Patent Grant
7807789
This is a National Stage Application of International Application No. PCT/US04/42940, filed Dec. 21, 2004 which is incorporated herein by reference in its entirety.
The invention relates generally to antibodies and peptide reagents for the detection of protein phosphorylation, and to protein phosphorylation in cancer.
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 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 (ErbB2/neu), the kinase dead HER3, and HER4 (ErbB4) (Casalini et al. (2004) J. Cell. Physiol 200: 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, 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 EGFRvIII), 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 al. 1998 Bioassays 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-Erk1/2 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 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. Iressa™ (ZD1839), a small-molecule inhibitor of EGFR, has recently entered clinical trials for the treatment of breast cancer, while another small molecule inhibitor, Tarceva™ (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-inhibitor, 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.
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.
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.
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 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 FIG. 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
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 FIG. 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
FIG. 5—is an exemplary mass spectrograph depicting the detection of the tyrosine 1328 phosphorylation site in HER-3 (see Row 126 in FIG. 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
FIG. 6—is an exemplary mass spectrograph depicting the detection of the tyrosine 539 phosphorylation site in STAT-3 (see Row 154 in FIG. 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
FIG. 7—is an exemplary mass spectrograph depicting the detection of the tyrosine 1238 phosphorylation site in Ron kinase (see Row 142 in FIG. 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
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/
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. 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/
In one embodiment, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a human 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 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/
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/
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 1 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 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.
(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 1 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 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 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 phosphorylation 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:
(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 phosphorylated 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 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 99-101, of Table 1, comprised within the 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: PI3K 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 1 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 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 ID 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:
(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 phosphorylated 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 only 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.
(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 C, 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 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 1 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 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 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/
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.
All 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.
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 1 above is identical to
The identification of these 168 phosphorylation sites is described in more detail in Part A below and in Example 1.
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 polypeptide derived therefrom) enumerated in Column A of Table 1/
“Heavy-isotope labeled peptide” (used interchangeably 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 al.), 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.
“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/
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 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, Mass., Cat #9411 (p-Tyr-100)) was used in the immunoaffinity step to isolate the widest possible number of phosphotyrosine 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 C18 columns to separate peptides from other cellular components. The solid phase extraction cartridges were eluted with varying steps of acetonitrile. Each lyophilized peptide fraction was redissolved in PBS and treated with phosphotyrosine antibody (P-Tyr-100, CST #9411) immobilized on protein G-Sepharose. Immunoaffinity-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×75 μm 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 phosphorylation 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/
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.
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/
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 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 1 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., A
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 only 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, C
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'l 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'l. 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 (for example, the p-38-delta 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'l. 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-related signaling protein phosphorylation 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., A
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 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 phosphorylated, 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., A
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% paraformaldehyde 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 phosphorylation-site specific antibody of the invention (which detects an 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 advantageously 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 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.
Generally, to develop a suitable internal standard, a particular 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 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 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 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 2H, 13C, 15N, 17O, 18O, 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 (MSn) 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 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 sample 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 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/
AQUA peptides of the invention may comprise all, or part of, a phosphorylation site peptide sequence disclosed herein (see Column E of Table 1/
The phosphorylation site peptide sequences disclosed herein (see Column E of Table 1/
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/
Certain particularly preferred subsets of AQUA peptides provided by the invention are described above (corresponding to particular protein 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/
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 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. Immunochemical 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 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.
Immunoassay 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, 125I, 131I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
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., A
Antibodies of the invention may be also be optimized for use in other clinically-suitable applications, for example bead-based multiplex-type assays, such as IGEN, Luminex™ and/or Bioplex™ 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/
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 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.
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.
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 2×108 cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented with 2.5 mM sodium pyrophosphate, 1 mM β-glycerol-phosphate) and sonicated.
Sonicated cell lysates were cleared by centrifugation at 20,000×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 μg/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 2×108 cells. Columns were washed with 15 volumes of 0.1% 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 III 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 lyophilized.
Peptides from each fraction corresponding to 2×108 cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HCl or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and insoluble matter (mainly in peptide fractions III) was removed by centrifugation. IAP was performed on each peptide fraction separately. The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Signaling Technology, Inc., catalog number 9411) was coupled at 4 mg/ml beads to protein G agarose (Roche). Immobilized antibody (15 μl, 60 μg) was added as 1:1 slurry in IAP buffer to 1 ml of each peptide fraction, and the mixture was incubated overnight at 4° 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 4° C. Peptides were eluted from beads by incubation with 75 μl of 0.1% TFA at room temperature for 10 min.
Alternatively, one single peptide fraction was obtained from Sep-Pak C18 columns by elution with 2 volumes each of 10%, 15%, 20%, 25%, 30%, 35% and 40% acetonitrile 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, mM sodium phosphate, 50 mM NaCl) and insoluble matter was removed by centrifugation. Immobilized antibody (40 μl, 160 μg) 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 4° C. Peptides were eluted from beads by incubation with 55 μl of 0.15% TFA at room temperature for 10 min (eluate 1), followed by a wash of the beads (eluate 2) with 45 μl of 0.15% TFA. Both eluates were combined.
Analysis by LC-MS/MS Mass Spectrometry.
40 μl of IAP eluate were purified by 0.2 μl StageTips or ZipTips. Peptides were eluted from the microcolumns with 1 μl of 40% MeCN, 0.1% TFA (fractions I and II) or 1 μl of 60% MeCN, 0.1% TFA (fraction III) into 7.6 μl of 0.4% acetic acid/0.005% heptafluorobutyric acid. This sample was loaded onto a 10 cm×75 μm 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 nl/min (Ultimate, Dionex), and tandem mass spectra were collected 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, 4×105; 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 IonQuest 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 Apr. 29, 2003 and containing 37,490 protein sequences). Cysteine carboxamidomethylation was 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 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/
Polyclonal antibodies that specifically bind an 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, as further described below. Production of exemplary 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 A
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 A
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 A
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 μg antigen per rabbit). The rabbits are boosted with same antigen in incomplete Freund adjuvant (250 μg 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 A
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. Cells 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 μl (10 μg 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 C
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 significantly cross-react with other phosphorylated signal transduction proteins, although 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.
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 A
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 A
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 A
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 μg antigen per mouse). The mice are boosted with same antigen in incomplete Freund adjuvant (e.g. 25 μg 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 1 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).
Heavy-isotope labeled peptides (AQUA peptides (internal standards)) for the detection and quantification of an EGFR-related signal transduction 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/15N-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.
B. PI3K p85-Beta (Tyrosine 467).
An AQUA peptide comprising the sequence, SREYDQLYEEy*TR (y*=phosphotyrosine; sequence incorporating 14C/15N-labeled leucine (indicated by bold L), which corresponds to the tyrosine 467 phosphorylation site in human PI3K 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 PI3K P85-beta(tyr467) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated PI3K 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 14C/15N-labeled leucine (indicated by bold L), which corresponds to the tyrosine 164 phosphorylation site in human Annexin A4 protein (see Row 61 in Table 1 (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 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, Calif.). Fmoc-derivatized stable-isotope monomers containing one 15N and five to nine 13C atoms may be obtained from Cambridge Isotope Laboratories (Andover, Mass.). Preloaded Wang resins may be obtained from Applied Biosystems. Synthesis scales may vary from 5 to 25 μmol. Amino acids are activated in situ with 1-H-benzotriazolium, 1-bis(dimethylamino) methylene]-hexafluorophosphate(1-),3-oxide:1-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, Mass.) 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 Ř150-220 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, Calif.) 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 1×108; 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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