WO2006068640A1 - Phosphorylation des protéines suivant des voies contrôlées par les egfr - Google Patents

Phosphorylation des protéines suivant des voies contrôlées par les egfr Download PDF

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WO2006068640A1
WO2006068640A1 PCT/US2004/042940 US2004042940W WO2006068640A1 WO 2006068640 A1 WO2006068640 A1 WO 2006068640A1 US 2004042940 W US2004042940 W US 2004042940W WO 2006068640 A1 WO2006068640 A1 WO 2006068640A1
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rows
protein
corresponding column
tyrosine
column
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PCT/US2004/042940
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Ailan Guo
Kimberly Lee
Klarisa Rikova
Charles Farnsworth
Yu Li
Albrecht Moritz
Roberto Polakiewicz
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Cell Signaling Technology, Inc.
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Priority to EP04815061A priority Critical patent/EP1841882A1/fr
Priority to CA002601700A priority patent/CA2601700A1/fr
Priority to PCT/US2004/042940 priority patent/WO2006068640A1/fr
Publication of WO2006068640A1 publication Critical patent/WO2006068640A1/fr
Priority to US11/821,130 priority patent/US7807789B2/en
Priority to US12/897,363 priority patent/US20110130547A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates generally to antibodies and peptide reagents for the detection of protein phosphorylation, and to protein phosphorylation in cancer.
  • Protein phosphorylation 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 _ 9
  • RTKs receptor tyrosine kinase family
  • RTKs 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 (EfbB2/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.)
  • 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.).
  • type III EGFR deletion mutant also known as EGFRvIII
  • EGFR overexpression in non-small cell lung cancers 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 ef al. 1998 Bioessays 20: 41-48).
  • Ligand binding induces homodimerization, leading to ATP-mediated autophosphorylation or transphosphorylation (by a partner kinase) of EGFR, which in turn activates its kinase function (Russo et al., 1985 J. Biol. Chem. 260: 5205-5208).
  • 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 ).
  • 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.)
  • Herceptin® an inhibitor of HER2/neu
  • IressaTM ZD1839
  • TarcevaTM another small molecule inhibitor
  • NSCLC non-small cell lung carcinoma
  • 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.
  • IAP immunoaffinity isolation and mass-spectrometric characterization methodology
  • FIG. 3 - is an exemplary mass spectrograph depicting the detection of the tyrosine 998 phosphorylation site in EGFR (see Row 123 in Figure 2/Table 1 ), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); pY and Y* indicate the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
  • FIG. 4 - is an exemplary mass spectrograph depicting the detection of the tyrosine 653 phosphorylation site in insulin receptor substrate-2 (IRS- 2) (see Row 14 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum; the purple M# indicates an oxidized methionine residue detected); pY and Y* indicate the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
  • FIG. 5 - is an exemplary mass spectrograph depicting the detection of the tyrosine 1328 phosphorylation site in HER-3 (see Row 126 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); pY and Y* indicate the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
  • FIG. 6 - is an exemplary mass spectrograph depicting the detection of the tyrosine 539 phosphorylation site in STAT-3 (see Row 154 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); pY and Y* indicate the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
  • FIG. 7 - is an exemplary mass spectrograph depicting the detection of the tyrosine 1238 phosphorylation site in Ron kinase (see Row 142 in Figure 2/ Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); pY and Y* indicate the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
  • EGFR Epidermal Growth Factor Receptor kinase
  • phosphorylation sites correspond to numerous different parent proteins (the full sequences of which (human) are all publicly available in SwissProt database and their Accession numbers listed in Column B of Table 1/Fig. 2), each of which fall into discrete protein type groups, for example Adaptor/Scaffold proteins, Cytoskeletal proteins, Receptor Tyrosine Kinases, and RNA Binding proteins, etc. (see Column C of Table 1 ), the phosphorylation of which is relevant to signal transduction activity downstream of EGFR, as disclosed herein.
  • the 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.
  • the invention provides an isolated phosphorylation site- specific antibody that specifically binds a given EGFR-related signaling protein only when phosphorylated (or not phosphorylated, respectively) at a particular tyrosine enumerated in Column D of Table 1/ Figure 2 comprised within the phosphorylatable peptide site sequence enumerated in corresponding Column E.
  • the invention provides a heavy-isotope labeled peptide (AQUA peptide) for the detection and quantification of a given EGFR-related signaling protein, the labeled peptide comprising a particular phosphorylatable peptide site/sequence enumerated in Column E of Table 1/ Figure 2 herein.
  • the reagents provided by the invention is an isolated phosphorylation site-specific antibody that specifically binds the STAT3 transcription factor only when phosphorylated (or only when not phosphorylated) at tyrosine 539 (see Row 154 (and Columns D and E) of Table 1/ Figure 2).
  • the group of reagents provided by the invention is an AQUA peptide for the quantification of phosphorylated STAT3 protein, the AQUA peptide comprising phosphorylatable peptide sequence listed in Column E, Row 154, of Table 1/ Figure 2 (which encompasses the phosphorylatable tyrosine at position 539).
  • the invention provides an isolated phosphorylation site-specific antibody that specifically binds a human EGFR-relat ⁇ d 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.
  • 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.
  • the immortalized cell line is a rabbit or mouse hybridoma.
  • 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.
  • 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 are provided in Column C of Table 1/ Figure 2, and include: Actin Binding proteins, Adaptor/Scaffold proteins, Adhesion proteins, Apoptosis proteins, Axon Guidance proteins, Calcium-binding proteins, Cell Cycle Regulation proteins, Cell Surface proteins, Cellular Metabolism enzymes, Chaperones, Cytoskeletal proteins, DNA Binding proteins, DNA Replication proteins, GTPase Activating proteins, Guanine Nucleotide Exchange Factors, Hydrolases, Immunoglobulin Superfamily proteins, Lipid Kinases, Lipid Binding proteins, Motor proteins, Oxidoreductases, Proteases, Protein Kinases, Protein Phosphatases,
  • Particularly preferred subsets of the phosphorylation sites (and their corresponding proteins) disclosed herein are those occurring on the following protein types/groups listed in Column C of Table 1/ Figure 2: Actin Binding proteins, Adaptor/Scaffold proteins, Calcium-Binding Proteins, Cell Cycle Regulation proteins, Cytoskeletal proteins, DNA Binding and Replication Proteins, GTPase Activating proteins, Guanine Nucleotide Exchange Factor proteins, Lipid Kinases, Receptor Tyrosine Kinases, Receptor Tyrosine Kinase ligands, Protein Kinases, Receptor and Protein Phosphatases, Transcription Factor proteins, Tumor Suppressor proteins, and Vesicle proteins. Accordingly, among preferred subsets of reagents provided by the invention are isolated antibodies and AQUA peptides useful for the detection and/or quantification of the foregoing preferred protein/phosphorylation site subsets.
  • 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).
  • Catenin delta-1 Y174, Y213, Y248, Y321 , Y335) (see SEQ ID NOs: 2-6).
  • Adaptor/Scaffold protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphorylated at the site).
  • a heavy-isotope labeled 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.
  • antibodies and AQUA peptides for the detection/quantification of the following Adaptor/Scaffold protein phosphorylation sites are particularly preferred: CbI (Y445), IRS-2 (Y653, Y675, Y823), STAM1 (Y198), and STAM2 (Y113) (see SEQ ID NOs: 9, 13-15, and 25-26).
  • 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.
  • 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 ).
  • 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).
  • 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.
  • 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).
  • a heavy-isotope labeled 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.
  • 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 ).
  • RhoGAP p190B Y1108
  • ARHGEF5 Y19
  • 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).
  • 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.
  • a Protein Kinase non-receptor
  • Non-receptor phosphorylation sites are particularly preferred: BRSK1
  • 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.
  • 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).
  • 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).
  • EGFR Y998)
  • HER2 Y923
  • HER3 Y1307, Y1328
  • 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.
  • 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).
  • a heavy-isotope labeled peptide (AQUA peptide) for the quantification of the FAT tumor suppressor protein 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.
  • the immortalized cell line is a rabbit hybridoma or a mouse hybridoma.
  • a heavy-isotope labeled peptide (AQUA peptide) of the invention comprises a disclosed site sequence wherein the phosphorylatable tyrosine is phosphorylated.
  • a heavy-isotope labeled peptide of the invention comprises a disclosed site sequence wherein the phosphorylatable tyrosine is not phosphorylated.
  • Also provided by the invention are' methods for detecting or quantifying an EGFR-related signaling protein that is tyrosine- phosphorylated 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.
  • the reagents comprise a subset of preferred reagents as described above.
  • Table 1 above is identical to Figure 2, except that the latter includes the cell type(s) in which the particular phosphorylation site was identified (Column F).
  • Antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including F a b or antigen-recognition fragments thereof, including chimeric, polyclonal, and monoclonal antibodies.
  • EGFR-related signaling protein means any protein (or polypeptide derived therefrom) enumerated in Column A of Table 1/ Figure 2, which is disclosed herein as being phosphorylated in one or more EGFR- activated cell line(s).
  • EGFR-related signaling proteins may be direct substrates of EGFR, or may be indirect substrates downstream of EGFR in signaling pathways, or may be EGFR itself.
  • An EGFR-related signaling protein may also be phosphorylated in other cell lines harboring activated kinase activity.
  • Heavy-isotope labeled peptide (used 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 ef a/.), further discussed below.
  • Protein is used interchangeably with polypeptide, and includes protein fragments and domains as well as whole protein.
  • Phosphorylatable amino acid means any amino acid that is capable of being modified by addition of a phosphate group, and includes both forms of such amino acid.
  • 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.
  • 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.
  • a general phosphotyrosine-specific monoclonal antibody (commercially available from Cell Signaling Technology, Inc., Beverly, MA, Cat #9411 (p-Tyr-100)) was used in the immunoaffinity step to isolate the widest possible number of phospho- tyrosine containing peptides from the EGFR-activated cell extracts.
  • Extracts from the following EGFR-activated, human carcinoma tumor cells lines were employed: A431 (skin), HCT116 (colon), HPAC (pancreas), MIAPACA2 (pancreas), PANIC- 1 (pancreas), A549 (lung), BxPC-3 (pancreas), DU 145 (prostate), HT-29 (colon), H460 (lung), and LNCaP (prostate).
  • A431 skin
  • HCT116 colon
  • HPAC pancreas
  • MIAPACA2 pancreas
  • PANIC- 1 pancreas
  • A549 lung
  • BxPC-3 pancreas
  • DU 145 prostate
  • HT-29 colon
  • H460 lung
  • LNCaP prostate
  • lysates were prepared from these cells line and digested with trypsin after treatment with DTT and iodoacetamide to alkylate cysteine residues.
  • peptides were pre-fractionated by reversed-phase solid phase extraction using Sep-Pak Cia 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 x 75 ⁇ m 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.
  • Isolated phosphorylation site-specific antibodies that specifically bind an EGFR-related signaling protein disclosed in Column A of Table 1 only when phosphorylated (or only when not phosphorylated) at the corresponding amino acid and phosphorylation site listed in Columns D and E of Table 1 may now be produced by standard antibody production methods, such as anti-peptide antibody methods, using the phosphorylation site sequence information provided in Column E of Table 1. For example, two previously unknown HER3 kinase phosphorylation sites (tyrosines 1307 and 1328) (see Rows 125-126 of Table 1/Fig. 2) are presently disclosed.
  • antibodies that specifically bind any either of these novel HER3 sites can now be produced by immunizing an animal with a peptide antigen comprising all or part of the amino acid sequence encompassing the respective phosphorylated residue (e.g. a peptide antigen comprising the sequence set forth in Row 126, Column E, of Table 1 (SEQ ID NO: 125) (which encompasses the phosphorylated tyrosine at position 1328 in HER3), to produce an antibody that only binds HER3 when phosphorylated at that site.
  • a peptide antigen comprising all or part of the amino acid sequence encompassing the respective phosphorylated residue
  • SEQ ID NO: 125 which encompasses the phosphorylated tyrosine at position 1328 in HER3
  • 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.
  • a suitable animal e.g., rabbit, goat, etc.
  • 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
  • a suitable animal e.g., rabbit, goat, etc.
  • 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.
  • the peptide antigen includes the phosphorylated form of the amino acid. Conversely, if an antibody that only binds the protein when not phosphorylated at the disclosed site is desired, the peptide antigen includes the non- phosphorylated form of the amino acid.
  • Peptide antigens suitable for producing antibodies of the invention may be designed, constructed and employed in accordance with well- known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 (1991 ); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962)).
  • 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, CURRENT PROTOCOLS IN MOLECULAR BiOLOGY, Ausubel et al. Eds. (1989). Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of diagnostic assay methods provided by the invention. For example, a solution containing the appropriate antigen may be injected into a mouse or other species and, after a sufficient time (in keeping with conventional techniques), the animal is sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • Rabbit fusion hybridomas may be produced as described in U.S Patent No. 5,675,063, C. Knight, Issued October 7, 1997.
  • the hybridoma cells are then grown in a suitable selection media, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below.
  • the secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.
  • Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'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. ScL 1 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.
  • 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.
  • 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 F ab 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 ai, Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Natl Acad. ScL 81- 6851 (1984); Neuberger et ai, 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 ai.)
  • 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.
  • hybridoma clones constructed as described above, that produce monoclonal antibodies to the EGFR- related signaling protein phosphorylation sitess disclosed herein are also provided.
  • the invention includes recombinant cells producing an antibody of the invention, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)
  • Phosphorylation site-specific antibodies of the invention 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 ).
  • 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.
  • 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.
  • 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 immunohistochemical staining may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g.
  • tumor tissue is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
  • 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).
  • samples may be centrifuged on Ficoll gradients to remove erythrocytes, and cells may then be fixed with 2% paraformaldehyde for 10 minutes at 37 0 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.
  • a flow cytometer e.g. a Beckman Coulter FC500
  • 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.
  • fluorescent dyes e.g. Alexa488, PE
  • CD34 cell marker
  • 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, perse.
  • 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.
  • 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 a/. Proc. Natl. Acad. Sci. U.S.A.
  • 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.
  • 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.
  • 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 ( 13 C, 15 N).
  • 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.
  • LC-SRM reaction monitoring
  • 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.
  • 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.
  • 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.
  • serine proteases e.g. trypsin, hepsin
  • metallo proteases e.g. PUMP1
  • chymotrypsin cathepsin
  • pepsin pepsin
  • thermolysin carboxypeptidases
  • 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.
  • the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins.
  • a peptide is preferably at least about 6 amino .
  • 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.
  • 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).
  • 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 (Ae. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods.
  • 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.
  • the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum.
  • the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.
  • the label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice.
  • the label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive.
  • the label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectrometric pattern at each labeled fragment position. Stable isotopes, such as 2 H, 13 C, 15 N, 17 0, 18 O, or 34 S, 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.
  • CID collision-induced dissociation
  • the fragments are then analyzed, for example by multi-stage mass spectrometry (MS ⁇ ) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature.
  • MS ⁇ multi-stage mass spectrometry
  • 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 MS 3 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.
  • 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.
  • 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 MS n 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.
  • AQUA internal peptide standards may now be produced, as described above, for any of the 168 novel EGFR-related signaling protein phosphorylation sites disclosed herein (see Table 1/ Figure 2).
  • Peptide standards for a given phosphorylation site e.g. the tyrosine 653 site in IRS-2 - see Row 114 of Table 1
  • may be produced for both the phosphorylated and non-phosphorylated forms of the site e.g. see IRS-2 site sequence in Column E, Row 114 of Table 1
  • such standards employed in the AQUA methodology employed in the AQUA methodology to detect and quantify both forms of such phosphorylation site in a biological sample.
  • AQUA peptides of the invention may comprise all, or part of, a phosphorylation site peptide sequence disclosed herein (see Column E of Table 1/ Figure 2).
  • an AQUA peptide of the invention comprises a phosphorylation site sequence disclosed herein in Table 1/ Figure 2.
  • Heavy-isotope labeled equivalents of an of the peptides enumerated in Table 1/ Figure 2 can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments.
  • the phosphorylation site peptide sequences disclosed herein are particularly well suited for development of corresponding AQUA peptides, since the IAP method by which they were identified (see Part A above and Example 1) inherently confirmed that such peptides are in fact produced by enzymatic digestion (trypsinization) and are in fact suitably fractionated/ionized in MS/MS.
  • heavy-isotope labeled equivalents of these peptides can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments.
  • the invention provides heavy-isotope labeled peptides (AQUA peptides) for the detection and/or quantification of any of the EGFR-related phosphorylation sites disclosed in Table 1 / Figure 2 (see Column E) and/or their corresponding parent proteins/polypeptides (see Column A).
  • a phosphopeptide sequence comprising any of the phosphorylation sequences listed in Table 1 may be considered a preferred AQUA peptide of the invention.
  • 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.
  • the above-described AQUA peptides corresponding to the both the phosphorylated and non-phosphorylated forms of the disclosed STAT3 tyrosine 593 phosphorylation site may be used to quantify the amount of phosphorylated STAT3(Tyr593) in a biological sample, e.g. a tumor cell sample (or a sample before or after treatment with a test drug).
  • AQUA peptides of the invention may also be employed within a kit that comprises one or multiple AQUA pepf ⁇ de(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.
  • 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.
  • Antibodies provided by the invention may be advantageously employed in a variety of standard immunological assays (the use of
  • Assays may be homogeneous assays or heterogeneous assays.
  • 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.
  • 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.
  • 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.
  • 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-lmmunoassay, (1980) (CRC Press, Inc., Boca Raton, FIa.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et al., "Methods for Modulating Ligand-Receptor Interactions and their
  • 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., 35 S, 125 1, 131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • radiolabels e.g., 35 S, 125 1, 131 I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescent labels e.g., fluorescein
  • 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.
  • 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.
  • 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.
  • 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 0 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 Coulter EPICS-XL
  • IHC immunohistochemical staining to detect differences in signal transduction or protein activity using normal and diseased tissues.
  • IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, supra. Briefly, paraffin-embedded tissue (e.g.
  • tumor tissue is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
  • Antibodies of the invention may be also be optimized for use in other clinically-suitable applications, for example bead-based multiplex- type assays, such as IGEN, LuminexTM and/or BioplexTM assay formats, or otherwise optimized for antibody arrays formats, such as reversed- phase array applications (see, e.g. Paweletz et al., Oncogene 20(16): 1981-89 (2001 )).
  • bead-based multiplex- type assays such as IGEN, LuminexTM and/or BioplexTM assay formats
  • antibody arrays formats such as reversed- phase array applications
  • the invention provides a method for the multiplex detection of EGFR-related protein phosphorylation in a biological sample, the method comprising utilizing at two or more antibodies or AQUA peptides of the invention to detect the presence of two or more phosphorylated EGFR-related signaling proteins enumerated in Column A of Table 1 / Figure 2.
  • two to five antibodies or AQUA peptides of the invention are employed in the method.
  • six to ten antibodies or AQUA peptides of the invention are employed, while in another preferred embodiment eleven to twenty such reagents are employed.
  • Antibodies and/or AQUA peptides of the invention may also be employed within a kit that comprises at least one phosphorylation site- specific antibody or AQUA peptide of the invention (which binds to or detects an EGFR-related signal transduction protein disclosed in Table 1), and, optionally, a second antibody conjugated to a detectable group.
  • 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 . .
  • 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.
  • 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.
  • 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.
  • Sonicated cell lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM.
  • protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK-trypsin (Worth ington) was added at 10-20 ⁇ g/mL Digestion was performed for 1-2 days at room temperature.
  • Trifluoroacetic acid was added to protein digests to a final concentration of 1%, precipitate was removed by centrifugation, and digests were loaded onto Sep-Pak C-is columns (Waters) equilibrated with 0.1% TFA. A column volume of 0.7-1.0 ml was used per 2 x 10 8 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.
  • TFA Trifluoroacetic acid
  • Fractions Il 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 x 10 8 cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HCI or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCI) and insoluble matter (mainly in peptide fractions III) was removed by centrifugation. IAP was performed on each peptide fraction separately.
  • the phosphotyrosine 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 ⁇ l, 60 ⁇ g) 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 ⁇ l of 0.1% TFA at room temperature for 10 min.
  • 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 % acetonitirile in 0.1% TFA and combination of all eluates.
  • IAP on this peptide fraction was performed as follows: After lyophilization, peptide was dissolved in 1.4 ml IAP buffer (MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCI) and insoluble matter was removed by centrifugation. Immobilized antibody (40 ⁇ l, 160 ⁇ g) 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 ⁇ l of 0.15% TFA at room temperature for 10 min (eluate 1 ), followed by a wash of the beads (eluate 2) with 45 ⁇ l of 0.15% TFA. Both eluates were combined.
  • IAP eluate 40 ⁇ l of IAP eluate were purified by 0.2 ⁇ l StageTips or ZipTips.
  • Peptides were eluted from the microcolumns with 1 ⁇ l of 40% MeCN, 0.1% TFA (fractions I and II) or 1 ⁇ l of 60% MeCN, 0.1% TFA (fraction III) into 7.6 ⁇ l of 0.4% acetic acid/0.005% heptafluorobutyric acid.
  • This sample was loaded onto a 10 cm x 75 ⁇ m 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).
  • 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 1 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4 x 10 5 ; 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.
  • 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).
  • Polyclonal antibodies that specifically bind an EGFR-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein 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.
  • BRSK1 specific BRSK1 (tyr121) polyclonal antibodies as described in Immunization/Screening below.
  • IRS-2 (tyrosine 823).
  • a synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and rabbits are injected intradermally (ID) on the back with antigen in complete Freunds adjuvant (500 ⁇ g antigen per rabbit). The rabbits are boosted with same antigen in incomplete Freund adjuvant (250 ⁇ g antigen per rabbit) every three weeks. After the fifth boost, bleeds are collected. The sera are purified by Protein A-affinity chromatography by standard methods (see ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, supra.).
  • the eluted immunoglobulins are further loaded onto a non-phosphorylated synthetic peptide antigen-resin Knotes column to pull out antibodies that bind the non-phdsphorylated form of the phosphorylation site.
  • the flow through fraction is collected and applied onto a phospho-synthetic peptide antigen-resin column to isolate antibodies that bind the phosphorylated form of the site.
  • the bound antibodies i.e. antibodies that bind a phosphorylated peptide described in A-C above, but do not bind the non-phosphorylated form of the peptide, are eluted and kept in antibody storage buffer.
  • the isolated antibody is then tested for phospho-specificity using Western blot assay using an appropriate cell line the expresses (or overexpresses) target phospho-protein (i.e. phosphorylated HER2, BRSK1 , or IRS-2, for example, HT-29, A431 and HCT-116 cells, respectively.
  • Cells are cultured in DMEM supplemented with 10% FCS. Before stimulation, the cells are starved in serum-free DMEM medium for 4 hours. The cells are then stimulated ligand (e.g. EGF 100 ng/ml) for 5 minutes. Cell are collected, washed with PBS and directly lysed in cell lysis buffer. The protein concentration of cell lysates are then measured.
  • ligand e.g. EGF 100 ng/ml
  • the loading buffer is added into cell lysate and the mixture is boiled at 100 0 C for 5 minutes. 20 ⁇ l (10 ⁇ g protein) of sample is then added onto 7.5% SDS-PAGE gel.
  • a standard Western blot may be performed according to the lmmunoblotting Protocol set out in the CELL SIGNALING TECHNOLOGY, INC. 2003-04 Catalogue, p. 390.
  • the isolated phospho-specific antibody is used at dilution 1 :1000. Phosphorylation-site specificity of the antibody will be shown by binding of only the phosphorylated form of the target protein.
  • Isolated phospho-specific polyclonal antibody does not recognize the target protein when not phosphorylated at the appropriate phosphorylation site in the non-stimulated cells (e.g. IRS-2 is not bound when not phosphorylated at tyrosine 823).
  • Monoclonal antibodies that specifically bind a EGFR-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein 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.
  • EphB4 (tyrosine 574).
  • This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal EphB4(tyr574) antibodies as described in Immunization/ Fusion/Screening below.
  • B. MINK tyrosine 906
  • This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal MINK(tyr906) antibodies as described in Immunization/ Fusion/Screening below.
  • This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal PTP-kappa (tyr858) antibodies as described in Immunization/Fusion/ Screening below.
  • a synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and BALB/C mice are injected intradermal ⁇ (ID) on the back with antigen in complete Freunds adjuvant (e.g. 50 ⁇ g antigen per mouse). The mice are boosted with same antigen in incomplete Freund adjuvant (e.g. 25 ⁇ g antigen per mouse) every three weeks. After the fifth boost, the animals are sacrificed and spleens are harvested.
  • ID intradermal ⁇
  • complete Freunds adjuvant e.g. 50 ⁇ g antigen per mouse
  • incomplete Freund adjuvant e.g. 25 ⁇ g antigen per mouse
  • 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.
  • the MS n 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.
  • a biological sample such as a digested cell extract.
  • 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.
  • 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.
  • 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.
  • the Taiin 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.
  • Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acid monomers may be obtained from AnaSpec (San Jose, CA).
  • Fluorenylmethoxycarbonyl (Fmoc)-derivatized stable- isotope monomers containing one 15 N and five to nine 13 C atoms may be obtained from Cambridge Isotope Laboratories (Andover, MA).
  • Preloaded Wang resins may be obtained from Applied Biosystems. Synthesis scales may vary from 5 to 25 ⁇ mol.
  • 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, MA) and ion-trap (ThermoFinnigan, LCQ DecaXP) MS.
  • MS/MS spectra for each AQUA peptide should exhibit a strong y- type ion peak as the most intense fragment ion that is suitable for use in an SRM monitoring/analysis.
  • Reverse-phase microcapillary columns (0.1 A ⁇ 150-220 mm) are prepared according to standard methods.
  • An Agilent 1100 liquid chromatograph may be used to develop and deliver a solvent gradient [0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA)/7% methanol and 0.4% acetic acid/0.005% HFBA/65% methanol/35% acetonitrile] to the microcapillary column by means of a flow splitter.
  • HFBA heptafluorobutyric acid
  • 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.
  • Target protein e.g. a phosphorylated protein of A-D above
  • AQUA peptide as described above.
  • the IAP method is then applied to the complex mixture of peptides derived from proteolytic cleavage of crude cell extracts to which the AQUA peptides have been spiked in.
  • LC-SRM of the entire sample is then carried out.
  • MS/MS may be performed by using a ThermoFinnigan (San Jose, CA) mass spectrometer (LCQ DecaXP ion trap or TSQ Quantum triple quadrupole).
  • LCQ DecaXP ion trap or TSQ Quantum triple quadrupole On the DecaXP, parent ions are isolated at 1.6 m/z width, the ion injection time being limited to 150 ms per microscan, with two microscans per peptide averaged, and with an AGC setting of 1 x 10 8 ; 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.
  • 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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Abstract

La présente invention décrit 168 nouveaux sites de phosphorylation identifiés parmi les protéines de transduction de signal ainsi que dans les voies en aval de la EGFR kinase, incluant la EGFR kinase elle-même. La présente invention concerne également des anticorps spécifiques des sites de phosphorylation et des peptides marqués à l'aide d'isotopes lourds (peptides AQUA) permettant la détection et la quantification sélectives de ces sites et/ou protéines phosphorylés, ainsi que des méthodes d'emploi de réactifs pour aboutir à ce résultat. Parmi les sites de phosphorylation identifiés se trouvent des sites contenus dans les types de protéines suivants : protéines Liant l'Actine, protéines Adaptatrices/Pièges, Protéines Liant le Calcium, protéines de Régulation du Cycle Cellulaire, protéines du Cytosquelette, protéines de Liaison et de Réplication de l'ADN, protéines d’Activation de la GTPase, protéines de type Facteur d'Échange Nucléotidique de la Guanine, Lipide Kinases, Récepteurs Tyrosine Kinases, ligands de Récepteurs Tyrosine Kinases, Protéine Kinases, Phosphatases de Protéines et de Récepteurs, protéines de type Facteur de Transcription, protéines de type Élimination de Tumeurs, et protéines de Vésicule.
PCT/US2004/042940 2004-12-21 2004-12-21 Phosphorylation des protéines suivant des voies contrôlées par les egfr WO2006068640A1 (fr)

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CA002601700A CA2601700A1 (fr) 2004-12-21 2004-12-21 Phosphorylation des proteines suivant des voies controlees par les egfr
PCT/US2004/042940 WO2006068640A1 (fr) 2004-12-21 2004-12-21 Phosphorylation des protéines suivant des voies contrôlées par les egfr
US11/821,130 US7807789B2 (en) 2004-12-21 2007-06-21 Reagents for the detection of protein phosphorylation in EGFR-signaling pathways
US12/897,363 US20110130547A1 (en) 2004-12-21 2010-10-04 Reagents For The Detection Of Protein Phosphorylation In EGFR Signaling Pathways

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WO2008008998A2 (fr) * 2006-07-13 2008-01-17 Cell Signaling Technology, Inc. Réactifs pour la détection de phosphorylation de protéines dans les chemins de signalisation
WO2008009004A3 (fr) * 2006-07-13 2008-10-30 Cell Signaling Technology Inc Réactifs pour la détection de la phosphorylation des protéines dans les voies de signalisation
WO2008008998A3 (fr) * 2006-07-13 2008-12-24 Cell Signaling Technology Inc Réactifs pour la détection de phosphorylation de protéines dans les chemins de signalisation
WO2008009004A2 (fr) * 2006-07-13 2008-01-17 Cell Signaling Technology, Inc. Réactifs pour la détection de la phosphorylation des protéines dans les voies de signalisation
US10416162B2 (en) 2007-12-20 2019-09-17 Monogram Biosciences, Inc. Her2 diagnostic methods
US9766242B2 (en) * 2009-01-15 2017-09-19 Laboratory Corporation Of America Holdings Methods of determining patient response by measurement of HER-3 and P95
US20130071859A1 (en) * 2009-01-15 2013-03-21 Laboratory Corporation Of America Holdings Methods of Determining Patient Response by Measurement of HER-3
US10775382B2 (en) 2009-01-15 2020-09-15 Laboratory Corporation Of America Holdings Methods of determining patient response by measurement of HER-3
US9110066B2 (en) * 2009-01-15 2015-08-18 Laboratory Corporation Of America Holdings HER-3 antibodies and methods of use
US20160061839A1 (en) * 2009-01-15 2016-03-03 Laboratory Corporation Of America Holdings Methods of Determining Patient Response by Measurement of HER-3
US9085622B2 (en) 2010-09-03 2015-07-21 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
WO2012092531A1 (fr) * 2010-12-29 2012-07-05 Expression Pathology, Inc. Analyse par srm/mrm de protéines her3
US9869680B2 (en) 2010-12-29 2018-01-16 Expression Pathology, Inc. HER3 protein SRM/MRM assay
AU2017201499B2 (en) * 2010-12-29 2018-09-13 Expression Pathology, Inc. Her3 protein SRM/MRM assay
US9128102B2 (en) 2010-12-29 2015-09-08 Expression Pathology, Inc. Her3 protein SRM/MRM assay
EP2658868A4 (fr) * 2010-12-29 2015-08-12 Expression Pathology Inc Analyse par srm/mrm de protéines her3

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