyrosine (non-
F n P06241 receptor) Y212 KLDNGGyYITTR SEQ ID NO: 117
Protein
119 phosphatase, IQNTGDYyDLYGGEK
tyrosine (non-
SHP-2 Q06124 rece tor Y63 SEQ ID NO: 118
Protein
120 acid phosphatase,
phospha tyrosine (non-
tase 1 P24666 receptor ; Y131 QLIIEDPyYGNDSDFETVYQQCVR SEQ ID NO: 119
Receptor protein
121 PTP- phosphatase,
kappa Q15262 tyrosine Y858 YLCEGTESPyQTGQLHPAIR SEQ ID NO: 120
similar Receptor protein
122 to phosphatase, AKVKKLTLGMDyMFQVKKVKGKGYSVSVMK
PTPRQ XP 291991 tyrosine Y630 KVPIK SEQ ID NO: 121
Receptor
123 EGFR P00533 tyrosine kinase Y998 MHLPSPTDSNFyR SEQ ID NO: 122
Receptor
124 HER2 P04626 tyrosine kinase Y923 FTHQSDVWSYGVTVWELMTFGAKP DGIPAR SEQ ID
NO: 123
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Receptor
125 HER3 P21860 tyrosine kinase Y1307 AFQGPGHQAPHVHyAR SEQ ID NO: 124
Receptor
126 HER3 P21860 tyrosine kinase Y1328 SLEATDSAFDNPDyWHSR SEQ ID NO: 125
Receptor
127 EphA2 P29317 tyrosine kinase Y575 QSPEDVyFSKSEQLKPLK SEQ ID NO: 126
Receptor
128 E hA2 P29317 t rosine kinase Y588 SEQLKPLKT VDPHTYEDPNQAVLK SEQ ID NO: 127
Receptor SEQLKPLKTYVDPHTyEDPNQAVLK
129 E hA2 P29317 t rosine kinase Y594 SEQ ID NO: 128
Receptor
130 EphA4 P54764 t rosine kinase Y596 TyVDPFTYEDPNQAVR SEQ ID NO: 129
Receptor
131 EphA4 P54764 tyrosine kinase Y602 TYVDPFTyEDPNQAVR SEQ ID NO: 130
Receptor
132 E hA5 P54756 tyrosine kinase Y650 T IDPHTYEDPNQAVHEFAK SEQ ID NO: 131
Receptor
133 EphA5 P54756 t rosine kinase Y656 TYIDPHTyEDPNQAVHEFAK SEQ ID NO: 132
Receptor
134 EphA7 Q15375 tyrosine kinase Y614 TYIDPETyEDPNR SEQ ID NO: 133
1 Receptor
135 EphB3 P54753 tyrosine kinase Y608 LQQYIAPGMKV IDPFTYEDPNEAVR SEQ ID NO:
134
Receptor
136 E hB3 P54753 tyrosine kinase Y792 FLEDDPSDPTyTSSLGGK SEQ ID NO: 135
Receptor
137 EphB4 P54760 tyrosine kinase Y574 EAEySDKHGQYLIGHGTK SEQ ID NO: 136
Receptor
138 EphB4 P54760 t rosine kinase Y590 HGQYLIGHGTKV IDPFTYEDPNEAVR SEQ ID NO:
137
Receptor
139 EphB4 P54760 t rosine kinase Y596 HGQYLIGHGTKVYIDPFTyEDPNEAVR SEQ ID NO:
138
Receptor
140 EphB4 P54760 tyrosine kinase Y774 FLEENSSDPTyTSSLGGK SEQ ID NO: 139
Receptor
141 E hB4 P54760 tyrosine kinase Y987 SQAKPGTPGGTGGPAPQy SEQ ID NO: 140
Receptor
142 Ron Q04912 tyrosine kinase Y1238 DILDREyYSVQQHR SEQ ID NO: 141
AYSQEEITQGFEETGDTLyAPYSTHFQLQNQ
143 RAIG1 095357 Receptor, GPCR Y317 PPQK SEQ ID NO: 142
AYSQEEITQGFEETGDTLYAPySTHFQLQNQ
144 RAIG1 095357 Receptor, GPCR Y320 PPQK SEQ ID NO: 143
145 LRP6 075581 Receptor, misc. Y1577 SQyLSAEENYESCPPSPYTER SEQ ID NO: 144
integrin Receptor, misc.;
146 beta-4 P16144 Adhesion Y1207 VCAYGAQGEGP SSLVSCR SEQ ID NO: 145
Receptor, misc.;
147 Cell surface;
DNA binding
APPL2 Q06481 rotein Y750 MQNHGyENPTYK SEQ ID NO: 146
Receptor, misc.;
148 Transcription
factor; Cell
APP P05067 surface; Y762 MQQNGYENPTyK SEQ ID NO: 147
hnRNP RNA binding
149 A2/B1 P22626 protein Y336 NMGGPYGGGNyGPGGSGGSGGYGGR SEQ ID NO: 148
RNA binding
150 hnRNP G P38159 protein Y214 DDGySTKDSYSSR SEQ ID NO: 149
RNA binding AGLQTADKyAALANLDNIFSAGQGGDQGSGF
151 RIP P52594 protein Y327 GTTGK SEQ ID NO: 150
Transcription
152 LISCH Q86X29 factor Y324 SSSAGGQGSyVPLLR SEQ ID NO: 151
Transcription
153 LISCH Q86X29 factor Y503 SRDPHyDDFR SEQ ID NO: 152
Transcription
154 STAT3 Q9BW54 factor Y539 LLGPGVN SGCQITWAK SEQ ID NO: 153
Transcription
155 TRIM29 Q14134 factor Y93 NSNyFSMDSMEGKR SEQ ID NO: 154
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Transcription
156 ZIM3 Q96PE6 factor Y251 QKSNLFQHQKMHTKEKPyQCKTCGK SEQ ID NO: 155
Transcription,
157 coactivator/corep
TRIP6 Q15654 ressor Y123 QAyEPPPPPAYR SEQ ID NO: 156
158 HNK1ST 043529 Transferase Y305 EAGIDHLVSyPTIPPGITVYNRTK SEQ ID NO: 157
159 ABCAIO Q7Z219 Trans orter, ABC Y46 YHEMVGVIFSDTFSyRLKFNWGYR SEQ ID NO: 158
160 ABCA10 Q7Z219 Transporter, ABC Y54 YHEMVGVIFSDTFSYRLKFNWGyR SEQ ID NO: 159
161 ABCB4 P21439 Trans orter, ABC Y279 ELERyQKHLENAKEIGIKK SEQ ID NO: 160
Transporter,
162 facilitator;
SLC20A2 Q08357 Rece tor, misc. Y377 IHIDRGPEEKPAQESNyR SEQ ID NO: 161
Tumor
163 FAT Q14517 suppressor Y4244 NI SDIPPQVPVRPISYTPSIPSDSR SEQ ID NO: 162
Ubiquitin
164 UBCE71 conjugating
P3 Q9BYM8 system Y320 NSQEAEVSCPFIDNTySCSGK SEQ ID NO: 163
165 epsin 2 095208 Vesicle protein Y186 GSSQPNLSTSHSEQE GK SEQ ID NO: 164
epsin 2
166 iso2 095208-2 Vesicle protein Y196 AGGSPASyHGSTSPR SEQ ID NO: 165
167 SCAMPI 015126 Vesicle protein Y73 MPNVPNTQPAIMKPTEEHPA TQIAK SEQ ID NO:
166
168 SCAMP3 014828 Vesicle protein Y83 NyGSYSTQASAAAATAELLK SEQ ID NO: 167
169 s ntaxi14 Q12846 Vesicle protein Y115 AIEPQKEEADENyNSVNTR SEQ ID NO: 168
The short name for each protein in which a phosphorylation site
has presently been identified is provided in Column A, and its SwissProt
accession number (human) is provided Column B. The protein
type/group into which each protein falls is provided in Column C. The
identified tyrosine residue at which phosphorylation occurs in a given
protein is identified in Column D, and the amino acid sequence of the
phosphorylation site encompassing the tyrosine residue is provided in
Column E (lower case y = the tyrosine (identified in Column D)) at which
phosphorylation occurs. Table I above is identical to Figure 2, except that
the latter includes the cell type(s) in which the particular phosphorylation
site was identified (Column F).
The identification of these 168 phosphorylation sites is described in
more detail in Part A below and in Example 1.
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Definitions.
As used herein, the following terms have the meanings indicated:
"Antibody" or "antibodies" refers to all types of immunoglobulins,
including IgG, IgM, IgA, IgD, and IgE, including Fab or antigen-recognition
fragments thereof, including chimeric, polyclonal, and monoclonal
antibodies. The term "does not bind" with respect to an antibody's binding
to one phospho-form of a sequence means does not substantially react
with as compared to the antibody's binding to the other phospho-form of
the sequence for which the antibody is specific.
"EGFR-related signaling protein" means any protein (or poly-
peptide derived therefrom) enumerated in Column A of Table 1/Figure 2,
which is disclosed herein as being phosphorylated in one or more EGFR-
activated cell line(s). EGFR-related signaling proteins may be direct
substrates of EGFR, or may be indirect substrates downstream of EGFR
in signaling pathways, or may be EGFR itself. An EGFR-related signaling
protein may also be phosphorylated in other cell lines harboring activated
kinase activity.
"Heavy-isotope labeled peptide" (used interchangeabiy with AQUA
peptide) means a peptide comprising at least one heavy-isotope label,
which is suitable for absolute quantification or detection of a protein as
described in WO/03016861, "Absolute Quantification of Proteins and
Modified Forms Thereof by Multistage Mass Spectrometry" (Gygi et a/.),
further discussed below.
"Protein" is used interchangeably with polypeptide, and includes
protein fragments and domains as well as whole protein.
"Phosphorylatable amino acid" means any amino acid that is
capable of being modified by addition of a phosphate group, and includes
both forms of such amino acid.
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"Phosphorylatable peptide sequence" means a peptide sequence
comprising a phosphorylatable amino acid.
"Phosphorylation site-specific antibody" means an antibody that
specifically binds a phosphorylatable peptide sequence/epitope only when
phosphorylated, or only when not phosphorylated, respectively. The term
is used interchangeably with "phospho-specific" antibody.
A. Identification of Novel EGFR-related Phosphorylation Sites.
The 168 novel EGFR-related signaling protein phosphorylation
sites disclosed herein and listed in Table 1/Figure 2 were discovered by
employing the modified peptide isolation and characterization techniques
described in described in "Immunoaffinity Isolation of Modified Peptides
From Complex Mixtures," U.S. Patent Publication No. 20030044848,
Rush et al. (the teaching of which is hereby incorporated herein by
reference, in its entirety) using cellular extracts from the following stably-
transfected cell lines expressing EGFR and stimulated with EGF: A431,
HCT116, HPAC, MIAPACA2, PANC-1, A549, BxPC-3, DU145, HT-29,
H460, and LNCaP. These cell lines are human carcinoma tumor cell
lines. The isolation and identification of phosphopeptides from these
EGFR-activated cell lines, using an immobilized general phosphotyrosine-
specific antibody, is described in detail in Example I below. In addition to
the 168 previously unknown protein phosphorylation sites discovered,
many known phosphorylation sites were also identified (not described
herein). The immunoaffinity/mass spectrometric technique described in
the 848 Patent Publication (the "IAP" method) -- and employed as
described in detail in the Examples -- is briefly summarized below.
The IAP method employed generally comprises the following
steps: (a) a proteinaceous preparation (e.g. a digested cell extract)
comprising phosphopeptides from two or more different proteins is
obtained from an organism; (b) the preparation is contacted with at least
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one immobilized general phosphotyrosine-specific antibody; (c) at least
one phosphopeptide specifically bound by the immobilized antibody in
step (b) is isolated; and (d) the modified peptide isolated in step (c) is
characterized by mass spectrometry (MS) and/or tandem mass
spectrometry (MS-MS). Subsequently, (e) a search program (e.g.
Sequest) may be utilized to substantially match the spectra obtained for
the isolated, modified peptide during the characterization of step (d) with
the spectra for a known peptide sequence. A quantification step
employing, e.g. SILAC or AQUA, may also be employed to quantify
isolated peptides in order to compare peptide levels in a sample to a
baseline.
In the IAP method as employed herein, a general phosphotyrosine-
specific monoclonal antibody (commercially available from Cell Signaling
Technology, Inc., Beverly, MA, Cat #9411 (p-Tyr-100)) was used in the
immunoaffinity step to isolate the widest possible number of phospho-
tyrosine containing peptides from the EGFR-activated cell extracts.
Extracts from the following EGFR-activated, human carcinoma
tumor cells lines (carcinoma type indicated in brackets) were employed:
A431 (skin), HCT116 (colon), HPAC (pancreas), MIAPACA2 (pancreas),
PANC-1 (pancreas), A549 (lung), BxPC-3 (pancreas), DU145 (prostate),
HT-29 (colon), H460 (lung), and LNCaP (prostate). Each of these cell
lines expresses EGFR, which has been activated by stimulation with
EGF, thus signaling pathways and proteins downstream of EGFR are
affected.
As described in more detail in the Examples, lysates were
prepared from these cells line and digested with trypsin after treatment
with DTT and iodoacetamide to alkylate cysteine residues. Before the
immunoaffinity step, peptides were pre-fractionated by reversed-phase
solid phase extraction using Sep-Pak C1$ columns to separate peptides
from other cellular components. The solid phase extraction cartridges
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were eluted with varying steps of acetonitrile. Each lyophilized peptide
fraction was redissolved in PBS and treated with phosphotyrosine
antibody (P-Tyr-1 00, CST #9411) immobilized on protein G-Sepharose.
lmmunoaffinity-purified peptides were eluted with 0.1 % TFA and a portion
of this fraction was concentrated with Stage tips and analyzed by LC-
MS/MS, using a ThermoFinnigan LCQ Deca XP Plus ion trap mass
spectrometer. Peptides were eluted from a 10 cm x 75 pm reversed-
phase column with a 45-min linear gradient of acetonitrile. MS/MS
spectra were evaluated using the program Sequest with the NCBI human
protein database.
This revealed a total of 168 novel tyrosine phosphoryiation sites in
signaling pathways affected by EGFR activation, including one novel site
on EGFR itself. The identified phosphorylation sites and their parent
proteins are enumerated in Table 1/Figure 2. The tyrosine (human
sequence) at which phosphorylation occurs is provided in Column D, and
the peptide sequence encompassing the phosphorylatable tyrosine
residue at the site is provided in Column E. Figure 2 also shows the
particular cell line(s) in which a particular phosphorylation site was
discovered (see Column F).
As a result of the discovery of these phosphorylation sites,
phospho-specific antibodies and AQUA peptides for the detection of and
quantification of these sites and their parent proteins may now be
produced by standard methods, described below. These new reagents
will prove highly useful in, e.g., studying the signaling pathways and
events underlying the progression of EGFR-mediated cancers and the
identification of new biomarkers and targets for diagnosis and treatment
of such diseases.
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B. Antibodies and Cell Lines
Isolated phosphorylation site-specific antibodies that specifically
bind an EGFR-related signaling protein disclosed in Column A of Table 1
only when phosphorylated (or only when not phosphorylated) at the
corresponding amino acid and phosphorylation site listed in Columns D
and E of Table 1 may now be produced by standard antibody production
methods, such as anti-peptide antibody methods, using the
phosphorylation site sequence information provided in Column E of
Table 1. For example, two previously unknown HER3 kinase
phosphorylation sites (tyrosines 1307 and 1328) (see Rows 125-126 of
Table 1/Fig. 2) are presently disclosed. Thus, antibodies that specifically
bind any either of these novel HER3 sites can now be produced by
immunizing an animal with a peptide antigen comprising all or part of the
amino acid sequence encompassing the respective phosphorylated
residue (e.g. a peptide antigen comprising the sequence set forth in Row
126, Column E, of Table 1(SEQ ID NO: 125) (which encompasses the
phosphorylated tyrosine at position 1328 in HER3), to produce an
antibody that oniy binds HER3 when phosphorylated at that site.
Polyclonal antibodies of the invention may be produced according
to standard techniques by immunizing a suitable animal (e.g., rabbit, goat,
etc.) with a peptide antigen corresponding to the EGFR-related
phosphorylation site of interest (i.e. a phosphorylation site enumerated in
Column E of Table 1, which comprises the corresponding
phosphorylatable amino acid listed in Column D of Table 1), collecting
immune serum from the animal, and separating the polyclonal antibodies
from the immune serum, in accordance with known procedures. For
example, a peptide antigen comprising the novel EGFR phosphorylation
site disclosed herein (SEQ ID NO: 122 = SPTDSNFyRALMDEE,
encompassing phosphorylated tyrosine 998 (see Row 123 of Table 1))
may be used to produce antibodies that only bind EGFR when
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phosphorylated at Tyr998. Similarly, a peptide comprising any of the
phosphorylation site sequences provided in Column E of Table 1 may
employed as an antigen to produce an antibody that only binds the '
corresponding protein listed in Column A of Table I when phosphorylated
(or when not phosphorylated) at the corresponding residue listed in
Column D. If an antibody that only binds the protein when
phosphorylated at the disclosed site is desired, the peptide antigen
includes the phosphorylated form of the amino acid. Conversely, if an
antibody that only binds the protein when not phosphorylated at the
disclosed site is desired, the peptide antigen includes the non-
phosphorylated form of the amino acid.
Peptide antigens suitable for producing antibodies of the invention
may be designed, constructed and employed in accordance with well-
known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL,
Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory
(1988); Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield,
J. Am. Chem. Soc. 85:21-49 (1962)).
It will be appreciated by those of skill in the art that longer or
shorter phosphopeptide antigens may be employed. See Id. For
example, a peptide antigen may consist of the full sequence disclosed in
Column E of Table 1, or it may comprise additional amino acids flanking
such disclosed sequence, or may comprise of oniy a portion of the
disclosed sequence immediately flanking the phosphorylatable amino
acid (indicated in Column E by lowercase "y"). Polyclonal antibodies
produced as described herein may be screened as further described
below.
Monoclonal antibodies of the invention may be produced in a
hybridoma cell line according to the well-known technique of Kohler and
Milstein. See Nature 265:495-97 (1975); Kohler and Milstein, Eur. J.
Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR
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BIOLOGY, Ausubel et al. Eds. (1989). Monoclonal antibodies so produced
are highly specific, and improve the selectivity and specificity of diagnostic
assay methods provided by the invention. For example, a solution
containing the appropriate antigen may be injected into a mouse or other
species and, after a sufficient time (in keeping with conventional
techniques), the animal is sacrificed and spleen cells obtained. The
spleen cells are then immortalized by fusing them with myeloma cells,
typically in the presence of polyethylene glycol, to produce hybridoma
cells. Rabbit fusion hybridomas, for example, may be produced as
described in U.S Patent No. 5,675,063, C. Knight, Issued October 7,
1997. The hybridoma cells are then grown in a suitable selection media,
such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant
screened for monoclonal antibodies having the desired specificity, as
described below. The secreted antibody may be recovered from tissue
culture supernatant by conventional methods such as precipitation, ion
exchange or affinity chromatography, or the like.
Monoclonal Fab fragments may also be produced in Escherichia
coli by recombinant techniques known to those skilled in the art. See,
e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'I
Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are
preferred for a particular application, particular isotypes can be prepared
directly, by selecting from the initial fusion, or prepared secondarily, from
a parental hybridoma secreting a monoclonal antibody of different isotype
by using the sib selection technique to isolate class-switch variants
(Steplewski, et al., Proc. Nat'1. Acad. Sci., 82: 8653 (1985); Spira et al.,
J.
Immunol. Methods, 74: 307 (1984)).
The preferred epitope of a phosphorylation-site specific antibody of
the invention is a peptide fragment consisting essentially of about 8 to 17
amino acids including the phosphorylatable tyrosine, wherein about 3 to 8
amino acids are positioned on each side of the phosphorylatable tyrosine
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(for example, the p-38-deita kinase tyrosine 182 phosphorylation site
sequence disclosed in Row 114, Column E of Table 1), and antibodies of
the invention thus specifically bind a target EGFR-related polypeptide
comprising such epitopic sequence. Particularly preferred epitopes
bound by the antibodies of the invention comprise all or part of a
phosphorylatable site sequence listed in Column E of Table 1, including
the phosphorylatable amino acid.
Included in the scope of the invention are equivalent non-antibody
molecules, such as protein binding domains or nucleic acid aptamers,
which bind, in a phospho-specific manner, to essentially the same
phosphorylatable epitope to which the phospho-specific antibodies of the
invention bind. See, e.g., Neuberger et al., Nature 312: 604 (1984). Such
equivalent non-antibody reagents may be suitably employed in the
methods of the invention further described below.
Antibodies provided by the invention may be any type of
immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including Fab or
antigen-recognition fragments thereof. The antibodies may be
monoclonal or polyclonal and may be of any species of origin, including
(for example) mouse, rat, rabbit, horse, or human, or may be chimeric
antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26:403-11
(1989); Morrision et al., Proc. Nat'1. Acad. Sci. 81: 6851 (1984);
Neuberger et al., Nature 312: 604 (1984)). The antibodies may be
recombinant monoclonal antibodies produced according to the methods
disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567
(Cabilly et al.) The antibodies may also be chemically constructed by
specific antibodies made according to the method disclosed in U.S. Pat.
No. 4,676,980 (Segel et al.)
The invention also provides immortalized cell lines that produce an
antibody of the invention. For example, hybridoma clones, constructed as
described above, that produce monoclonal antibodies to the EGFR-
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related signaling protein phosphoryiation sties disclosed herein are also
provided. Similarly, the invention includes recombinant cells producing an
antibody of the invention, which cells may be constructed by well known
techniques; for example the antigen combining site of the monoclonal
antibody can be cloned by PCR and single-chain antibodies produced as
phage-displayed recombinant antibodies or soluble antibodies in E. coli
(see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press,
Sudhir Paul editor.)
Phosphorylation site-specific antibodies of the invention, whether
polyclonal or monoclonal, may be screened for epitope and phospho-
specificity according to standard techniques. See, e.g. Czernik et al.,,
Methods in Enzymology, 201: 264-283 (1991). For example, the
antibodies may be screened against the phospho and non-phospho
peptide library by ELISA to ensure specificity for both the desired antigen
(i.e. that epitope including a phosphorylation site sequence enumerated in
Column E of Table 1) and for reactivity only with the phosphorylated (or
non-phosphorylated) form of the antigen. Peptide competition assays
may be carried out to confirm lack of reactivity with other phospho-
epitopes on the given EGFR-related signaling protein. The antibodies
may also be tested by Western blotting against cell preparations
containing the signaling protein, e.g. cell lines over-expressing the target
protein, to confirm reactivity with the desired phosphorylated
epitope/target.
Specificity against the desired phosphorylated epitope may also be
examined by constructing mutants lacking phosphorylatable residues at
positions outside the desired epitope that are known to be
phosphorylated, or by mutating the desired phospho-epitope and
confirming lack of reactivity. Phosphorylation-site specific antibodies of
the invention may exhibit some limited cross-reactivity related epitopes in
non-target proteins. This is not unexpected as most antibodies exhibit
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some degree of cross-reactivity, and anti-peptide antibodies will often
cross-react with epitopes having high homology to the immunizing
peptide. See, e.g., Czernik, supra. Cross-reactivity with non-target
proteins is readily characterized by Western blotting alongside markers of
known molecular weight. Amino acid sequences of cross-reacting
proteins may be examined to identify sites highly homologous to the
EGFR-related signaling protein epitope for which the antibody of the
invention is specific.
In certain cases, polyclonal antisera may be exhibit some
undesirable general cross-reactivity to phosphotyrosine, which may be
removed by further purification of antisera, e.g. over a phosphotyramine
column. Antibodies of the invention specifically bind their target protein
(i.e. a protein listed in Column A of Table 1) only when phosphorylated (or
only when not phosphoryiated, as the case may be) at the site disclosed
in corresponding Columns D/E, and do not (substantially) bind to the
other form (as compared to the form for which the antibody is specific).
Antibodies may be further characterized via immunohistochemical
(IHC) staining using normal and diseased tissues to examine EGFR-
related phosphorylation and activation status in diseased tissue. IHC may
be carried out according to well-known techniques. See, e.g., ANTIBODIES:
A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring
Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g. tumor
tissue) is prepared for immunohistochemical staining by deparaffinizing
tissue sections with xylene followed by ethanol; hydrating in water then
PBS; unmasking antigen by heating slide in sodium citrate buffer;
incubating sections in hydrogen peroxide; blocking in blocking solution;
incubating siide in primary antibody and secondary antibody; and finally
detecting using ABC avidin/biotin method according to manufacturer's
instructions.
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Antibodies may be further characterized by flow cytometry carried
out according to standard methods. See Chow et al., Cytometry
(Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by
way of example, the following protocol for cytometric analysis may be
employed: samples may be centrifuged on Ficoll gradients to remove
erythrocytes, and cells may then be fixed with 2% paraformaidehyde for
minutes at 37 C followed by permeabilization in 90% methanol for 30
minutes on ice. Cells may then be stained with the primary
phosphorylation-site specific antibody of the invention (which detects an
10 EGFR-related signal transduction protein enumerated in Table 1), washed
and labeled with a fluorescent-labeled secondary antibody. Additional
fluorochrome-conjugated marker antibodies (e.g. CD45, CD34) may also
be added at this time to aid in the subsequent identification of specific
hematopoietic cell types. The cells would then be analyzed on a flow
cytometer (e.g. a Beckman Coulter FC500) according to the specific
protocols of the instrument used.
Antibodies of the invention may also be advantageousiy
conjugated to fluorescent dyes (e.g. Alexa488, PE) for use in multi-
parametric analyses along with other signal transduction (phospho-CrkL,
phospho-Erk 1/2) and/or cell marker (CD34) antibodies.
Phosphorylation-site specific antibodies of the invention specifically
bind to a human EGFR-related signal transduction protein or polypeptide
only when phosphorylated at a disclosed site, but are not limited only to
binding the human species, per se. The invention includes antibodies
that also bind conserved and highly homologous or identical
phosphorylation sites in respective EGFR-related proteins from other
species (e.g. mouse, rat, monkey, yeast), in addition to binding the human
phosphorylation site. Highly homologous or identical sites conserved in
other species can readily be identified by standard sequence
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comparisons, such as using BLAST, with the human EGFR-related signal
transduction protein phosphorylation sites disclosed herein.
C. Heavy-Isotope Labeled Peptides (AQUA Peptides).
The novel EGFR-related signaling protein phosphorylation sites
disclosed herein now enable the production of corresponding heavy-
isotope labeled peptides for the absolute quantification of such signaling
proteins (both phosphorylated and not phosphorylated at a disclosed site)
in biological samples. The production and use of AQUA peptides for the
absolute quantification of proteins (AQUA) in complex mixtures has been
described. See WO/03016861, "Absolute Quantification of Proteins and
Modified Forms Thereof by Multistage Mass Spectrometry," Gygi et al.
and also Gerber et al. Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 (2003)
(the teachings of which are hereby incorporated herein by reference, in
their entirety).
The AQUA methodology employs the introduction of a known
quantity of at least one heavy-isotope labeled peptide standard (which
has a unique signature detectable by LC-SRM chromatography) into a
digested biological sample in order to determine, by comparison to the
peptide standard, the absolute quantity of a peptide with the same
sequence and protein modification in the biological sample. Briefly, the
AQUA methodology has two stages: peptide internal standard selection
and validation and method development; and implementation using
validated peptide internal standards to detect and quantify a target protein
in sample. The method is a powerful technique for detecting and
quantifying a given peptide/protein within a complex biological mixture,
such as a cell lysate, and may be employed, e.g., to quantify change in
protein phosphorylation as a result of drug treatment, or to quantify
differences in the level of a protein in different biological states.
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Generally, to develop a suitable internal standard, a particuiar
peptide (or modified peptide) within a target protein sequence is chosen
based on its amino acid sequence and the particular protease to be used
to digest. The peptide is then generated by solid-phase peptide synthesis
such that one residue is replaced with that same residue containing stable
isotopes (13C, 15N) The result is a peptide that is chemically identical to
its
native counterpart formed by proteolysis, but is easily distinguishable by
MS via a 7-Da mass shift. A newly synthesized AQUA internal standard
peptide is then evaluated by LC-MS/MS. This process provides
qualitative information about peptide retention by reverse-phase
chromatography, ionization efficiency, and fragmentation via collision-
induced dissociation. Informative and abundant fragment ions for sets of
native and internal standard peptides are chosen and then specifically
monitored in rapid succession as a function of chromatographic retention
to form a selected reaction monitoring (LC-SRM) method based on the
unique profile of the peptide standard.
The second stage of the AQUA strategy is its implementation to
measure the amount of a protein or modified protein from complex
mixtures. Whole cell lysates are typically fractionated by SDS-PAGE gel
electrophoresis, and regions of the gel consistent with protein migration
are excised. This process is followed by in-gel proteolysis in the presence
of the AQUA peptides and LC-SRM analysis. (See Gerber et al. supra.)
AQUA peptides are spiked in to the complex peptide mixture obtained by
digestion of the whole cell lysate with a proteolytic enzyme and subjected
to immunoaffinity purification as described above. The retention time and
fragmentation pattern of the native peptide formed by digestion (e.g.
trypsinization) is identical to that of the AQUA internal standard peptide
determined previously; thus, LC-MS/MS analysis using an SRM
experiment results in the highly specific and sensitive measurement of
both internal standard and analyte directly from extremely complex
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peptide mixtures. Because an absolute amount of the AQUA peptide is
added (e.g. 250 fmol), the ratio of the areas under the curve can be used
to determine the precise expression levels of a protein or phosphorylated
form of a protein in the original cell lysate. In addition, the internal
standard is present during in-gel digestion as native peptides are formed,
such that peptide extraction efficiency from gel pieces, absolute losses
during sample handling (including vacuum centrifugation), and variability
during introduction into the LC-MS system do not affect the determined
ratio of native and AQUA peptide abundances.
An AQUA peptide standard is developed for a known
phosphorylation site sequence previously identified by the IAP-LC-MS/MS
method within in a target protein. One AQUA peptide incorporating the
phosphorylated form of the particular residue within the site may be
developed, and a second AQUA peptide incorporating the non-
phosphorylated form of the residue developed. In this way, the two
standards may be used to detect and quantify both the phosphorylated
and non-phosphorylated forms of the site in a biological sample.
Peptide internal standards may also be generated by examining
the primary amino acid sequence of a protein and determining the
boundaries of peptides produced by protease cleavage. Alternatively, a
protein may actually be digested with a protease and a particular peptide
fragment produced can then sequenced. Suitable proteases include, but
are not limited to, serine proteases (e.g. trypsin, hepsin), metallo
proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin,
carboxypeptidases, etc.
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. Thus, a peptide is preferably at least about 6 amino
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acids. The size of the peptide is also optimized to maximize ionization
frequency. Thus, peptides longer than about 20 amino acids are not
preferred. The preferred ranged is about 7 to 15 amino acids. A peptide
sequence is also selected that is not likely to be chemically reactive
during mass spectrometry, thus sequences comprising cysteine,
tryptophan, or methionine are avoided.
A peptide sequence that does not include a modified region of the
target region may be selected so that the peptide internal standard can be
used to determine the quantity of all forms of the protein. Alternatively, a
peptide internal standard encompassing a modified amino acid may be
desirable to detect and quantify only the modified form of the target
protein. Peptide standards for both modified and unmodified regions can
be used together, to determine the extent of a modification in a particular
sample (i.e. to determine what fraction of the total amount of protein is
represented by the modified form). For example, peptide standards for
both the phosphorylated and unphosphorylated form of a protein known to
be phosphorylated at a particular site can be used to quantify the amount
of phosphorylated form in a sample.
The peptide is labeled using one or more labeled amino acids (i.e.
the label is an actual part of the peptide) or less preferably, labels may be
attached after synthesis according to standard methods. Preferably, the
label is a mass-altering label selected based on the following
considerations: The mass should be unique to shift fragments 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
that preferably exhibits a unique ion mass signature in MS analysis; 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 ones by the ion/mass pattern in the resulting mass
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spectrum. Preferably, the ion mass signature component imparts a mass
to a protein fragment that does not match the residue mass for any of the
20 natural amino acids.
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. The label preferably does not suppress the
ionization efficiency of the protein and is not chemically reactive. The
label may contain a mixture of two or more isotopically distinct species to
generate a unique mass spectrometric pattern at each labeled fragment
position. Stable isotopes, such as 2 H,'3C, 15N, 170, 180, or 34S, are
among preferred labels. Pairs of peptide internal standards that
incorporate a different isotope label may also be prepared. Preferred
amino acid residues into which a heavy isotope label may be incorporated
include leucine, proline, valine, and phenylalanine.
Peptide internal standards are characterized according to their
mass-to-charge (m/z) ratio, and preferably, also according to their
retention time on a chromatographic column (e.g. an HPLC column).
Internal standards that co-elute with unlabeled peptides of identical
sequence are selected as optimal internal standards. The internal
standard is then analyzed by fragmenting the peptide by any suitable
means, for example by collision-induced dissociation (CID) using, e.g.,
argon or helium as a collision gas. The fragments are then analyzed, for
example by multi-stage mass spectrometry (MS") to obtain a fragment ion
spectrum, to obtain a peptide fragmentation signature. Preferably,
peptide fragments have significant differences in m/z ratios to enable
peaks corresponding to each fragment to be well separated, and a
signature is that is unique for the target peptide is obtained. If a suitable
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fragment signature is not obtained at the first stage, additional stages of
MS are performed until a unique signature is obtained.
Fragment ions in the MS/MS and MS3 spectra are typically highly
specific for the peptide of interest, and, in conjunction with LC methods,
allow a highly selective means of detecting and quantifying a target
peptide/protein in a complex protein mixture, such as a cell lysate,
containing many thousands or tens of thousands of proteins. Any
biological sample potentially containing a target protein/peptide of interest
may be assayed. Crude or partially purified cell extracts are preferably
employed. Generally, the sampie has at least 0.01 mg of protein, typically
a concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer
concentration and pH.
A known amount of a labeled peptide internal standard, preferably
about 10 femtomoles, corresponding to a target protein to be
detected/quantified is then added to a biological sample, such as a cell
lysate. The spiked sample is then digested with one or more protease(s)
for a suitable time period to allow digestion. A separation is then
performed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis,
ion exchange chromatography, etc.) to isolate the labeled internal
standard and its corresponding target peptide from other peptides in the
sample. Microcapillary LC is a preferred method.
Each isolated peptide is then examined by monitoring of a selected
reaction in the MS. This involves using the prior knowledge gained by the
characterization of the peptide internal standard and then requiring the
MS to continuously monitor a specific ion in the MS/MS or MSn spectrum
for both the peptide of interest and the internal standard. After elution, the
area under the curve (AUC) for both peptide standard and target peptide
peaks are calculated. 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
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number of copies of the protein per cell. Further details of the AQUA
methodology are described in Gygi et al., and Gerber et al. supra.
In accordance with the present invention, AQUA internal peptide
standards (heavy-isotope labeled peptides) may now be produced, as
described above, for any of the 168 novel EGFR-related signaling protein
phosphorylation sites disclosed herein (see Table 1/Figure 2). Peptide
standards for a given phosphorylation site (e.g. the tyrosine 653 site in
IRS-2 - see Row 114 of Table 1) may be produced for both the
phosphorylated and non-phosphorylated forms of the site (e.g. see IRS-2
site sequence in Column E, Row 114 of Table 1) and such standards
employed in the AQUA methodology to detect and quantify both forms of
such phosphorylation site in a biological sample.
AQUA peptides of the invention may comprise all, or part of, a
phosphorylation site peptide sequence disclosed herein (see Column E of
Table 1/Figure 2). In a preferred embodiment, an AQUA peptide of the
invention comprises a phosphorylation site sequence disclosed herein in
Table 1/Figure 2. For example, an AQUA peptide of the invention for
detection/quantification of Fyn kinase when phosphorylated at tyrosine
Y212 may comprise the sequence KLDNGGyYITTR (y=phosphotyrosine),
which comprises phosphorylatable tyrosine 212 (see Row 118, Column E;
SEQ ID NO: 117). Heavy-isotope labeled equivalents of an of the
peptides enumerated in Table 1/Figure 2 (both in phosphorylated and
unphosphorylated form) can be readily synthesized and their unique MS
and LC-SRM signature determined, so that the peptides are validated as
AQUA peptides and ready for use in quantification experiments.
The phosphorylation site peptide sequences disclosed herein (see
Column E of Table 1/Figure 2) are particularly well suited for development
of corresponding AQUA peptides, since the IAP method by which they
were identified (see Part A above and Example 1) inherently confirmed
that such peptides are in fact produced by enzymatic digestion
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(trypsinization) and are in fact suitably fractionated/ionized in MS/MS.
Thus, heavy-isotope labeled equivalents of these peptides (both in
phosphorylated and unphosphorylated form) can be readily synthesized
and their unique MS and LC-SRM signature determined, so that the
peptides are validated as AQUA peptides and ready for use in
quantification experiments.
Accordingly, the invention provides heavy-isotope labeled peptides
(AQUA peptides) for the detection and/or quantification of any of the
EGFR-related phosphorylation sites disclosed in Table 1/Figure 2 (see
Column E) and/or their corresponding parent proteins/polypeptides (see
Column A). A phosphopeptide sequence comprising any of the
phosphorylation sequences listed in Table 1 may be considered a
preferred AQUA peptide of the invention. For example, an AQUA peptide
comprising the sequence LLGPGVNySGCQITWAK (SEQ ID NO: 153)
(where y may be either phosphotyrosine or tyrosine, and where V
labeled valine (e.g. 14C)) is provided for the quantification of
phosphorylated (or non-phosphorylated) STAT3 (Tyr539) in a biological
sample (see Row 154 of Table 1, tyrosine 539 being the phosphorylatable
residue within the site). However, it will be appreciated that a larger
AQUA peptide comprising a disclosed phosphorylation site sequence
(and additional residues downstream or upstream of it) may also be
constructed. Similarly, a smaller AQUA peptide comprising less than all
of the residues of a disclosed phosphorylation site sequence (but still
comprising the phosphorylatable residue enumerated in Column D of
Table 1/Figure 2) may alternatively be constructed. Such larger or shorter
AQUA peptides are within the scope of the present invention, and the
selection and production of preferred AQUA peptides may be carried out
as described above (see Gygi et al., Gerber et al. supra.).
Certain particularly preferred subsets of AQUA peptides provided
by the invention are described above (corresponding to particular protein
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types/groups in Table 1, for example, Receptor Tyrosine Kinases or
Transcription Factor proteins). Example 4 is provided to further illustrate
the construction and use, by standard methods described above, of
exemplary AQUA peptides provided by the invention. For example, the
above-described AQUA peptides corresponding to the both the
phosphorylated and non-phosphorylated forms of the disclosed STAT3
tyrosine 593 phosphorylation site (see Row 154 of Table 1/Figure 2) may
be used to quantify the amount of phosphorylated STAT3(Tyr593) in a
biological sample, e.g. a tumor cell sample (or a sample before or after
treatment with a test drug).
AQUA peptides of the invention may also be employed within a kit
that comprises one or multiple AQUA peptide(s) provided herein (for the
quantification of an EGFR-related signal transduction protein disclosed in
Table 1), and, optionally, a second detecting reagent conjugated to a
detectable group. For example, a kit may include AQUA peptides for both
the phosphorylation and non-phosphorylated form of a phosphorylation
site disclosed herein. The reagents may also include ancillary agents
such as buffering agents and protein stabilizing agents, e.g.,
polysaccharides and the like. The kit may further include, where
necessary, other members of the signal-producing system of which
system the detectable group is a member (e.g., enzyme substrates),
agents for reducing background interference in a test, control reagents,
apparatus for conducting a test, and the like. The test kit may be
packaged in any suitable manner, typically with all elements in a single
container along with a sheet of printed instructions for carrying out the
test.
AQUA peptides provided by the invention will be highly useful in
the further study of signal transduction anomalies underlying cancer,
including EGFR-mediated cancers, and in identifying diagnostic/bio-
markers of these diseases, new potential drug targets, and/or in
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monitoring the effects of test compounds on EGFR-related signal
transduction proteins and pathways.
D. Immunoassay Formats
Antibodies provided by the invention may be advantageously
employed in a variety of standard immunological assays (the use of
AQUA peptides provided by the invention is described separately above).
Assays may be homogeneous assays or heterogeneous assays. In a
homogeneous assay the immunological reaction usually involves a
phosphorylation-site specific antibody of the invention), a labeled analyte,
and the sample of interest. The signal arising from the label is modified,
directly or indirectly, upon the binding of the antibody to the labeled
analyte. Both the immunological reaction and detection of the extent
thereof are carried out in a homogeneous solution. lmmunochemical
labels that may be employed include free radicals, radioisotopes,
fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
In a heterogeneous assay approach, the reagents are usually the
specimen, a phosphorylation-site specific antibody of the invention, and
suitable means for producing a detectable signal. Similar specimens as
described above may be used. The antibody is generally immobilized on
a support, such as a bead, plate or slide, and contacted with the
specimen suspected of containing the antigen in a liquid phase. The
support is then separated from the liquid phase and either the support
phase or the liquid phase is examined for a detectable signal employing
means for producing such signal. The signal is related to the presence of
the analyte in the specimen. Means for producing a detectable signal
include the use of radioactive labels, fluorescent labels, enzyme labels,
and so forth. For example, if the antigen to be detected contains a
second binding site, an antibody which binds to that site can be
conjugated to a detectable group and added to the liquid phase reaction
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solution before the separation step. The presence of the detectable group
on the solid support indicates the presence of the antigen in the test
sample. Examples of suitable immunoassays are the radioimmunoassay,
immunofluorescence methods, enzyme-linked immunoassays, and the
like.
lmmunoassay formats and variations thereof that may be useful for
carrying out the methods disclosed herein are well known in the art. See
generally E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc.,
Boca Raton, Fla.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et al.,
"Methods for Modulating Ligand-Receptor Interactions and their
Application"); U.S. Pat. No. 4,659,678 (Forrest et al., "Immunoassay of
Antigens"); U.S. Pat. No. 4,376,110 (David et al., "Immunometric Assays
Using Monoclonal Antibodies"). Conditions suitable for the formation of
reagent-antibody complexes are well described. See id. Monoclonal
antibodies of the invention may be used in a "two-site" or "sandwich"
assay, with a single cell line serving as a source for both the labeled
monoclonal antibody and the bound monoclonal antibody. Such assays
are described in U.S. Pat. No. 4,376,110. The concentration of detectable
reagent should be sufficient such that the binding of a target EGFR-
related signal transduction protein is detectable compared to background.
Phosphorylation site-specific antibodies disclosed herein may be
conjugated to a solid support suitable for a diagnostic assay (e.g., beads,
plates, slides or wells formed from materials such as latex or polystyrene)
in accordance with known techniques, such as precipitation. Antibodies,
or other target protein or target site-binding reagents, may likewise be
conjugated to detectable groups such as radiolabels (e.g., 35S 1251, 1311),
enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and
fluorescent labels (e.g., fluorescein) in accordance with known
techniques.
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Antibodies of the invention may also be optimized for use in a flow
cytometry assay to determine the activation/phosphorylation status of a
target EGFR-related signal transduction protein in patients before, during,
and after treatment with a drug targeted at inhibiting phosphorylation at
such a protein at the phosphorylation site disclosed herein. For example,
bone marrow cells or peripheral blood cells from patients may be
analyzed by flow cytometry for target EGFR-related signal transduction
protein phosphorylation, as well as for markers identifying various
hematopoietic cell types. In this manner, activation status of the
malignant cells may be specifically characterized. Flow cytometry may be
carried out according to standard methods. See, e.g. Chow et al.,
Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001).
Briefly and by way of example, the following protocol for cytometric
analysis may be employed: fixation of the cells with 1 % para-
formaldehyde for 10 minutes at 37 C followed by permeabilization in 90%
methanol for 30 minutes on ice. Cells may then be stained with the
primary antibody (a phospho-specific antibody of the invention), washed
and labeled with a fluorescent-labeled secondary antibody. Alternatively,
the cells may be stained with a fluorescent-labeled primary antibody. The
cells would then be analyzed on a flow cytometer (e.g. a Beckman
Coulter EPICS-XL) according to the specific protocols of the instrument
used. Such an analysis would identify the presence of activated EGFR-
related signal transduction protein(s) in the malignant cells and reveal the
drug response on the targeted protein.
Alternatively, antibodies of the invention may be employed in
immunohistochemical (IHC) staining to detect differences in signal
transduction or protein activity using normal and diseased tissues. IHC
may be carried out according to well-known techniques. See, e.g.,
ANTIBODIES: A LABORATORY MANUAL, supra. Briefly, paraffin-embedded
tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by
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deparaffinizing tissue sections with xylene followed by ethanol; hydrating
in water then PBS; unmasking antigen by heating slide in sodium citrate
buffer; incubating sections in hydrogen peroxide; blocking in blocking
solution; incubating,slide in primary antibody and secondary antibody; and
finally detecting using ABC avidin/biotin method according to
manufacturer's instructions.
Antibodies of the invention may be also be optimized for use in
other clinically-suitable applications, for exampie bead-based multiplex-
type assays, such as IGEN, LuminexTM and/or BioplexTM assay formats,
or otherwise optimized for antibody arrays formats, such as reversed-
phase array applications (see, e.g. Paweletz et aL, Oncogene 20(16):
1981-89 (2001)). Accordingly, in another embodiment, the invention
provides a method for the multiplex detection of EGFR-related protein
phosphorylation in a biological sample, the method comprising utilizing at
two or more antibodies or AQUA peptides of the invention to detect the
presence of two or more phosphorylated EGFR-related signaling proteins
enumerated in Column A of Table 1/Figure 2. In one preferred
embodiment, two to five antibodies or AQUA peptides of the invention are
employed in the method. In another preferred embodiment, six to ten
antibodies or AQUA peptides of the invention are employed, while in
another preferred embodiment eleven to twenty such reagents are
employed.
Antibodies and/or AQUA peptides of the invention may also be
employed within a kit that comprises at least one phosphorylation site-
specific antibody or AQUA peptide of the invention (which binds to or
detects an EGFR-related signal transduction protein disclosed in Table 1),
and, optionally, a second antibody conjugated to a detectable group. In
some embodies, the kit is suitable for multiplex assays and comprises two
or more antibodies or AQUA peptides of the invention, and in some
embodiments, comprises two to five, six to ten, or eleven to twenty
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reagents of the invention. The kit may also include ancillary agents such
as buffering agents and protein stabilizing agents, e.g., polysaccharides
and the like. The kit may further include, where necessary, other
members of the signal-producing system of which system the detectable
group is a member (e.g., enzyme substrates), agents for reducing
background interference in a test, control reagents, apparatus for
conducting a test, and the like. The test kit may be packaged in any
suitable manner, typically with all elements in a single container along
with a sheet of printed instructions for carrying out the test.
The following Examples are provided only to further illustrate the
invention, and are not intended to limit its scope, except as provided in
the claims appended hereto. The present invention encompasses
modifications and variations of the methods taught herein which would be
obvious to one of ordinary skill in the art.
EXAMPLE 1
Isolation of Phosphotyrosine-Containing Peptides from Extracts of
EGFR-Activated Tumor Cell Lines and Identification of Novel
Phosphorylation Sites.
In order to discover previously unknown EGFR-related signal
transduction protein phosphorylation sites, IAP isolation techniques were
employed to identify phosphotyrosine-containing peptides in cell extracts
from the following human carcinoma tumor cell lines, each of which has
activated EGFR kinase: A431, HCT116, HPAC, MIAPACA2, PANC-1,
A549, BxPC-3, DU145, HT-29, H460, and LNCaP. Increased expression
of EGFR has been demonstrated in a variety of human cancers, including
breast, colon, pancreatic, ovarian, lung, esophogeal, and neural. See,
e.g., Yeatman, supra. Thus, the cancer cell lines expressing elevated
levels of EGFR and stimulated with EGF were chosen to mimic signaling
pathway activity in cancers involving activated EGFR.
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Tryptic phosphotyrosine peptides were purified and analyzed from
extracts of the each of the eleven cell lines mentioned above as follows.
Cells were cultured in DMEM medium or RPMI 1640 medium
supplemented with 10% fetal bovine serum and penicillin/streptomycin.
Cells at about 80% confluency were starved in medium without serum for
16 hours and stimulated with 100 ng/ml EGF for 5 minutes. After
complete aspiration of medium from the plates, cells were scraped off the
plate in 10 ml lysis buffer per 2 x 108 cells (20 mM HEPES pH 8.0, 9 M
urea, 1 mM sodium vanadate, supplemented with 2.5 mM sodium
pyrophosphate, 1 mM 9-glycerol-phosphate) and sonicated.
Sonicated cell lysates were cleared by centrifugation at 20,000 x g,
and proteins were reduced with DTT at a final concentration of 4.1 mM
and alkylated with iodoacetamide at 8.3 mM. For digestion with trypsin,
protein extracts were diluted in 20 mM HEPES pH 8.0 to a final
concentration of 2 M urea and soluble TLCK-trypsin (Worthington) was
added at 10-20 g/mL. Digestion was performed for 1-2 days at room
temperature.
Trifluoroacetic acid (TFA) was added to protein digests to a final
concentration of 1%, precipitate was removed by centrifugation, and
digests were loaded onto Sep-Pak C18 columns (Waters) equilibrated with
0.1 % TFA. A column volume of 0.7-1.0 ml was used per 2 x 108 cells.
Columns were washed with 15 volumes of 0.1 /o TFA, followed by 4
volumes of 5% acetonitrile (MeCN) in 0.1 % TFA. Peptide fraction I was
obtained by eluting columns with 2 volumes each of 8, 12, and 15%
MeCN in 0.1 % TFA and combining the eluates. Fractions II and I I I were a
combination of eluates after eluting columns with 18, 22, 25% MeCN in
0.1 % TFA and with 30, 35, 40% MeCN in 0.1 % TFA, respectively. All
peptide fractions were lyophiiized.
Peptides from each fraction corresponding to 2 x 108 cells were
dissolved in 1 ml of IAP buffer (20 mM Tris/HCI or 50 mM MOPS pH 7.2,
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mM sodium phosphate, 50 mM NaCI) and insoluble matter (mainly in
peptide fractions III) was removed by centrifugation. IAP was performed
on each peptide fraction separately. The phosphotyrosine monoclorial
antibody P-Tyr-100 (Cell Signaling Technology, Inc., catalog number
5 9411) was coupled at 4 mg/mI beads to protein G agarose (Roche).
Immobilized antibody (15 pI, 60 pg) was added as 1:1 slurry in IAP buffer
to 1 ml of each peptide fraction, and the mixture was incubated overnight
at 40 C with gentle rotation. The immobilized antibody beads were washed
three times with 1 ml IAP buffer and twice with 1 ml water, all at 40 C.
10 Peptides were eluted from beads by incubation with 75 lal of 0.1 % TFA at
room temperature for 10 min.
Alternatively, one single peptide fraction was obtained from Sep-Pak
C 18 columns by elution with 2 volumes each of 10%, 15%, 20 1 , 25 l ,
30 %, 35 % and 40 % acetonitirile in 0.1 % TFA and combination of all
eluates. IAP on this peptide fraction was performed as follows: After
lyophilization, peptide was dissolved in 1.4 ml IAP buffer (MOPS pH 7.2,
10 mM sodium phosphate, 50 mM NaCI) and insoluble matter was
removed by centrifugation. Immobilized antibody (40 pl, 160 pg) was
added as 1:1 slurry in IAP buffer, and the mixture was incubated
overnight at 4 C with gentle shaking. The immobilized antibody beads
were washed three times with 1 ml IAP buffer and twice with 1 ml water,
all at 40 C. Peptides were eluted from beads by incubation with 55 PI of
0.15% TFA at room temperature for 10 min (eluate 1), followed by a wash
of the beads (eluate 2) with 45 ,ul of 0.15% TFA. Both eluates were
combined.
Analysis by LC-MS/MS Mass Spectrometry.
40 pl of IAP eluate were purified by 0.2 pl StageTips or ZipTips.
Peptides were eluted from the microcolumns with 1pl of 40% MeCN,
0.1 % TFA (fractions I and II) or 1 pl of 60% MeCN, 0.1 % TFA (fraction III)
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into 7.6 pl of 0.4% acetic acid/0.005% heptafluorobutyric acid. This
sample was loaded onto a 10 cm x 75 pm PicoFrit capillary column (New
Objective) packed with Magic C18 AQ reversed-phase resin (Michrom
Bioresources) using a Famos autosampler with an inert sample injection
valve (Dionex). The column was then developed with a 45-min linear
gradient of acetonitrile delivered at 200 ni/min (Ultimate, Dionex), and
tandem mass spectra were coliected in a data-dependent manner with an
LCQ Deca XP Plus ion trap mass spectrometer essentially as described
by Gygi et al., supra.
Database Analysis & Assignments.
MS/MS spectra were evaluated using TurboSequest in the
Sequest Browser package (v. 27, rev. 12) supplied as part of BioWorks
3.0 (ThermoFinnigan). Individual MS/MS spectra were extracted from the
raw data file using the Sequest Browser program CreateDta, with the
following settings: bottom MW, 700; top MW, 4,500; minimum number of
ions, 20; minimum TIC, 4 x 105; and precursor charge state, unspecified.
Spectra were extracted from the beginning of the raw data file before
sample injection to the end of the eluting gradient. The lonQuest and
VuDta programs were not used to further select MS/MS spectra for
Sequest analysis. MS/MS spectra were evaluated with the following
TurboSequest parameters: peptide mass tolerance, 2.5; fragment ion
tolerance, 0.0; maximum number of differential amino acids per
modification, 4; mass type parent, average; mass type fragment, average;
maximum number of internal cleavage sites, 10; neutral losses of water
and ammonia from b and y ions were considered in the correlation
analysis. Proteolytic enzyme was specified except for spectra collected
from elastase digests.
Searches were performed against the NCBI human protein
database (for all other studies) (released on April 29, 2003 and containing
37,490 protein sequences). Cysteine carboxamidomethylation was
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specified as a static modification, and phosphorylation was allowed as a
variable modification on serine, threonine, and tyrosine residues or on
tyrosine residues alone. It was determined that restricting phosphorylation
to tyrosine residues had little effect on the number of phosphorylation
sites assigned.
In proteomics research, it is desirable to validate protein
identifications based solely on the observation of a single peptide in one
experimental result, in order to indicate that the protein is, in fact,
present
in a sample. This has led to the development of statistical methods for
validating peptide assignments, which are not yet universally accepted,
and guidelines for the publication of protein and peptide identification
results (see Carr et al., Mol. Cell Proteomics 3: 531-533 (2004)), which
were followed in this Example. However, because the immunoaffinity
strategy separates phosphorylated peptides from unphosphorylated
peptides, observing just one phosphopeptide from a protein is a common
result, since many phosphorylated proteins have only one tyrosine-
phosphorylated site. For this reason, it is appropriate to use additional
criteria to validate phosphopeptide assignments. Assignments are likely to
be correct if any of these additional criteria are met: (i) the same
sequence is assigned to co-eluting ions with different charge states, since
the MS/MS spectrum changes markedly with charge state; (ii) the site is
found in more than one peptide sequence context due to sequence
overlaps from incomplete proteolysis or use of proteases other than
trypsin; (iii) the site is found in more than one peptide sequence context
due to homologous but not identical protein isoforms; (iv) the site is found
in more than one peptide sequence context due to homologous but not
identical proteins among species; and (v) sites validated by MS/MS
analysis of synthetic phosphopeptides corresponding to assigned
sequences, since the ion trap mass spectrometer produces highly
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reproducible MS/MS spectra. The last criterion is routinely employed to
confirm novel site assignments of particular interest.
All spectra and all sequence assignments made by Sequest were
imported into a relational database. Assigned sequences were accepted
or rejected following a conservative, two-step process. In the first step, a
subset of high-scoring sequence assignments was selected by filtering for
XCorr values of at least 1.5 for a charge state of +1, 2.2 for +2, and 3.3
for +3, allowing a maximum RSp value of 10. Assignments in this subset
were rejected if any of the following criteria were satisfied: (i) the
spectrum contained at least one major peak (at least 10% as intense as
the most intense ion in the spectrum) that could not be mapped to the
assigned sequence as an a, b, or y ion, as an ion arising from neutral-loss
of water or ammonia from a b or y ion, or as a multiply protonated ion; (ii)
the spectrum did not contain an series of b or y ions equivalent to at least
six uninterrupted residues; or (iii) the sequence was not observed at least
five times in all the studies we have conducted (except for overlapping
sequences due to incomplete proteolysis or use of proteases other than
trypsin). In the second step, assignments with below-threshold scores
were accepted if the low-scoring spectrum showed a high degree of
similarity to a high-scoring spectrum collected in another study, which
simulates a true reference library-searching strategy. All spectra
supporting the final list of 168 assigned sequences enumerated in
Table 1/Figure 2 herein were reviewed by at least three people to
establish their credibility.
EXAMPLE 2
Production of Phospho-specific Polyclonal Antibodies for the
Detection of EGFR-related Signaling Protein Phosphorylation
Polyclonal antibodies that specifically bind an EGFR-related signal
transduction protein only when phosphorylated at the respective
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phosphorylation site disclosed herein (see Table 1) are produced
according to standard methods by first constructing a synthetic peptide
antigen comprising the phosphorylation site sequence and then
immunizing an animal to raise antibodies against the antigen, as further
described below. Production of exempiary polyclonal antibodies is
provided below.
A. HER2 (tyrosine 923).
A 15 amino acid phospho-peptide antigen, MTFGAKPy*DGIPAR
(where y*= phosphotyrosine) that corresponds to the sequence
encompassing the tyrosine 923 phosphorylation site in human HER2
receptor kinase (see Row 124 of Table 1; SEQ ID NO: 123), plus cysteine
on the C-terminal for coupling, is constructed according to standard
synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc.,
Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL,
supra.; Merrifield, supra. This peptide is then coupled to KLH and used to
immunize animals to produce (and subsequently screen) phospho-
specific HER2(tyr923) polyclonal antibodies as described in
Immunization/ Screening below.
B. BRSK1 (tyrosine 121).
A 13 amino acid phospho-peptide antigen, VYENKKy*LYLVLE
(where y*= phosphotyrosine) that corresponds to the sequence
encompassing the tyrosine 121 phosphorylation site in human BRSK1
kinase (see Row 112 of Table 1(SEQ ID NO: 111)), plus cysteine on the
C-terminal for coupling, is constructed according to standard synthesis
techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony
peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.;
Merrifield, supra. This peptide is then coupled to KLH and used to
immunize animals to produce (and subsequently screen) phospho-
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specific BRSKI(tyrl2l) polyclonal antibodies as described in
Immunization/Screening below.
C. IRS-2 (tyrosine 823).
A 15 amino acid phospho-peptide antigen, CGGDSDQy*VLMSSPV
(where y*= phosphotyrosine) that corresponds to the sequence
encompassing the tyrosine 823 phosphorylation site in human IRS-2
protein (see Row 16 of Table 1(SEQ ID NO: 15), plus cysteine on the C-
terminal for coupling, is constructed according to standard synthesis
techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony
peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.;
Merrifield, supra. This peptide is then coupled to KLH and used to
immunize animals to produce (and subsequently screen) phospho-specific
IRS-2 (tyr823) antibodies as described in Immunization/Screening below.
Immunization/Screening.
A synthetic phospho-peptide antigen as described in A-C above is
coupled to KLH, and rabbits are injected intradermally (ID) on the back
with antigen in complete Freunds adjuvant (500 g antigen per rabbit).
The rabbits are boosted with same antigen in incomplete Freund adjuvant
(250 g antigen per rabbit) every three weeks. After the fifth boost,
bleeds are collected. The sera are purified by Protein A-affinity
chromatography by standard methods (see ANTIBODIES: A LABORATORY
MANUAL, Cold Spring Harbor, supra.). The eluted immunoglobulins are
further loaded onto a non-phosphorylated synthetic peptide antigen-resin
Knotes column to pull out antibodies that bind the non-phosphorylated
form of the phosphorylation site. The flow through fraction is collected
and applied onto a phospho-synthetic peptide antigen-resin column to
isolate antibodies that bind the phosphorylated form of the site. After
washing the column extensively, the bound antibodies (i.e. antibodies that
bind a phosphorylated peptide described in A-C above, but do not bind
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the non-phosphorylated form of the peptide, are eluted and kept in
antibody storage buffer.
The isolated antibody is then tested for phospho-specificity using
Western blot assay using an appropriate cell line the expresses (or
overexpresses) target phospho-protein (i.e. phosphorylated HER2,
BRSK1, or IRS-2, for example, HT-29, A431 and HCT-116 cells,
respectively. Cells are cultured in DMEM supplemented with 10% FCS.
Before stimulation, the cells are starved in serum-free DMEM medium for
4 hours. The cells are then stimulated ligand (e.g. EGF 100 ng/ml) for 5
minutes. Cell are collected, washed with PBS and directly lysed in cell
lysis buffer. The protein concentration of cell lysates are then measured.
The loading buffer is added into cell lysate and the mixture is boiled at
100 C for 5 minutes. 20 ul (10 g protein) of sample is then added onto
7.5% SDS-PAGE gel.
A standard Western blot may be performed according to the
Immunoblotting Protocol set out in the CELL SIGNALING TECHNOLOGY, INC.
2003-04 Catalogue, p. 390. The isolated'phospho-specific antibody is
used at dilution 1:1000. Phosphorylation-site specificity of the antibody
will be shown by binding of only the phosphorylated form of the target
protein. Isolated phospho-specific polyclonal antibody does not recognize
the target protein when not phosphorylated at the appropriate
phosphorylation site in the non-stimulated cells (e.g. IRS-2 is not bound
when not phosphorylated at tyrosine 823).
In order to confirm the specificity of the isolated antibody, different
cell lysates containing various phosphorylated signal transduction
proteins other than the target protein are prepared. The Western blot
assay is preformed again using these cell lysates. The phospho-specific
polyclonal antibody isolated as described above is used (1:1000 dilution)
to test reactivity with the different phosphorylated non-target proteins on
Western blot membrane. The phospho-specific antibody does not
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significantly cross-react with other phosphorylated signal transduction
proteins, aithough occasionally slight binding with a highly homologous
phosphorylation-site on another protein may be observed. In such case
the antibody may be further purified using affinity chromatography, or the
specific immunoreactivity cloned by rabbit hybridoma technology.
Ex,4nnPLE 3
Production of Phospho-specific Monoclonal Antibodies for the
Detection of EGFR-related Signaling Protein Phosphorylation
Monoclonal antibodies that specifically bind a EGFR-related signal
transduction protein only when phosphorylated at the respective
phosphorylation site disclosed herein (see Table 1) are produced
according to standard methods by first constructing a synthetic peptide
antigen comprising the phosphorylation site sequence and then
immunizing an animal to raise antibodies against the antigen, and
harvesting spleen cells from such animals to produce fusion hybridomas,
as further described below. Production of exemplary monoclonal
antibodies is provided below.
A. EphB4 (tyrosine 574).
A 13 amino acid phospho-peptide antigen, NGREAEy*SDKHGQ
(where y*= phosphotyrosine) that corresponds to the sequence
encompassing the tyrosine 574 phosphorylation site in human EphB4
kinase (see Row 137 of Table 1(SEQ ID NO: 136)), plus cysteine on the
C-terminal for coupling, is constructed according to standard synthesis
techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony
peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.;
Merrifield, supra. This peptide is then coupled to KLH and used to
immunize animals and harvest spleen cells for generation (and
subsequent screening) of phospho-specific monoclonal EphB4(tyr574)
antibodies as described in Immunization/ Fusion/Screening below.
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B. MINK (tyrosine 906).
A 15 amino acid phospho-peptide antigen, LHADSNGy*TNLPDVV
(where y*= phosphotyrosine) that corresponds to the sequence
encompassing the tyrosine 906 phosphorylation site in human MINK
kinase (see Row 116 of Table 1(SEQ ID NO: 115)), plus cysteine on the
C-terminal for coupling, is constructed according to standard synthesis
techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony
peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.;
Merrifield, supra. This peptide is then coupled to KLH and used to
immunize animals and harvest spleen cells for generation (and
subsequent screening) of phospho-specific monoclonal MINK(tyr9O6)
antibodies as described in Immunization/ Fusion/Screening below.
C. PTP-kappa (tyrosine 858).
A 14 amino acid phospho-peptide antigen, CEGTESPy*YGNDSD
(where y*= phosphotyrosine) that corresponds to the sequence
encompassing the tyrosine 858 phosphorylation site in human PTP-kappa
phosphatase (see Row 121 of Table 1(SEQ ID NO: 120)), plus cysteine
on the C-terminal for coupling, is constructed according to standard
synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc.,
Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL,
supra.; Merrifield, supra. This peptide is then coupled to KLH and used to
immunize animals and harvest spleen cells for generation (and
subsequent screening) of phospho-specific monoclonal PTP-kappa
(tyr858) antibodies as described in Immunization/Fusion/ Screening
below.
Immunization/Fusion/Screening.
A synthetic phospho-peptide antigen as described in A-C above is
coupled to KLH, and BALB/C mice are injected intradermally (ID) on the
back with antigen in complete Freunds adjuvant (e.g. 50 g antigen per
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mouse). The mice are boosted with same antigen in incomplete Freund
adjuvant (e.g. 25 g antigen per mouse) every three weeks. After the fifth
boost, the animals are sacrificed and spleens are harvested.
Harvested spleen cells are fused to SP2/0 mouse myeloma fusion
partner cells according to the standard protocol of Kohler and Milstein
(1975). Colonies originating from the fusion are screened by ELISA for
reactivity to the phospho-peptide and non-phospho-peptide forms of the
antigen and by Western blot analysis (as described in Example I above).
Colonies found to be positive by ELISA to the phospho-peptide while
negative to the non-phospho-peptide are further characterized by
Western blot analysis. Colonies found to be positive by Western blot
analysis are subcloned by limited dilution. Mouse ascites are produced
from a single clone obtained from subcloning, and tested for phospho-
specificity (against the EphB4, MINK, or PTP-delta phospho-peptide
antigen, as the case may be) on ELISA. Clones identified as positive on
Western blot analysis using cell culture supernatant as having phospho-
specificity, as indicated by a strong band in the induced lane and a weak
band in the uninduced lane of the blot, are isolated and subcloned as
clones producing monoclonal antibodies with the desired specificity.
Ascites fluid from isolated clones may be further tested by Western
blot analysis. The ascites fluid should produce similar results on Western
blot analysis as observed previously with the cell culture supernatant,
indicating phospho-specificity against the phosphorylated target (e.g.
MINK phosphorylated at tyrosine 906).
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EXAMPLE 4
Production and Use of AQUA Peptides for the Quantification of
EGFR-related Signaling Protein Phosphorylation
Heavy-isotope labeled peptides (AQUA peptides (internal
standards)) for the detection and quantification of an EGFR-related signal
trans'duction protein only when phosphorylated at the respective
phosphorylation site disclosed herein (see Table 1) are produced
according to the standard AQUA methodology (see Gygi et al., Gerber et
al., supra.) methods by first constructing a synthetic peptide standard
corresponding to the phosphorylation site sequence and incorporating a
heavy-isotope label. Subsequently, the MSn and LC-SRM signature of
the peptide standard is validated, and the AQUA peptide is used to
quantify native peptide in a biological sample, such as a digested cell
extract. Production and use of exemplary AQUA peptides is provided
below.
A. Ron (tyrosine 1238).
An AQUA peptide comprising the sequence, DILDREy*YSVQQHR
(y*= phosphotyrosine; sequence incorporating 14C/1 5N-labeled leucine
(indicated by bold L), which corresponds to the tyrosine 1238
phosphorylation site in human Ron kinase (see Row 142 in Table 1(SEQ
ID NO: 141)), is constructed according to standard synthesis techniques
using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide
synthesizer (see Merrifield, supra.) as further described below in
Synthesis & MS/MS Signature. The Ron(tyr1238) AQUA peptide is then
spiked into a biological sample to quantify the amount of phosphorylated
Ron(tyr1238) in the sample, as further described below in Analysis &
Quantification.
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B. P13K p85-beta (tyrosine 467).
An AQUA peptide comprising the sequence, SREYDQLYEEy*TR
(y*= phosphotyrosine; sequence incorporating14C/'5N-labeled leucine
(indicated by bold L), which corresponds to the tyrosine 467
phosphorylation site in human P13K p85-beta kinase (see Row 100 in
Table 1(SEQ ID NO: 99)), is constructed according to standard synthesis
techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony
peptide synthesizer (see Merrifield, supra.) as further described below in
Synthesis & MS/MS Signature. The P13K P85-befia(tyr467) AQUA
peptide is then spiked into a biological sample to quantify the amount of
phosphorylated P13K P85-beta(tyr467) in the sample, as further described
below in Analysis & Quantification.
C. Annexin A4 (tyrosine 164)
An AQUA peptide comprising the sequence,
VLVSLSAGGRDEGNy*LDDALVR (y*= phosphotyrosine; sequence
incorporating '4C/1 5N-labeled leucine (indicated by bold L), which
corresponds to the tyrosine 164 phosphorylation site in human Annexin
A4 protein (see Row 61 in Table 9(SEQ ID NO: 60)), is constructed
according to standard synthesis techniques using, e.g., a Rainin/Protein
Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.)
as further described below in Synthesis & MS/MS Signature. The
Annexin A4(tyr164) AQUA peptide is then spiked into a biological sample
to quantify the amount of phosphorylated Annexin A4(tyr164) in the
sample, as further described below in Analysis & Quantification.
D. Talin 1(tyrosine 26).
An AQUA peptide comprising the sequence,
TMQFEPSTMVy*DACR (y*= phosphotyrosine; sequence incorporating
14C/15N-labeled valine (indicated by bold V), which corresponds to THE
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tyrosine 26 phosphorylation site in human Talin 1 protein (see Row 78 in
Table 1(SEQ ID NO: 77)), is constructed according to standard synthesis
techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony
peptide synthesizer (see Merrifield, supra.) as further described below in
Synthesis & MS/MS Signature. The Talin 1(tyr26) AQUA peptide is then
spiked into a biological sample to quantify the amount of phosphorylated
Talin 1(tyr26) in the sample, as further described below in Analysis &
Quantification.
Synthesis & MS/MS Spectra.
Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acid monomers
may be obtained from AnaSpec (San Jose, CA). Fmoc-derivatized stable-
isotope monomers containing one 15N and five to nine 13C atoms may be
obtained from Cambridge Isotope Laboratories (Andover, MA). Preloaded
Wang resins may be obtained from Applied Biosystems. Synthesis scales
may vary from 5 to 25 pmol. Amino acids are activated in situ with 1-H-
benzotriazolium, 1-bis(dimethylamino) methylene]-hexafluorophosphate(1-
),3-oxide:l-hydroxybenzotriazole hydrate and coupled at a 5-fold molar
excess over peptide. Each coupling cycle is followed by capping with acetic
anhydride to avoid accumulation of one-residue deletion peptide
byproducts. After synthesis peptide-resins are treated with a standard
scavenger-containing trifluoroacetic acid (TFA)-water cleavage solution, and
the peptides are precipitated by addition to cold ether. Peptides (i.e. a
desired AQUA peptide described in A-D above) are purified by reversed-
phase C18 HPLC using standard TFA/acetonitrile gradients and
characterized by matrix-assisted laser desorption ionization-time of flight
(Biflex III, Bruker Daltonics, Billerica, MA) and ion-trap (ThermoFinnigan,
LCQ DecaXP) MS.
MS/MS spectra for each AQUA peptide should exhibit a strong y-
type ion peak as the most intense fragment ion that is suitable for use in
an SRM monitoring/analysis. Reverse-phase microcapillary columns (0.1
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A- 150-220 mm) are prepared according to standard methods. An
Agilent 1100 liquid chromatograph may be used to develop and deliver a
solvent gradient [0.4% acetic acid/0.005% heptafluorobutyric acid
(HFBA)/7% methanol and 0.4% acetic acid/0.005% HFBA/65%
methanol/35% acetonitrile] to the microcapillary column by means of a
flow splitter. Samples are then directly loaded onto the microcapillary
column by using a. FAMOS inert capillary autosampler (LC Packings, San
Francisco) after the flow split. Peptides are reconstituted in 6% acetic
acid/0.01% TFA before injection.
Analysis & Quantification.
Target protein (e.g. a phosphorylated protein of A-D above) in a
biological sample is quantified using a validated AQUA peptide (as
described above). The IAP method is then applied to the complex
mixture of peptides derived from proteolytic cleavage of crude cell
extracts to which the AQUA peptides have been spiked in.
LC-SRM of the entire sample is then carried out. MS/MS may be
performed by using a ThermoFinnigan (San Jose, CA) mass
spectrometer (LCQ DecaXP ion trap or TSQ Quantum triple quadrupole).
On the DecaXP, parent ions are isolated at 1.6 m/z width, the ion injection
time being limited to 150 ms per microscan, with two microscans per
peptide averaged, and with an AGC setting of 1 x 108; on the Quantum,
Q1 is kept at 0.4 and Q3 at 0.8 m/z with a scan time of 200 ms per
peptide. On both instruments, analyte and internal standard are analyzed
in alternation within a previously known reverse-phase retention window;
well-resolved pairs of internal standard and analyte are analyzed in
separate retention segments to improve duty cycle. Data are processed
by integrating the appropriate peaks in an extracted ion chromatogram
(60.15 m/z from the fragment monitored) for the native and internal
standard, followed by calculation of the ratio of peak areas multiplied by
the absolute amount of internal standard (e.g., 500 fmol).
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