WO2007027957A2 - Reagents for the detection of protein phosphorylation in leukemia signaling pathways - Google Patents

Reagents for the detection of protein phosphorylation in leukemia signaling pathways Download PDF

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WO2007027957A2
WO2007027957A2 PCT/US2006/034126 US2006034126W WO2007027957A2 WO 2007027957 A2 WO2007027957 A2 WO 2007027957A2 US 2006034126 W US2006034126 W US 2006034126W WO 2007027957 A2 WO2007027957 A2 WO 2007027957A2
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rows
corresponding column
protein
phosphorylated
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WO2007027957A3 (en
WO2007027957A9 (en
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Roberto Polakiewicz
Valerie Goss
Kimberly Lee
Ting-Lei Gu
Albrecht Moritz
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Cell Signaling Technology, Inc.
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Priority to EP06802749A priority Critical patent/EP1929305A2/en
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Publication of WO2007027957A9 publication Critical patent/WO2007027957A9/en
Publication of WO2007027957A3 publication Critical patent/WO2007027957A3/en

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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/008—Peptides; Proteins
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
    • 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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • 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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • 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
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

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.
  • Leukemia is a malignant disease of the bone marrow and blood, characterized by abnormal accumulation of blood cells, and is divided in four major categories. An estimated 33,500 new cases of leukemia will be diagnosed in the U.S. alone this year, affecting roughly 30,000 adults and 3,000 children, and close to 24,000 patients will die from the disease (Source: The Leukemia & Lymphoma Society (2004)). Depending of the cell type involved and the rate by which the disease progresses it can be defined as acute or chronic myelogenous leukemia (AML or CML), or acute and chronic lymphocytic leukemia (ALL or CLL).
  • AML or CML acute or chronic myelogenous leukemia
  • ALL or CLL acute and chronic lymphocytic leukemia
  • the acute forms of the disease rapidly progress, causing the accumulation of immature, functionless cells in the marrow and blood, which in turn results in anemia, immunodeficiency and coagulation deficiencies, respectively.
  • Chronic forms of leukemia progress more slowly, allowing a greater number of mature, functional cells to be produced, which amass to high concentration in the blood over time.
  • AML acute myelogenous leukemia
  • ALL acute lymphocytic leukemia
  • CLL Chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • the resulting BCR-AbI kinase protein is constitutively active and elicits characteristic signaling pathways that have been shown to drive the proliferation and survival of CML cells (see Daley, Science 247: 824-830 (1990); Raitano et al., Biochim. Biophys. Acta. Dec 9; 1333(3): F201-16 (1997)).
  • lmanitib also known as STI571 or Gleevec®
  • the first molecularly targeted compound designed to specifically inhibit the tyrosine kinase activity of BCR-AbI provided critical confirmation of the central role of BCR-AbI signaling in the progression of CML (see Schindler et al., Science 289: 1938-1942 (2000); Nardi et al., Curr. Opin. Hematol. 11: 35-43 (2003)).
  • Gleevec® now serves as a paradigm for the development of targeted drugs designed to block the activity of other tyrosine kinases known to be involved in leukemias and other malignancies (see, e.g., Sawyers, Curr. Opin. Genet. Dev. Feb; 12(1): 111-5 (2002); Druker, Adv. Cancer Res. 911-30 (2004)).
  • tyrosine kinases known to be involved in leukemias and other malignancies
  • FLT3 Fms-like tyrosine kinase 3
  • RTK class III receptor tyrosine kinase family including FMS, platelet-derived growth factor receptor (PDGFR) and c- KIT
  • PDGFR platelet-derived growth factor receptor
  • c- KIT c- KIT
  • FLT3 is the single most common activated gene in AML known to date. This evidence has triggered an intensive search for FLT3 inhibitors for clinical use leading to at least four compounds in advanced stages of clinical development, including: PKC412 (by Novartis), CEP- 701 (by Cephalon), MLN518 (by Millenium Pharmaceuticals), and SU5614 (by Sugen/Pfizer) (see Stone et al., Blood (in press)(2004); Smith et al., Blood 103: 3669-3676 (2004); Clark etal., Blood 104: 2867-2872 (2004); and Spiekerman et al., Blood 101: 1494-1504 (2003)).
  • diagnosis of leukemia is made by tissue biopsy and detection of different cell surface markers.
  • misdiagnosis can occur since some leukemia cases can be negative for certain markers, and because these markers may not indicate which genes or protein kinases may be deregulated.
  • the genetic translocations and/or mutations characteristic of a particular form of leukemia can be sometimes detected, it is clear that other downstream effectors of constitutively active kinases having potential diagnostic, predictive, or therapeutic value, remain to be elucidated. Accordingly, identification of downstream signaling molecules and phosphorylation sites involved in different types of leukemia 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 this disease.
  • the invention discloses nearly 480 novel phosphorylation sites identified in signal transduction proteins and pathways underlying human Leukemias 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, quantification, and profiling of the disclosed phosphorylation sites.
  • FIG. 1 - I 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 330 phosphorylation site in DOK2 (see Row 24 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y * indicates the phosphorylated tyrosine (shown as lowercase "y” in Figure 2).
  • FIG. 4 - is an exemplary mass spectrograph depicting the detection of the tyrosine 630 phosphorylation site in FLT3 (see Row 286 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase "y” in Figure 2).
  • FIG. 3 - is an exemplary mass spectrograph depicting the detection of the tyrosine 330 phosphorylation site in DOK2 (see Row 24 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected
  • FIG. 5 - is an exemplary mass spectrograph depicting the detection of the tyrosine 1736 phosphorylation site in TSC2 (see Row 87 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase "y” in Figure 2).
  • FIG. 6 - is an exemplary mass spectrograph depicting the detection of the tyrosine 260 phosphorylation site in ICAM (see Row 71 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase "y” in Figure 2).
  • 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, Protein Kinases, and Adhesion proteins, etc. (see Column C of Table 1), the phosphorylation of which is relevant to signal transduction activity underlying Leukemias (AML, CML, CLL, and ALL), as disclosed herein.
  • AML, CML, CLL, and ALL Leukemias
  • the invention provides novel reagents - phospho-specific antibodies and AQUA peptides - for the specific detection and/or quantification of a Leukemia-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 Leukemia-related signaling proteins using the phosphorylation-site specific antibodies and AQUA peptides of the invention, and methods of obtaining a phosphorylation profile of such proteins (e.g. Kinases).
  • the invention provides an isolated phosphorylation site- specific antibody that specifically binds a given Leukemia-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 Leukemia-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 MELK tyrosine kinase only when phosphorylated (or only when not phosphorylated) at tyrosine 438 (see Row 244 (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 MELK tyrosine kinase, the AQUA peptide comprising the phosphorylatable peptide sequence listed in Column E, Row 244, of Table 1 / Figure 2 (which encompasses the phosphorylatable tyrosine at position 438).
  • the invention provides an isolated phosphorylation site-specific antibody that specifically binds a human Leukemia-related signaling protein selected from Column A of Table 1 (Rows 2-481) only when phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21, 23, 26-30, 32-34, 36-45, 48- 52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182-193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319- 336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440- 474, and 476-480), wherein said antibody does not
  • the invention provides an isolated phosphorylation site- specific antibody that specifically binds a Leukemia-related signaling protein selected from Column A of Table 1 only when not phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1 , comprised within the peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182- 193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319-336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440-474, and 476-480), wherein said antibody does not bind said signaling protein when phosphorylated at said t
  • 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 a Leukemia- 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: 1-7, 9-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182-193, 195-197, 199-208, 210-221 , 223-279, 281- 294, 296-297, 299-316, 319-336, 339-345, 347-356, 358, 360-366, 368- 378, 380-417, 419-438, 440-474, and 476-480), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D of
  • the phosphorylatable tyrosine within the labeled peptide is phosphorylated, while in other preferred embodiments, the phosphorylatable residue within the labeled peptide is not phosphorylated.
  • Reagents (antibodies and AQUA peptides) provided by the invention may conveniently be grouped by the type of Leukemia-related signaling protein in which a given phosphorylation site (for which reagents are provided) occurs.
  • each respective protein includes: adaptor/scaffold proteins, acetyltransferases, actin binding proteins, adhesion proteins, apoptosis proteins, calcium-binding proteins, cell cycle regulation proteins, cell surface proteins, channel proteins, chaperone proteins, contractile proteins, cytokine proteins, cytoskeletal proteins, G protein regulators and GTPase activating proteins, guanine nucleotide exchange factors, helicase proteins, immunoglobulin superfamily proteins, inhibitor proteins, protein kinases, lipid kinases, ligases, lipid binding proteins, methytransferases, motor proteins, oxidoreductases, phosphotases, phosphodiesterases, phospholipases, proteases, receptor proteins, transcription factors, transferases, translation/transporter proteins, and ubiquitin conjugating system proteins.
  • adaptor/scaffold proteins include: adaptor/scaffold proteins, acetyltransferases, actin binding proteins, adhesion proteins,
  • 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, are the protein kinases adaptor/scaffold proteins, adhesion proteins, cell cycle regulation proteins, cell surface proteins, transcription proteins, phosphatases, phospholipases, phosphodiesterases, receptor proteins, cytoskeltal proteins, G protein regulators, and lipid kinases. 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 protein kinase phosphorylation sites are particularly preferred: ATM (Y2129), MELK (Y438), MAPK14 (Y24), BLK (Y187), BTK (Y344), SYK (Y296), ZAP70 (Y69), FLT3 (Y630), FLT3 (Y726), FLT3 (Y768), and ROS1 (Y363) (see SEQ ID NOs: 229, 243, 249, 270-271 , 273, 283, and 285-288).
  • antibodies and AQUA peptides for the detection/quantification of the following adaptor/scaffold protein phosphorylation sites are particularly preferred: ABI2 (Y192), PIK3AP1 (Y594), DOK2 (Y330), LAT2 (Y40), SIT1 (Y127), STAM (Y384), SCAP1 (Y142) (see SEQ ID NOs: 10, 14, 23, 30, 37, 40-41).
  • ABI2 Y192
  • PIK3AP1 Y594
  • DOK2 Y330
  • LAT2 Y40
  • SIT1 Y127
  • STAM STAM
  • SCAP1 SCAP1
  • ADAM18 Y197
  • ICAM2 Y260
  • PECAM 1 Y663
  • TSC2 Y1736
  • SEQ ID NO: 86 see SEQ ID NO: 86.
  • antibodies and AQUA peptides for the detection/quantification of the following cell surface protein phosphorylation sites are particularly preferred: CD72 (Y39) and CD84 (Y299) (see SEQ ID NOs: 89 and 93).
  • antibodies and AQUA peptides for the detection/quantification of the following trascription factor/coactivor/corepressor phosphorylation sites are particularly preferred: STAT5A (Y22), STAT5A (Y90), STAT5A (Y114), SMAD2 (Y102), and NSEP1 (Y208) (see SEQ ID NOs: 410-413 and 417).
  • antibodies and AQUA peptides for the detection/quantification of the following phosphatase phosphorylation sites are particularly preferred: INPPL1 (Y831), INPPL1 (Y1135) and PTPRC (Y705) (see SEQ ID NOs: 336-337, and 347).
  • An isolated phosphorylation site-specific antibody specifically binds a phosphodiesterase/phospholipase selected from Column A, Rows 1 351- 359, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 351-359, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 351-359, of Table 1 (SEQ ID NOs: 350-356, and 358), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • antibodies and AQUA peptides for the detection/quantification of the following phosphodiesterase/phospholipase phosphorylation sites are particularly preferred: PLCG1 (Y481), PLCG2 (Y680) and PLCG2 (Y1264) (see SEQ ID NOs: 352, 354 and 358).
  • antibodies and AQUA peptides for the detection/quantification of the following receptor protein phosphorylation sites are particularly preferred: LEPR (Y795) (see SEQ ID NO: 365).
  • a heavy-isotope labeled peptide for the quantification of a Leukemia-related signaling protein that cytoskeletal protein selected from Column A, Rows 114-170, said labeled peptide comprising the phosphoryiatable peptide sequence listed in corresponding Column E, Rows 114-170, of Table 1 (SEQ ID NOs: 113-119, 121-124, 129-151 , 153-160, and 163-169), which sequence comprises the phosphoryiatable tyrosine or serine listed in corresponding Column D, Rows 114-170, of Table 1.
  • GNAI2 Y61
  • SEQ ID NO: 177 SEQ ID NO: 177
  • antibodies and AQUA peptides for the detection/quantification of the following lipid kinase phosphorylation sites are particularly preferred: PIK3CB (Y962) (see SEQ ID NO: 293).
  • An isolated phosphorylation site-specific antibody that specifically binds a protein selected from the group consisting of EIF4EBP2, EIF4G2 and EIF4B (Column A, Rows 446, 448 and 460 of Table 1) only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 191 , 199, 446, 448 and 460 of Table 1), said tyrosine comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 446, 448 and 460, of Table 1 (SEQ ID NOs: 445, 447 and 459), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
  • 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 (for example, an AQUA peptide within any of the foregoing preferred subsets of AQUA peptides) 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 a Leukemia-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 Leukemia-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.
  • Also provided by the invention is a method for obtaining a phosphorylation profile of protein kinases that are phosphorylated in Leukemia signaling pathways, said method comprising the step of utilizing one or more isolated antibody that specifically binds a protein inase selected from Column A, Rows 210-291 , of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 210-291 , of Table 1, comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: SEQ ID NOs: 210-221 , 223-280, and 281-290), to detect the phosphorylation of one or more of said protein kinases, thereby obtaining a phosphorylation profile for said kinases.
  • Table 1 above is identical to Figure 2, except that the latter includes the disease and cell type(s) in which the particular phosphorylation site was identified (Columns F and G).
  • 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.
  • Leukemia-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 leukemia cell line(s).
  • Leukemia-related signaling proteins may be tyrosine kinases, such as Flt-3 or BCR-AbI, or serine/threonine kinases, or direct substrates of such kinases, or may be indirect substrates downstream of such kinases in signaling pathways.
  • a Leukemia-related signaling protein may also be phosphorylated in other cell lines (non-leukemic) 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 et a/.), further discussed below.
  • Protein is used interchangeably with polypeptide, and includes protein fragments and domains as well as whole protein.
  • Phosphorylatable amino acid means any amino acid that is capable of being modified by addition of a phosphate group, and includes both forms of such amino acid.
  • 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 cell extracts.
  • Extracts from the following human Leukemia cell lines were employed: Jurkat, K562, SEM, HT-93, CTV-1 , MOLT15, CLL-9, H 1993, OCL-ly3, KBM-3, UT-7, SUPT-13,
  • 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 Cis 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 a phosphotyrosine antibody (P-Tyr-100, CST #9411) immobilized on protein G-Sepharose or Protein A-Sepharose.
  • Immunoaffinity-purified peptides were eluted with 0.1 % TFA and a portion of this fraction was concentrated with Stage or Zip tips and analyzed by LC-MS/MS, using a ThermoFinnigan LTQ 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 a Leukemia-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/ Figure 2 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 BCR kinase phosphorylation sites (tyrosines 58 and 231) (see Rows 225-226 of Table 1/Fig. 2) are presently disclosed.
  • antibodies that specifically bind either of these novel BCR kinase sites can now be produced, e.g. 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 225, Column E, of Table 1 (SEQ ID NO: 224) (which encompasses the phosphorylated tyrosine at position 58 in BCR), to produce an antibody that only binds BCR kinase when phosphorylated at that site.
  • 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 225, Column E, of Table 1 (SEQ ID NO: 224) (which encompasses the phosphorylated tyrosine at position 58 in BCR), to produce an antibody that only binds BCR kinase when phospho
  • 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 Leukemia-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 Leukemia-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 all or part of any one 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,
  • a peptide antigen may comprise the full sequence disclosed in Column E of Table 1/ Figure 2, 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 an uppercase "Y").
  • a desirable peptide antigen will comprise four or more amino acids flanking each side of the phosphorylatable amino acid and encompassing it.
  • 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.
  • 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 MELK tyrosine 438 phosphorylation site sequence disclosed in Row 244, Column E of Table 1), and antibodies of the invention thus specifically bind a target Leukemia-related signaling 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.
  • 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 F a b 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. Natl Acad. ScL 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.
  • hybridoma clones constructed as described above, that produce monoclonal antibodies to the Leukemia- 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. coll (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 Leukemia-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 to 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 Leukemia-related signaling protein epitope for which the antibody of the invention is specific.
  • polyclonal antisera may exhibit some undesirable general cross-reactivity to phosphotyrosine itself, 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 Leukemia- related phosphorylation and activation status in diseased tissue.
  • IHC immunohistochemical
  • IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g.
  • tumor tissue is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating 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
  • 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 a Leukemia-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.
  • fluorescent dyes e.g. Alexa488, PE
  • CD34 cell marker
  • Phosphorylation-site specific antibodies of the invention specifically bind to a human Leukemia-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 Leukemia-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 Leukemia-related signal transduction protein phosphorylation sites disclosed herein.
  • the novel Leukemia-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.
  • 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).
  • 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.
  • 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 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.
  • 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.
  • 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 acids.
  • the size of the peptide is also optimized to maximize ionization frequency.
  • 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 (Ae. 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 fragment masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety 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 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.
  • AQUA internal peptide standards may now be produced, as described above, for any of the nearly 480 novel Leukemia-related signaling protein phosphorylation sites disclosed herein (see Table 1/ Figure 2).
  • Peptide standards for a given phosphorylation site e.g. the tyrosine 187 in BLK - see Row 271 of Table 1
  • BLK site sequence in Column E 1 Row 271 of Table 1 SEQ ID NO: 270
  • 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 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 Leukemia-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 peptide comprising less than all of the residues of a disclosed phosphorylation site sequence (but still comprising the phosphorylatable residue enumerated in Column D of Table 1/ Figure 2) may alternatively be constructed.
  • Such larger or shorter AQUA peptides are within the scope of the present invention, and the selection and production of preferred AQUA peptides may be carried out as described above (see Gygi et al., Gerber et al. supra.).
  • AQUA peptides provided by the invention are described above (corresponding to particular protein types/groups in Table 1 , for example, Tyrosine Protein Kinases or Protein Phosphatases).
  • 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 FLT3 kinase tyrosine 630 phosphorylation site may be used to quantify the amount of phosphorylated FLT3 (Tyr630) in a biological sample, e.g.
  • 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 a Leukemia-related signal transduction protein disclosed in Table 1/ Figure 2), and, optionally, a second detecting reagent conjugated to a detectable group.
  • a kit may include AQUA peptides for both the phosphorylated 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 leukemias, and in identifying diagnostic/bio-markers of these diseases, new potential drug targets, and/or in monitoring the effects of test compounds on Leukemia-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 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.
  • 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. lmmunoassay formats and variations thereof that may be useful for carrying out the methods disclosed herein are well known in the art. See generally E.
  • 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 (FC) assay to determine the activation/phosphorylation status of a target Leukemia-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.
  • FC flow cytometry
  • bone marrow cells or peripheral blood cells from patients may be analyzed by flow cytometry for target Leukemia-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.
  • cytometric analysis may be employed: fixation of the cells with 1% 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 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 Leukemia-related signal transduction protein(s) in the malignant cells and reveal the drug response on the targeted protein.
  • a flow cytometer e.g. a Beckman Coulter EPICS-XL
  • antibodies of the invention may be employed in immunohistochemical (IHC) staining to detect differences in signal transduction or protein activity using normal and diseased tissues.
  • IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, supra. Briefly, paraffin-embedded tissue (e.g.
  • tumor tissue is prepared for immunohistochemical staining by 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- 5 phase array applications (see, e.g.
  • the invention provides a method for the multiplex detection of Leukemia-related protein phosphorylation in a biological sample, the method comprising utilizing two or more antibodies or AQUA peptides of the invention to detect the
  • 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
  • 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
  • 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),
  • 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- and/or phosphoserine- containing peptides in cell extracts from the following human Leukemia cell lines and patient cell lines: Jurkat, K562, SEM, HT-93, CTV-1 , MOLT15, CLL-9, H1993, OCL-ly3, KBM-3, UT-7, SUPT-13, MKPL-1 , HU-3, M-07e, HU-3, EHEB, SU-DHL1 , OCI-IyI , DU-528, CMK, OCI-ly ⁇ , ELF-153, OCI-IyI 8, MEC-1 , Karpas 299, CLL23LB4, OCI-ly12, M01043, CLL-10, HL60, MoIm 14, MV4-11 , CLL-1202, EOL-1 , CLL-19, CV-1, PL21 ; or from the following cell lines expressing
  • Tryptic phosphotyrosine - containing peptides were purified and analyzed from extracts of each of the 29 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. Suspension cells were harvested by low speed centrifugation. After complete aspiration of medium, cells were resuspended in 1 ml_ lysis buffer per 1.25 x 10 8 cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or not with 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerol-phosphate) and sonicated.
  • Sonicated cell lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM.
  • protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK-trypsin (Worthington) was added at 10-20 ⁇ g/mL. Digestion was performed for 1-2 days at room temperature.
  • Trifluoroacetic acid was added to protein digests to a final concentration of 1%, precipitate was removed by centrifugation, and digests were loaded onto Sep-Pak Ci 8 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. 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 1 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 centrif ligation. 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 or protein A agarose (Roche), respectively.
  • 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 minutes.
  • 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 //I, 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.
  • 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 LTQ ion trap mass spectrometer essentially as described by Gygi et ah, supra.
  • MS/MS spectra were evaluated using TurboSequest in the Sequest Browser package (v. 27, rev. 12) supplied as part of BioWorks 3.0 (ThermoFinnigan). Individual MS/MS spectra were extracted from the raw data file using the Sequest Browser program CreateDta, with the following settings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4 x 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.
  • Sequest scoring thresholds were used to select phosphopeptide assignments that are likely to be correct: RSp ⁇ 6, XCorr > 2.2, and DeltaCN > 0.099. Further, the assigned sequences could be accepted or rejected with respect to accuracy by using the following conservative, two-step process.
  • a subset of high-scoring sequence assignments should be 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 should be rejected if any of the following criteria were satisfied: (i) the spectrum contains at least one major peak (at least 10% as intense as the most intense ion in the spectrum) that can 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 does not contain a series of b or y ions equivalent to at least six uninterrupted residues; or (iii) the sequence is not observed at least five times in all the studies conducted (except for overlapping sequences due to incomplete proteolysis or use of proteases other than trypsin).
  • Polyclonal antibodies that specifically bind a Leukemia-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.
  • MELK tyrosine 438
  • B- SIT1 (tyrosine 127).
  • a synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and rabbits are injected intradermal ⁇ (ID) on the back with antigen in complete Freunds adjuvant (500 ⁇ g antigen per rabbit). The rabbits are boosted with same antigen in incomplete Freund adjuvant (250 ⁇ g antigen per rabbit) every three weeks. After the fifth boost, bleeds are collected. The sera are purified by Protein A-affinity chromatography by standard methods (see ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, supra.).
  • the eluted immunoglobulins are further loaded onto a non-phosphorylated synthetic peptide antigen-resin Knotes column to pull out antibodies that bind the non-phosphorylated form of the phosphorylation site.
  • the flow through fraction is collected and applied onto a phospho-synthetic peptide antigen-resin column to isolate antibodies that bind the phosphorylated form of the site.
  • 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
  • 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
  • the isolated antibody is then tested for phospho-specificity using Western blot assay using an appropriate cell line that expresses (or overexpresses) target phospho-protein (i.e. phosphorylated MELK, SIT1 or PECAM1), for example, SEM, Jurkat and MKPL-1 cells, respectively.
  • Cells are cultured in DMEM or RPMI supplemented with 10% FCS.
  • Cell are collected, washed with PBS and directly lysed in cell lysis buffer. The protein concentration of cell lysates is then measured.
  • 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 (substantially) recognize the target protein when not phosphorylated at the appropriate phosphorylation site in the non-stimulated cells (e.g. PECAM1 is not bound when not phosphorylated at tyrosine 663).
  • Monoclonal antibodies that specifically bind a Leukemia-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.
  • TSC2 (tyrosine 1736).
  • 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 TSC2(tyr1736) antibodies as described in Immunization/Fusion/Screening below.
  • CD84 (tyrosine 279).
  • 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 CD84(tyr279) 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 STAT5A (tyr22) antibodies as described in Immunization/Fusion/Screening below. Immunization/Fusion/Screening.
  • a synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and BALB/C mice are injected 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 TSC2, CD84, or STAT5A phospho-peptide. antigen, as the case may be) on ELISA.
  • 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. STAT5A phosphorylated at tyrosine 22).
  • Heavy-isotope labeled peptides (AQUA peptides (internal standards)) for the detection and quantification of a Leukemia-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/ Figure 2) 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.
  • the ZAP70(tyr164) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated ZAP70(tyr164) in the sample, as further described below in Analysis & Quantification.
  • SCAP1 tyrosine 1412.
  • the SCAP1 (tyr142) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated SCAP1(tyr142) in the sample, as further described below in Analysis & Quantification.
  • the CFL1(tyr68) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated CFL1(tyr68) in the sample, as further described below in Analysis & Quantification.
  • the BLK(tyr187) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated BLK(tyr187) 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, i-bis(dimethylamino) methylenej-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 by- products. 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.
  • TFA trifluoroacetic acid
  • 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.
  • MS/MS may be performed by using a ThermoFinnigan (San Jose, CA) mass spectrometer (LTQ ion trap or TSQ Quantum triple quadrupole).
  • LTQ ThermoFinnigan
  • parent ions are isolated at 1.6 m/z width, the ion injection time being limited to 100 ms per microscan, with one microscans per peptide, and with an AGC setting of 1 x 10 5 ; 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).

Abstract

The invention discloses nearly 480 novel phosphorylation sites identified in signal transduction proteins and pathways underlying human Leukemia, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites/proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: adaptor/scaffold proteins, acetyltransferases, actin binding proteins, adhesion proteins, apoptosis proteins, calcium-binding proteins, cell cycle regulation proteins, cell surface proteins, channel proteins, chaperone proteins, contractile proteins, cytokine proteins, cytoskeletal proteins, G protein regulators and GTPase activating proteins, guanine nucleotide exchange factors, helicase proteins, immunoglobulin superfamily proteins, inhibitor proteins, protein kinases, lipid kinases, ligases, lipid binding proteins, methytransferases, motor proteins, oxidoreductases, phosphotases, phosphodiesterases, phospholipases, proteases, receptor proteins, trascription factors, transferases, translation/transporter proteins, and ubiquitin conjugating system proteins.

Description

REAGENTS FOR THE DETECTION OF PROTEIN PHOSPHORYLATION IN LEUKEMIA SIGNALING PATHWAYS
RELATED APPLICATIONS
This application claims the benefit of, and priority to, U. S. S. N. 60/712,776, filed August 31 , 2005, presently pending, the disclosure of which is incorporated herein, in its entirety, by reference.
FIELD OF THE INVENTION
The invention relates generally to antibodies and peptide reagents for the detection of protein phosphorylation, and to protein phosphorylation in cancer.
BACKGROUND OF THE INVENTION
The activation of proteins by post-translational modification is an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. Protein phosphorylation, for example, plays a critical role in the etiology of many pathological conditions and diseases, including cancer, developmental disorders, autoimmune diseases, and diabetes. Yet, in spite of the importance of protein modification, it is not yet well understood at the molecular level, due to the extraordinary complexity of signaling pathways, and the slow development of technology necessary to unravel it.
Protein phosphorylation on a proteome-wide scale is extremely complex as a result of three factors: the large number of modifying proteins, e.g. kinases, encoded in the genome, the much larger number of sites on substrate proteins that are modified by these enzymes, and the dynamic nature of protein expression during growth, development, disease states, and aging. The human genome, for example, encodes over 520 different protein kinases, making them the most abundant class of enzymes known. See Hunter, Nature 411: 355-65 (2001). Most kinases phosphorylate many different substrate proteins, at distinct tyrosine, serine, and/or threonine residues. Indeed, it is estimated that 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. One form of cancer in which underlying signal transduction events are involved, but still poorly understood, is leukemia. Leukemia is a malignant disease of the bone marrow and blood, characterized by abnormal accumulation of blood cells, and is divided in four major categories. An estimated 33,500 new cases of leukemia will be diagnosed in the U.S. alone this year, affecting roughly 30,000 adults and 3,000 children, and close to 24,000 patients will die from the disease (Source: The Leukemia & Lymphoma Society (2004)). Depending of the cell type involved and the rate by which the disease progresses it can be defined as acute or chronic myelogenous leukemia (AML or CML), or acute and chronic lymphocytic leukemia (ALL or CLL). The acute forms of the disease rapidly progress, causing the accumulation of immature, functionless cells in the marrow and blood, which in turn results in anemia, immunodeficiency and coagulation deficiencies, respectively. Chronic forms of leukemia progress more slowly, allowing a greater number of mature, functional cells to be produced, which amass to high concentration in the blood over time.
More than half of adult leukemias occur in patients 67 years of age or older, and leukemia accounts for about 30% of all childhood cancers. The most common type of adult leukemia is acute myelogenous leukemia (AML), with an estimated 11 ,920 new cases annually. Without treatment patients rarely survive beyond 6-12 months, and despite continued development of new therapies, it remains fatal in 80% of treated patients (Source: The Leukemia & Lymphoma Society (2004)). The most common childhood leukemia is acute lymphocytic leukemia (ALL), but it can develop at any age. Chronic lymphocytic leukemia (CLL) is the second most prevalent adult leukemia, with approximately 8,200 new cases of CLL diagnosed annually in the U.S. The course of the disease is typically slower than acute forms, with a five-year relative survival of 74%. Chronic myelogenous leukemia (CML) is less prevalent, with about 4,600 new cases diagnosed each year in the U.S., and is rarely observed in children.
Most varieties of leukemia are generally characterized by genetic alterations associated with the etiology of the disease, and it has recently become apparent that, in many instances, such alterations (chromosomal translocations, deletions or point mutations) result in the constitutive activation of protein kinase genes, and their products, particularly tyrosine kinases. The most well known alteration is the oncogenic role of the chimeric BCR-AbI gene, which is generated by translocation of chromosome 9 to chromosome 22, creating the so-called Philadelphia chromosome characteristic of CML (see Nowell, Science 132: 1497 (1960)). The resulting BCR-AbI kinase protein is constitutively active and elicits characteristic signaling pathways that have been shown to drive the proliferation and survival of CML cells (see Daley, Science 247: 824-830 (1990); Raitano et al., Biochim. Biophys. Acta. Dec 9; 1333(3): F201-16 (1997)). The recent success of lmanitib (also known as STI571 or Gleevec®), the first molecularly targeted compound designed to specifically inhibit the tyrosine kinase activity of BCR-AbI, provided critical confirmation of the central role of BCR-AbI signaling in the progression of CML (see Schindler et al., Science 289: 1938-1942 (2000); Nardi et al., Curr. Opin. Hematol. 11: 35-43 (2003)).
The success of Gleevec® now serves as a paradigm for the development of targeted drugs designed to block the activity of other tyrosine kinases known to be involved in leukemias and other malignancies (see, e.g., Sawyers, Curr. Opin. Genet. Dev. Feb; 12(1): 111-5 (2002); Druker, Adv. Cancer Res. 911-30 (2004)). For example, recent studies have demonstrated that mutations in the FLT3 gene occur in one third of adult patients with AML. FLT3 (Fms-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase (RTK) family including FMS, platelet-derived growth factor receptor (PDGFR) and c- KIT (see Rosnet et al., Crit. Rev. Oncog. 4: 595-613 (1993). In 20-27% of patients with AML, an internal tandem duplication in the juxta-membrane region of FLT3 can be detected (see Yokota et al., Leukemia 11: 1605- 1609 (1997)). Another 7% of patients have mutations within the active loop of the second kinase domain, predominantly substitutions of aspartate residue 835 (D835), while additional mutations have been described (see Yamamoto et al., Blood 97: 2434-2439 (2001); Abu-Duhier et al., Br. J. Haematol. 113: 983-988 (2001)). Expression of mutated FLT3 receptors results in constitutive tyrosine phosphorylation of FLT3, and subsequent phosphorylation and activation of downstream molecules such as STAT5, Akt and MAPK, resulting in factor-independent growth of hematopoietic cell lines.
Altogether, FLT3 is the single most common activated gene in AML known to date. This evidence has triggered an intensive search for FLT3 inhibitors for clinical use leading to at least four compounds in advanced stages of clinical development, including: PKC412 (by Novartis), CEP- 701 (by Cephalon), MLN518 (by Millenium Pharmaceuticals), and SU5614 (by Sugen/Pfizer) (see Stone et al., Blood (in press)(2004); Smith et al., Blood 103: 3669-3676 (2004); Clark etal., Blood 104: 2867-2872 (2004); and Spiekerman et al., Blood 101: 1494-1504 (2003)).
There is also evidence indicating that kinases such as FLT3, c-KIT and AbI are implicated in some cases of ALL (see Cools et al., Cancer Res. 64: 6385-6389 (2004); Hu, Nat.Genet 36: 453-461 (2004); and Graux et al., Nat. Genet. 36: 1084-1089 (2004)). In contrast, very little is know regarding any causative role of protein kinases in CLL, except for a high correlation between high expression of the tyrosine kinase ZAP70 and the more aggressive form of the disease (see Rassenti et al., N. Eng. J. Med. 351: 893-901 (2004)).
Despite the identification of a few key molecules involved in progression of leukemia, the vast majority of signaling protein changes underlying this disease remains unknown. There is, therefore, relatively scarce information about kinase-driven signaling pathways and phosphorylation sites relevant to the different types of leukemia. This has hampered a complete and accurate understanding of how protein activation within signaling pathways is driving these complex cancers. Accordingly, there is a continuing and pressing need to unravel the molecular mechanisms of kinase-driven oncogenesis in leukemia by identifying the downstream signaling proteins mediating cellular transformation in this disease. Identifying particular phosphorylation sites on such signaling proteins and providing new reagents, such as phospho- - fc> -
specific antibodies and AQUA peptides, to detect and quantify them remains particularly important to advancing our understanding of the biology of this disease.
Presently, diagnosis of leukemia is made by tissue biopsy and detection of different cell surface markers. However, misdiagnosis can occur since some leukemia cases can be negative for certain markers, and because these markers may not indicate which genes or protein kinases may be deregulated. Although the genetic translocations and/or mutations characteristic of a particular form of leukemia can be sometimes detected, it is clear that other downstream effectors of constitutively active kinases having potential diagnostic, predictive, or therapeutic value, remain to be elucidated. Accordingly, identification of downstream signaling molecules and phosphorylation sites involved in different types of leukemia 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 this disease.
SUMMARY OF THE INVENTION
The invention discloses nearly 480 novel phosphorylation sites identified in signal transduction proteins and pathways underlying human Leukemias 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, quantification, and profiling of the disclosed phosphorylation sites.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 - Is a diagram broadly depicting the immunoaffinity isolation and mass-spectrometric characterization methodology (IAP) employed to identify the novel phosphorylation sites disclosed herein.
FIG. 2 - Is a table (corresponding to Table 1) enumerating the Leukemia 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 or serine 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; Column F = the type of leukemia in which the phosphorylation site was discovered; and Column G = the cell type(s), tissue(s) and/or patient tissue(s) in which the phosphorylation site was discovered. FIG. 3 - is an exemplary mass spectrograph depicting the detection of the tyrosine 330 phosphorylation site in DOK2 (see Row 24 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase "y" in Figure 2). FIG. 4 - is an exemplary mass spectrograph depicting the detection of the tyrosine 630 phosphorylation site in FLT3 (see Row 286 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase "y" in Figure 2). FIG. 5 - is an exemplary mass spectrograph depicting the detection of the tyrosine 1736 phosphorylation site in TSC2 (see Row 87 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase "y" in Figure 2). FIG. 6 - is an exemplary mass spectrograph depicting the detection of the tyrosine 260 phosphorylation site in ICAM (see Row 71 in Figure 2/Table 1), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine (shown as lowercase "y" in Figure 2).
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, nearly 480 novel protein phosphorylation sites in signaling proteins and pathways underlying human Leukemia 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 ai, using cellular extracts from a variety of leukemia-derived cell lines, e.g. SEM, HT-93, etc., as further described below. The novel phosphorylation sites (tyrosine), and their corresponding parent proteins, disclosed herein are listed in Table 1. These phosphorylation sites correspond to numerous different parent proteins (the full sequences of which (human) are all publicly available in SwissProt database and their Accession numbers listed in Column B of Table 1/Fig. 2), each of which fall into discrete protein type groups, for example Adaptor/Scaffold proteins, Cytoskeletal proteins, Protein Kinases, and Adhesion proteins, etc. (see Column C of Table 1), the phosphorylation of which is relevant to signal transduction activity underlying Leukemias (AML, CML, CLL, and ALL), as disclosed herein. The discovery of the nearly 480 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 Leukemia. Accordingly, the invention provides novel reagents - phospho-specific antibodies and AQUA peptides - for the specific detection and/or quantification of a Leukemia-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 Leukemia-related signaling proteins using the phosphorylation-site specific antibodies and AQUA peptides of the invention, and methods of obtaining a phosphorylation profile of such proteins (e.g. Kinases).
In part, the invention provides an isolated phosphorylation site- specific antibody that specifically binds a given Leukemia-related signaling protein only when phosphorylated (or not phosphorylated, respectively) at a particular tyrosine enumerated in Column D of Table 1 /Figure 2 comprised within the phosphorylatable peptide site sequence enumerated in corresponding Column E. In further part, the invention provides a heavy-isotope labeled peptide (AQUA peptide) for the detection and quantification of a given Leukemia-related signaling protein, the labeled peptide comprising a particular phosphorylatable peptide site/sequence enumerated in Column E of Table 1/Figure 2 herein. For example, among the reagents provided by the invention is an isolated phosphorylation site-specific antibody that specifically binds the MELK tyrosine kinase only when phosphorylated (or only when not phosphorylated) at tyrosine 438 (see Row 244 (and Columns D and E) of Table 1/Figure 2). By way of further example, among the group of reagents provided by the invention is an AQUA peptide for the quantification of phosphorylated MELK tyrosine kinase, the AQUA peptide comprising the phosphorylatable peptide sequence listed in Column E, Row 244, of Table 1 /Figure 2 (which encompasses the phosphorylatable tyrosine at position 438).
In one embodiment, the invention provides an isolated phosphorylation site-specific antibody that specifically binds a human Leukemia-related signaling protein selected from Column A of Table 1 (Rows 2-481) only when phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21, 23, 26-30, 32-34, 36-45, 48- 52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182-193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319- 336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440- 474, and 476-480), 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 a Leukemia-related signaling protein selected from Column A of Table 1 only when not phosphorylated at the tyrosine residue listed in corresponding Column D of Table 1 , comprised within the peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182- 193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319-336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440-474, and 476-480), 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 a Leukemia- 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: 1-7, 9-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182-193, 195-197, 199-208, 210-221 , 223-279, 281- 294, 296-297, 299-316, 319-336, 339-345, 347-356, 358, 360-366, 368- 378, 380-417, 419-438, 440-474, and 476-480), 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 residue within the labeled peptide is not phosphorylated. Reagents (antibodies and AQUA peptides) provided by the invention may conveniently be grouped by the type of Leukemia-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/Figure 2, and include: adaptor/scaffold proteins, acetyltransferases, actin binding proteins, adhesion proteins, apoptosis proteins, calcium-binding proteins, cell cycle regulation proteins, cell surface proteins, channel proteins, chaperone proteins, contractile proteins, cytokine proteins, cytoskeletal proteins, G protein regulators and GTPase activating proteins, guanine nucleotide exchange factors, helicase proteins, immunoglobulin superfamily proteins, inhibitor proteins, protein kinases, lipid kinases, ligases, lipid binding proteins, methytransferases, motor proteins, oxidoreductases, phosphotases, phosphodiesterases, phospholipases, proteases, receptor proteins, transcription factors, transferases, translation/transporter proteins, and ubiquitin conjugating system proteins. Each of these distinct protein groups is considered a preferred subset of Leukemia-related signal transduction protein phosphorylation sites disclosed herein, and reagents for their detection/quantification may be considered a preferred subset of reagents provided by the invention.
Particularly preferred subsets of the phosphorylation sites (and their corresponding proteins) disclosed herein are those occurring on the following protein types/groups listed in Column C of Table 1/Figure 2, are the protein kinases adaptor/scaffold proteins, adhesion proteins, cell cycle regulation proteins, cell surface proteins, transcription proteins, phosphatases, phospholipases, phosphodiesterases, receptor proteins, cytoskeltal proteins, G protein regulators, and lipid kinases. 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.
In one subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a protein kinase selected from Column A, Rows 210-291 , of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 210-291 , of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: 210-221 , 223-280, and 281-290), 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 a protein kinase selected from Column A, Rows 210-291 , said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: 210-221 , 223-280, and 281-290), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 210- 291 , of Table t
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following protein kinase phosphorylation sites are particularly preferred: ATM (Y2129), MELK (Y438), MAPK14 (Y24), BLK (Y187), BTK (Y344), SYK (Y296), ZAP70 (Y69), FLT3 (Y630), FLT3 (Y726), FLT3 (Y768), and ROS1 (Y363) (see SEQ ID NOs: 229, 243, 249, 270-271 , 273, 283, and 285-288).
In a second subset of preferred embodiments there is provided: (i) An isolated phosphorylation site-specific antibody that specifically binds an adaptor/scaffold protein selected from Column A, Rows 11-59, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 11-59, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 11-59, of Table 1 (SEQ ID NOs: 10-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, and 56-58), 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 Leukemia-related signaling protein that is a adaptor/scaffold protein selected from Column A, Rows 11-59, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 11-59, of Table 1 (SEQ ID NOs: 10-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, and 56-58), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 11-59, 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: ABI2 (Y192), PIK3AP1 (Y594), DOK2 (Y330), LAT2 (Y40), SIT1 (Y127), STAM (Y384), SCAP1 (Y142) (see SEQ ID NOs: 10, 14, 23, 30, 37, 40-41). In another subset of preferred embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds an adhesion protein selected from Column A, Rows 60-79, of Table 1 only when phosphorylated at the tyrosine or serine listed in corresponding Column D, Rows 60-79, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 60-79, of Table 1 (SEQ ID NOs: 60-78), 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 adhesion 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 Leukemia-related signaling protein that is an adhesion protein selected from Column A, Rows 60-79, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 60-79, of Table 1 (SEQ ID NOs: 60-78), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 60-79, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following adhesion protein phosphorylation sites are particularly preferred: ADAM18 (Y197), ICAM2 (Y260) and PECAM 1 (Y663) (see SEQ ID NOs: 60, 70 and 72).
In still another subset of preferred embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a cell cycle regulation protein selected from Column A, Rows 83-87, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 83-87, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 83-87, of Table 1 (SEQ ID NOs: 82-86), 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 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 Leukemia-related signaling protein that is a cell cycle regulation protein selected from Column A, Rows 83-87, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 83-87, of Table 1 (SEQ ID NOs: 82-86), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 83-87, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following cell cycle regulation protein phosphorylation sites are particularly preferred: TSC2 (Y1736) (see SEQ ID NO: 86). In still another subset of preferred embodiments there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a cell surface protein selected from Column A, Rows 88-94, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 88-94, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 88-94, of Table 1 (SEQ ID NOs: 87-90, and 93), 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 cell surface protein when not phosphorylated at the disclosed site (and does not bind the protein when it is phosphoryiated at the site).
(iii) A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a Leukemia-related signaling protein that is a cell surface protein selected from Column A, Rows 88-94, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 88-94, of Table 1 (SEQ ID NOs: 87-90, and 93), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 88-94, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following cell surface protein phosphorylation sites are particularly preferred: CD72 (Y39) and CD84 (Y299) (see SEQ ID NOs: 89 and 93).
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 selected from Column A, Rows 387-425, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 387-425, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 387-425of Table 1 (SEQ ID NOs: 386-417, and 419-424), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
(ii) An equivalent antibody to (i) above that only binds transcription factor/coactivator/corepressor 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 Leukemia-related signaling protein that is a transcription factor/coactivator/corepressor selected from Column A, Rows 387-425, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 387-425, of Table 1 (SEQ ID NOs: 386-417, and 419-424), which sequence comprises the phosphorylatable tyrosine or serine listed in corresponding Column D, Rows 387-425, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following trascription factor/coactivor/corepressor phosphorylation sites are particularly preferred: STAT5A (Y22), STAT5A (Y90), STAT5A (Y114), SMAD2 (Y102), and NSEP1 (Y208) (see SEQ ID NOs: 410-413 and 417).
In yet another subset of preferred embodiments, there is provided: (i) An isolated phosphorylation site-specific antibody that specifically binds a phophatase selected from Column A, Rows 331-350, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 331-350, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 331-350, of Table 1 (SEQ ID NOs: 330-349), 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 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 a Leukemia-related signaling protein that is a phophatase selected from Column A, Rows 331-350, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 331-350, of Table 1 (SEQ ID NOs: 330-349), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 331-350, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following phosphatase phosphorylation sites are particularly preferred: INPPL1 (Y831), INPPL1 (Y1135) and PTPRC (Y705) (see SEQ ID NOs: 336-337, and 347).
In yet another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody specifically binds a phosphodiesterase/phospholipase selected from Column A, Rows1351- 359, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 351-359, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 351-359, of Table 1 (SEQ ID NOs: 350-356, and 358), 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 Tyrosine 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 a Leukemia-related signaling protein that is a phosphodiesterase/phospholipase selected from Column A, Rows 351- 359, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 351-359, of Table 1 (SEQ ID NOs: 350-356, and 358), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 351- 359, of Table t
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following phosphodiesterase/phospholipase phosphorylation sites are particularly preferred: PLCG1 (Y481), PLCG2 (Y680) and PLCG2 (Y1264) (see SEQ ID NOs: 352, 354 and 358).
In yet another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a receptor protein selected from Column A, Rows 366-386, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 366-386, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 366-386, of Table 1 (SEQ ID NOs: 365-366, 368-378, and 380-385), 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 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 Leukemia-related signaling protein that is a receptor protein selected from Column A, Rows 366-386, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 366-386, of Table 1 (SEQ ID NOs: 365-366, 368-378, arid 380-385), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 366-386, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following receptor protein phosphorylation sites are particularly preferred: LEPR (Y795) (see SEQ ID NO: 365).
In still 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 114-170, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 114-170, of Table 1, comprised within the phosphoryiatable peptide sequence listed in corresponding Column E, Rows 114-170, of Table 1 (SEQ ID NOs: 113-119, 121-124, 129-151 , 153- 160, and 163-169), 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 Leukemia-related signaling protein that cytoskeletal protein selected from Column A, Rows 114-170, said labeled peptide comprising the phosphoryiatable peptide sequence listed in corresponding Column E, Rows 114-170, of Table 1 (SEQ ID NOs: 113-119, 121-124, 129-151 , 153-160, and 163-169), which sequence comprises the phosphoryiatable tyrosine or serine listed in corresponding Column D, Rows 114-170, of Table 1.
In still another subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a G protein selected from Column A, Rows 174-180, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 174-180, of Table 1 , comprised within the phosphoryiatable peptide sequence listed in corresponding Column E, Rows 174-180, of Table 1 (SEQ ID NOs: 173-189), 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 G 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 Leukemia-related signaling protein that is an G protein selected from Column A, Rows 174-180, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 174-180, of Table 1 (SEQ ID NOs: 173-179), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 174-180, of Table 1.
Among this preferred subset of reagents, antibodies and AQUA peptides for the detection/quantification of the following G protein phosphorylation sites are particularly preferred: GNAI2 (Y61) (see SEQ ID NO: 177). 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 292-298, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 292-298, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 292-298, of Table 1 (SEQ ID NOs: 291-297), 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 Leukemia-related signaling protein that is an lipid kinase selected from Column A, Rows 292-298, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 292-298, of Table 1 (SEQ ID NOs: 291-297), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 292-298, 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: PIK3CB (Y962) (see SEQ ID NO: 293).
In yet a further subset of preferred embodiments, there is provided:
(i) An isolated phosphorylation site-specific antibody that specifically binds a protein selected from the group consisting of EIF4EBP2, EIF4G2 and EIF4B (Column A, Rows 446, 448 and 460 of Table 1) only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 191 , 199, 446, 448 and 460 of Table 1), said tyrosine comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 446, 448 and 460, of Table 1 (SEQ ID NOs: 445, 447 and 459), 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 EIF4EBP2, EIF4G2 and EIF4B 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 protein selected from the group consisting of EIF4EBP2, EIF4G2 and EIF4B (Column A, Rows 446, 448 and 460 of Table 1), said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 446, 448 and 460, of Table 1 (SEQ ID NOs: 445, 447 and 459), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 446, 448 and 460, 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 any 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/Figure 2.
Also provided by the invention are methods for detecting or quantifying a Leukemia-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 Leukemia-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.
Also provided by the invention is a method for obtaining a phosphorylation profile of protein kinases that are phosphorylated in Leukemia signaling pathways, said method comprising the step of utilizing one or more isolated antibody that specifically binds a protein inase selected from Column A, Rows 210-291 , of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 210-291 , of Table 1, comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: SEQ ID NOs: 210-221 , 223-280, and 281-290), to detect the phosphorylation of one or more of said protein kinases, thereby obtaining a phosphorylation profile for said kinases.
The identification of the disclosed novel Leukemia-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.
Table 1. Newly Discovered Leukemia-related Phosphorylation Sites.
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
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 or serine 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
10 phosphorylation occurs. Table 1 above is identical to Figure 2, except that the latter includes the disease and cell type(s) in which the particular phosphorylation site was identified (Columns F and G).
The identification of these 480 phosphorylation sites is described in more detail in Part A below and in Example 1.
15 Definitions.
As used herein, the following terms have the meanings indicated:
"Antibody" or "antibodies" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including Fab or antigen-recognition fragments thereof, including chimeric, polyclonal, and monoclonal antibodies. The term "does not bind" with respect to an antibody's binding to one phospho-form of a sequence means does not substantially react with as compared to the antibody's binding to the other phospho-form of the sequence for which the antibody is specific. "Leukemia-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 leukemia cell line(s). Leukemia-related signaling proteins may be tyrosine kinases, such as Flt-3 or BCR-AbI, or serine/threonine kinases, or direct substrates of such kinases, or may be indirect substrates downstream of such kinases in signaling pathways. A Leukemia-related signaling protein may also be phosphorylated in other cell lines (non-leukemic) 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 et a/.), further discussed below. "Protein" is used interchangeably with polypeptide, and includes protein fragments and domains as well as whole protein. "Phosphorylatable amino acid" means any amino acid that is capable of being modified by addition of a phosphate group, and includes both forms of such amino acid.
"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 Leukemia-related Protein Phosphorylation Sites.
The nearly 480 novel Leukemia-related signaling protein phosphorylation sites disclosed herein and listed in Table 1 /Figure 2 were discovered by employing the modified peptide isolation and characterization techniques described in "Immunoaffinity Isolation of Modified Peptides From Complex Mixtures," U.S. Patent Publication No. 20030044848, Rush et a/, (the teaching of which is hereby incorporated herein by reference, in its entirety) using cellular extracts from the following human Leukemia (AML, ALL, CML and CLL) derived cell lines and patient samples: Jurkat, K562, SEM, HT-93, CTV-1 , MOLT15, CLL-9, H1993, OCL-ly3, KBM-3, UT-7, SUPT-13, MKPL-1 , HU-3, M-07e, HU-3, EHEB, SU-DHL1 , OCI-IyI , DU-528, CMK, OCI-lyβ, ELF-153, OCI-ly18, MEC-1 , Karpas 299, CLL23LB4, OCI-ly12, M01043, CLL-10, HL60, MoIm 14, MV4-11 , CLL-1202, EOL-1 , CLL-19, CV-1 , PL21 ; or from the following cell lines expressing activated BCR-AbI wild type and mutant kinases such as: Baf3-p210 BCR-AbI, Baf3-M351 T-BCR-ABL, Baf3-E255K-BCR- AbI, Baf3-Y253F-BCR-Abl, Baf3-T315l-BCR-ABI, 3T3-v-Abl; or activated Flt3 kinase such as Baf3-FLT3 or FLT3-ITD; or JAK2 such as Baf3/Jak2; or mutant JAK2 V617F such as Baf3-V617F -JAK2, or Tyk2 such as Baf3/Tyk2; or TEL-FGFR3 such as Baf3-Tel/FGFR3; or TpoR such as Baf3/TpoR and Baf3/cc-TpoR-IV; or FGFR1 such as 293T-FGFR. The isolation and identification of phosphopeptides from these cell lines, using an immobilized general phosphotyrosine-specific antibody, or an antibody recognizing the phosphorylated motif PXpSP is described in detail in Example 1 below. In addition to the 80 previously unknown protein phosphorylation sites (tyrosine) discovered, many known phosphorylation sites were also identified (not described herein). The immunoaffinity/mass spectrometric technique described in the '848 Patent Publication (the "IAP" method) - and employed as described in detail in the Examples - is briefly summarized below.
The IAP method employed generally comprises the following steps: (a) a proteinaceous preparation (e.g. a digested cell extract) comprising phosphopeptides from two or more different proteins is obtained from an organism; (b) the preparation is contacted with at least one immobilized general phosphotyrosine-specific antibody; (c) at least one phosphopeptide specifically bound by the immobilized antibody in step (b) is isolated; and (d) the modified peptide isolated in step (c) is characterized by mass spectrometry (MS) and/or tandem mass spectrometry (MS-MS). Subsequently, (e) a search program (e.g.
Sequest) may be utilized to substantially match the spectra obtained for the isolated, modified peptide during the characterization of step (d) with the spectra for a known peptide sequence. A quantification step employing, e.g. SILAC or AQUA, may also be employed to quantify isolated peptides in order to compare peptide levels in a sample to a baseline.
In the IAP method as employed herein, a general phosphotyrosine- specific monoclonal antibody (commercially available from Cell Signaling Technology, Inc., Beverly, MA, Cat #9411 (p-Tyr-100)), was used in the immunoaffinity step to isolate the widest possible number of phospho- tyrosine containing peptides from the cell extracts.
Extracts from the following human Leukemia cell lines (ALL, AML, CLL, CML, respectively) were employed: Jurkat, K562, SEM, HT-93, CTV-1 , MOLT15, CLL-9, H 1993, OCL-ly3, KBM-3, UT-7, SUPT-13,
MKPL-1 , HU-3, M-07e, HU-3, EHEB, SU-DHL1, OCI-IyI , DU-528, CMK, OCI-lyβ, ELF-153, OCI-IyI 8, MEC-1 , Karpas 299, CLL23LB4, OCI-IyI 2, M01043, CLL-10, HL60, MoIm 14, MV4-11 , CLL-1202, EOL-1 , CLL-19, CV-1 , PL21 ; or from the following cell lines expressing activated BCR-AbI wild type and mutant kinases such as: Baf3-p210 BCR-AbI, Baf3-M351T- BCR-ABL, Baf3-E255K-BCR-Abl, Baf3-Y253F-BCR-Abl, Baf3-T315l- BCR-ABI, 3T3-v-Abl; or activated Flt3 kinase such as Baf3-FLT3 or FLT3- ITD; or JAK2 such as Baf3/Jak2; or mutant JAK2 V617F such as Baf3- V617F -JAK2, or Tyk2 such as Baf3/Tyk2; or TEL-FGFR3 such as Baf3- Tel/FGFR3; or TpoR such as Baf3/TpoR and Baf3/cc-TpoR-IV; or FGFR1 such as 293T-FGFR.
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 Cis 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 a phosphotyrosine antibody (P-Tyr-100, CST #9411) immobilized on protein G-Sepharose or Protein A-Sepharose. Immunoaffinity-purified peptides were eluted with 0.1 % TFA and a portion of this fraction was concentrated with Stage or Zip tips and analyzed by LC-MS/MS, using a ThermoFinnigan LTQ ion trap mass spectrometer. Peptides were eluted from a 10 cm x 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 nearly 480 novel tyrosine phosphorylation sites in signaling pathways affected by kinase activation or active in leukemia cells. The identified phosphorylation sites and their parent proteins are enumerated in Table 1 /Figure 2. The tyrosine (human sequence) at which phosphorylation occurs is provided in Column D, and the peptide sequence encompassing the phosphorylatable tyrosine residue at the site is provided in Column E. Figure 2 also shows the particular type of leukemic disease (see Column G) and cell line(s) (see Column F) in which a particular phosphorylation site was discovered.
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 leukemias 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 a Leukemia-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/Figure 2 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 BCR kinase phosphorylation sites (tyrosines 58 and 231) (see Rows 225-226 of Table 1/Fig. 2) are presently disclosed. Thus, antibodies that specifically bind either of these novel BCR kinase sites can now be produced, e.g. 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 225, Column E, of Table 1 (SEQ ID NO: 224) (which encompasses the phosphorylated tyrosine at position 58 in BCR), to produce an antibody that only binds BCR kinase when phosphorylated at that site.
Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with a peptide antigen corresponding to the Leukemia-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 corresponding to all or part of the novel ATM kinase phosphorylation site disclosed herein (SEQ ID NO: 229 = EVEGTSYH ESLyNALQSLRDREFYESLKYAR, encompassing phosphorylated tyrosine 2129 (see Row 230 of Table 1)) may be used to produce antibodies that only bind ATM when phosphorylated at Tyr2129. Similarly, a peptide comprising all or part of any one 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., ANTIBODIES: A LABORATORY MANUAL,
Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962)).
It will be appreciated by those of skill in the art that longer or shorter phosphopeptide antigens may be employed. See Id. For example, a peptide antigen may comprise the full sequence disclosed in Column E of Table 1/Figure 2, 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 an uppercase "Y"). Typically, a desirable peptide antigen will comprise four or more amino acids flanking each side of the phosphorylatable amino acid and encompassing it. 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, for example, may be produced as described in U.S Patent No. 5,675,063, C. Knight, Issued October 7, 1997. The hybridoma cells are then grown in a suitable selection media, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below. The secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like. Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'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, 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 MELK tyrosine 438 phosphorylation site sequence disclosed in Row 244, Column E of Table 1), and antibodies of the invention thus specifically bind a target Leukemia-related signaling 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. lncluded 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. Natl Acad. ScL 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 Leukemia- 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. coll (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)
Phosphorylation site-specific antibodies of the invention, whether polyclonal or monoclonal, may be screened for epitope and phospho- specificity according to standard techniques. See, e.g. Czernik et al., Methods in Enzymology, 201: 264-283 (1991). For example, the antibodies may be screened against the phospho and non-phospho peptide library by ELISA to ensure specificity for both the desired antigen (i.e. that epitope including a phosphorylation site sequence enumerated in Column E of Table 1) and for reactivity only with the phosphorylated (or non-phosphorylated) form of the antigen. Peptide competition assays may be carried out to confirm lack of reactivity with other phospho- epitopes on the given Leukemia-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 to 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 Leukemia-related signaling protein epitope for which the antibody of the invention is specific.
In certain cases, polyclonal antisera may exhibit some undesirable general cross-reactivity to phosphotyrosine itself, 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 Leukemia- related phosphorylation and activation status in diseased tissue. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating 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). 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 0C 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 a Leukemia-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 Leukemia-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 Leukemia-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 Leukemia-related signal transduction protein phosphorylation sites disclosed herein.
C. Heavy-Isotope Labeled Peptides (AQUA Peptides).
The novel Leukemia-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 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 (Ae. 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 (Ae. 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 fragment masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety 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, 170, 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 (MS") to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature. Preferably, peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature 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 at., 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 nearly 480 novel Leukemia-related signaling protein phosphorylation sites disclosed herein (see Table 1/Figure 2). Peptide standards for a given phosphorylation site (e.g. the tyrosine 187 in BLK - see Row 271 of Table 1) may be produced for both the phosphorylated and non-phosphorylated forms of the site (e.g. see BLK site sequence in Column E1 Row 271 of Table 1 (SEQ ID NO: 270) and such standards employed in the AQUA methodology to detect and quantify both forms of such phosphorylation site in a biological sample.
AQUA peptides of the invention may comprise all, or part of, a phosphorylation site peptide sequence disclosed herein (see Column E of Table 1/Figure 2). In a preferred embodiment, an AQUA peptide of the invention comprises a phosphorylation site sequence disclosed herein in Table 1/Figure 2. For example, an AQUA peptide of the invention for detection/quantification of SYK kinase when phosphorylated at tyrosine Y296 may comprise the sequence IKSySFPKPGHR (y=phosphotyrosine), which comprises phosphorylatable tyrosine 296 (see Row 274, Column E; (SEQ ID NO: 273)). Heavy-isotope labeled equivalents of the peptides enumerated in Table 1/Figure 2 (both in phosphorylated and unphosphorylated form) can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments.
The phosphorylation site peptide sequences disclosed herein (see Column E of Table 1/Figure 2) are particularly well suited for development of corresponding AQUA peptides, since the IAP method by which they were identified (see Part A above and Example 1) inherently confirmed that such peptides are in fact produced by enzymatic digestion (trypsinization) and are in fact suitably fractionated/ionized in MS/MS. Thus, heavy-isotope labeled equivalents of these peptides (both in phosphorylated and unphosphorylated form) can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments. Accordingly, the invention provides heavy-isotope labeled peptides (AQUA peptides) for the detection and/or quantification of any of the Leukemia-related phosphorylation sites disclosed in Table 1 /Figure 2 (see Column E) and/or their corresponding parent proteins/polypeptides (see Column A). A phosphopeptide sequence comprising any of the phosphorylation sequences listed in Table 1 may be considered a preferred AQUA peptide of the invention. For example, an AQUA peptide comprising the sequence VMNATAyGISK (SEQ ID NO:285) (where y may be either phosphotyrosine or tyrosine, and where V = labeled valine (e.g. 14C)) is provided for the quantification of phosphorylated (or non- phosphorylated) FLT3 kinase (Tyr630) in a biological sample (see Row 286 of Table 1, tyrosine 630 being the phosphorylatable residue within the site). However, it will be appreciated that a larger AQUA peptide comprising a disclosed phosphorylation site sequence (and additional residues downstream or upstream of it) may also be constructed.
Similarly, a smaller AQUA peptide comprising less than all of the residues of a disclosed phosphorylation site sequence (but still comprising the phosphorylatable residue enumerated in Column D of Table 1/Figure 2) may alternatively be constructed. Such larger or shorter AQUA peptides are within the scope of the present invention, and the selection and production of preferred AQUA peptides may be carried out as described above (see Gygi et al., Gerber et al. supra.).
Certain particularly preferred subsets of AQUA peptides provided by the invention are described above (corresponding to particular protein types/groups in Table 1 , for example, Tyrosine Protein Kinases or Protein Phosphatases). 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 FLT3 kinase tyrosine 630 phosphorylation site (see Row 286 of Table 1/Figure 2) may be used to quantify the amount of phosphorylated FLT3 (Tyr630) in a biological sample, e.g. a tumor cell sample (or a sample before or after treatment with a test drug). AQUA peptides of the invention may also be employed within a kit that comprises one or multiple AQUA peptide(s) provided herein (for the quantification of a Leukemia-related signal transduction protein disclosed in Table 1/Figure 2), and, optionally, a second detecting reagent conjugated to a detectable group. For example, a kit may include AQUA peptides for both the phosphorylated 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 leukemias, and in identifying diagnostic/bio-markers of these diseases, new potential drug targets, and/or in monitoring the effects of test compounds on Leukemia-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. lmmunoassay formats and variations thereof that may be useful for carrying out the methods disclosed herein are well known in the art. See generally E. Maggio, Enzyme-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
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 Leukemia- related signal transduction protein is detectable compared to background.
Phosphorylation site-specific antibodies disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies, or other target protein or target site-binding reagents, may likewise be conjugated to detectable groups such as radiolabels (e.g., 35S, 1251, 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 (FC) assay to determine the activation/phosphorylation status of a target Leukemia-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 Leukemia-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 a/., 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% paraformaldehyde for 10 minutes at 37 0C 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 Leukemia-related signal transduction protein(s) in the malignant cells and reveal the drug response on the targeted protein.
Alternatively, antibodies of the invention may be employed in immunohistochemical (IHC) staining to detect differences in signal transduction or protein activity using normal and diseased tissues. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, supra. Briefly, paraffin-embedded tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by 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, Luminex™ and/or Bioplex™ assay formats, or otherwise optimized for antibody arrays formats, such as reversed- 5 phase array applications (see, e.g. Paweletz et a/., Oncogene 20(16): 1981-89 (2001)). Accordingly, in another embodiment, the invention provides a method for the multiplex detection of Leukemia-related protein phosphorylation in a biological sample, the method comprising utilizing two or more antibodies or AQUA peptides of the invention to detect the
10. presence of two or more phosphorylated Leukemia-related signaling proteins enumerated in Column A of Table 1/Figure 2. In one preferred embodiment, two to five antibodies or AQUA peptides of the invention are employed in the method. In another preferred embodiment, six to ten antibodies or AQUA peptides of the invention are employed, while in
15 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
20 detects a Leukemia-related signal transduction protein disclosed in Table 1/Figure 2), 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
25 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),
30 agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.
The following Examples are provided only to further illustrate the invention, and are not intended to limit its scope, except as provided in the claims appended hereto. The present invention encompasses modifications and variations of the methods taught herein which would be obvious to one of ordinary skill in the art.
EXAMPLE 1
Isolation of Phosphotyrosine-Containing Peptides from Extracts of
Leukemia Cell Lines and Identification of Novel
Phosphorylation Sites.
In order to discover previously unknown Leukemia-related signal transduction protein phosphorylation sites, IAP isolation techniques were employed to identify phosphotyrosine- and/or phosphoserine- containing peptides in cell extracts from the following human Leukemia cell lines and patient cell lines: Jurkat, K562, SEM, HT-93, CTV-1 , MOLT15, CLL-9, H1993, OCL-ly3, KBM-3, UT-7, SUPT-13, MKPL-1 , HU-3, M-07e, HU-3, EHEB, SU-DHL1 , OCI-IyI , DU-528, CMK, OCI-lyβ, ELF-153, OCI-IyI 8, MEC-1 , Karpas 299, CLL23LB4, OCI-ly12, M01043, CLL-10, HL60, MoIm 14, MV4-11 , CLL-1202, EOL-1 , CLL-19, CV-1, PL21 ; or from the following cell lines expressing activated BCR-AbI wild type and mutant kinases such as: Baf3-p210 BCR-AbI, Baf3-M351 T-BCR-ABL, Baf3-E255K-BCR- AbI, Baf3-Y253F-BCR-Abl, Baf3-T315l-BCR-ABI, 3T3-v-Abl; or activated Flt3 kinase such as Baf3-FLT3 or FLT3-ITD; or JAK2 such as Baf3/Jak2; or mutant JAK2 V617F such as Baf3-V617F -JAK2, or Tyk2 such as Baf3/Tyk2; or TEL-FGFR3 such as Baf3-Tel/FGFR3; or TpoR such as Baf3/TpoR and Baf3/cc-TpoR-IV; or FGFR1 such as 293T-FGFR. Tryptic phosphotyrosine - containing peptides were purified and analyzed from extracts of each of the 29 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. Suspension cells were harvested by low speed centrifugation. After complete aspiration of medium, cells were resuspended in 1 ml_ lysis buffer per 1.25 x 108 cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or not with 2.5 mM sodium pyrophosphate, 1 mM β-glycerol-phosphate) and sonicated. Sonicated cell lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM. For digestion with trypsin, protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK-trypsin (Worthington) was added at 10-20 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 Ci8 columns (Waters) equilibrated with 0.1 % TFA. A column volume of 0.7-1.0 ml was used per 2 x 108 cells. Columns were washed with 15 volumes of 0.1% 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 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% TFA1 respectively. All peptide fractions were lyophilized.
Peptides from each fraction corresponding to 2 x 108 cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HCI or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCI) and insoluble matter (mainly in peptide fractions III) was removed by centrif ligation. 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 or protein A agarose (Roche), respectively. 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 minutes.
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 % 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 //I, 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 bv LC-MS/MS Mass Spectrometry. 40 μl or more 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 x 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 LTQ ion trap mass spectrometer essentially as described by Gygi et ah, supra.
Database Analysis & Assignments.
MS/MS spectra were evaluated using TurboSequest in the Sequest Browser package (v. 27, rev. 12) supplied as part of BioWorks 3.0 (ThermoFinnigan). Individual MS/MS spectra were extracted from the raw data file using the Sequest Browser program CreateDta, with the following settings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4 x 105; and precursor charge state, unspecified. Spectra were extracted from the beginning of the raw data file before sample injection to the end of the eluting gradient. The 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 (as released on February 23, 2004 and containing 27,175 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 restricted phosphorylation to tyrosine residues had little effect on the number of phosphorylation sites assigned. Furthermore it should be noted that certain peptides were originally isolated in mouse and later normalized to human sequences as shown by Table1/Figure2.
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., MoI. 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. The following Sequest scoring thresholds were used to select phosphopeptide assignments that are likely to be correct: RSp < 6, XCorr > 2.2, and DeltaCN > 0.099. Further, the assigned sequences could be accepted or rejected with respect to accuracy by using the following conservative, two-step process.
In the first step, a subset of high-scoring sequence assignments should be 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 should be rejected if any of the following criteria were satisfied: (i) the spectrum contains at least one major peak (at least 10% as intense as the most intense ion in the spectrum) that can 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 does not contain a series of b or y ions equivalent to at least six uninterrupted residues; or (iii) the sequence is not observed at least five times in all the studies 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 should be accepted if the low-scoring spectrum shows a high degree of similarity to a high-scoring spectrum collected in another study, which simulates a true reference library-searching strategy. EXAWIPLE 2
Production of Phospho-specific Polyclonal Antibodies for the Detection of Leukemia-related Signaling Protein Phosphorylation
Polyclonal antibodies that specifically bind a Leukemia-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/Figure 2) 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. MELK (tyrosine 438).
A 15 amino acid phospho-peptide antigen, SAVKNEEy*FMFPEPK (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 438 phosphorylation site in human MELK kinase (see Row 244 of Table 1 ; SEQ ID NO: 243), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho- specific MELK(tyr438 ) polyclonal antibodies as described in Immunization/ Screening below.
B- SIT1 (tyrosine 127). A 15 amino acid phospho-peptide antigen, AAEEVMCyTSLQLRP
(where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 127 phosphorylation site in human SIT1 adaptor/scaffold protein (see Row 38 of Table 1 (SEQ ID NO: 37)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho-specific SIT1(tyr127) polyclonal antibodies as described in Immunization/Screening below.
C. P ECA M 1 (tyrosine 663).
A 13 amino acid phospho-peptide antigen, MEANSHy*GHNDDV (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 663 phosphorylation site in human PECAM 1 adhesion protein (see Row 73 of Table 1 (SEQ ID NO: 72), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho-specific PECAM1 (tyr663) antibodies as described in Immunization/Screening below.
Immunization/Screening.
A synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and rabbits are injected intradermal^ (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 ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, supra.). The eluted immunoglobulins are further loaded onto a non-phosphorylated synthetic peptide antigen-resin Knotes column to pull out antibodies that bind the non-phosphorylated form of the phosphorylation site. The flow through fraction is collected and applied onto a phospho-synthetic peptide antigen-resin column to isolate antibodies that bind the phosphorylated form of the site. After washing the column extensively, the bound antibodies (i.e. antibodies that bind a phosphorylated peptide described in A-C above, but do not bind 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 that expresses (or overexpresses) target phospho-protein (i.e. phosphorylated MELK, SIT1 or PECAM1), for example, SEM, Jurkat and MKPL-1 cells, respectively. Cells are cultured in DMEM or RPMI supplemented with 10% FCS. Cell are collected, washed with PBS and directly lysed in cell lysis buffer. The protein concentration of cell lysates is then measured. The loading buffer is added into cell lysate and the mixture is boiled at 100 0C 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 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 (substantially) recognize the target protein when not phosphorylated at the appropriate phosphorylation site in the non-stimulated cells (e.g. PECAM1 is not bound when not phosphorylated at tyrosine 663).
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 performed 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.
EXAMPLE 3 Production of Phospho-specific Monoclonal Antibodies for the
Detection of Leukemia-related Signaling Protein Phosphorylation
Monoclonal antibodies that specifically bind a Leukemia-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1 /Figure 2) 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. TSC2 (tyrosine 1736).
A 10 amino acid phospho-peptide antigen, PTDIy*PSKW (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 1736 phosphorylation site in human TSC2 GTPase activating protein (see Row 87 of Table 1 (SEQ ID NO: 86)), plus cysteine on the C- terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal TSC2(tyr1736) antibodies as described in Immunization/Fusion/Screening below.
B. CD84 (tyrosine 279). A 10 amino acid phospho-peptide antigen, ESRIy*DEIL (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 279 phosphorylation site in human CD84 cell surface protein (see Row 93 of Table 1 (SEQ ID NO: 92)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal CD84(tyr279) antibodies as described in Immunization/Fusion/Screening below.
C. STAT5A (tyrosine 22).
A 14 amino acid phospho-peptide antigen, QMQVLy*GQHFPIEV (where Y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 22 phosphorylation site in human STAT5A transcription factor (see Row 411 of Table 1 (SEQ ID NO: 410)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho-specific monoclonal STAT5A (tyr22) antibodies as described in Immunization/Fusion/Screening below. Immunization/Fusion/Screening.
A synthetic phospho-peptide antigen as described in A-C above is coupled to KLH, and BALB/C mice are injected intradermal^ (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 TSC2, CD84, or STAT5A 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. STAT5A phosphorylated at tyrosine 22). EXAMPLE 4
Production and Use of AQUA Peptides for the Quantification of Leukemia-related Signaling Protein Phosphorylation
Heavy-isotope labeled peptides (AQUA peptides (internal standards)) for the detection and quantification of a Leukemia-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 1/Figure 2) 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. ZAP70 (tyrosine 164).
An AQUA peptide comprising the sequence, MPWy*HSSLTR (y*= phosphotyrosine; sequence incorporating 14C/15N-labeled leucine (indicated by bold L), which corresponds to the tyrosine 164 phosphorylation site in human ZAP70 kinase (see Row 284 in Table 1 (SEQ ID NO: 283)), 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 ZAP70(tyr164) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated ZAP70(tyr164) in the sample, as further described below in Analysis & Quantification. B. SCAP1 (tyrosine 142).
An AQUA peptide comprising the sequence GLFYy*YANEK (y*= phosphotyrosine; sequence incorporating 14C/1δN-labeled leucine (indicated by bold L), which corresponds to the tyrosine 142 phosphorylation site in human SCAP1 adaptor/scaffold protein (see Row 42 in Table 1 (SEQ ID NO: 41)), 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 SCAP1 (tyr142) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated SCAP1(tyr142) in the sample, as further described below in Analysis & Quantification.
C. CFL1 (tyrosine 68)
An AQUA peptide comprising the sequence, GQTVDDPy*ATFVKML (y*= phosphotyrosine; sequence incorporating 14C/15N-labeled phenylalanine (indicated by bold F), which corresponds to the tyrosine 211 phosphorylation site in human CFL1 adaptor/scaffold protein {see Row 121 in Table 1 (SEQ ID NO: 120)), 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 CFL1(tyr68) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated CFL1(tyr68) in the sample, as further described below in Analysis & Quantification.
D. BLK (tyrosine 187).
An AQUA peptide comprising the sequence, CLDEGGy*YISPR (y*= phosphotyrosine; sequence incorporating 14C/15N-labeled proline (indicated by bold P), which corresponds to the tyrosine 187 phosphorylation site in human BLK kinase (see Row 271 in Table 1 (SEQ ID NO: 270)), 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 BLK(tyr187) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated BLK(tyr187) in the sample, as further described below in Analysis & Quantification.
Synthesis & MS/MS Spectra. Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acid monomers may be obtained from AnaSpec (San Jose, CA). Fmoc-derivatized stable- isotope monomers containing one 15N and five to nine 13C atoms may be obtained from Cambridge Isotope Laboratories (Andover, MA). Preloaded Wang resins may be obtained from Applied Biosystems. Synthesis scales may vary from 5 to 25 μmol. Amino acids are activated in situ with 1-H- benzotriazolium, i-bis(dimethylamino) methylenej-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 by- products. 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. Samples are then directly loaded onto the microcapillary column by using a FAMOS inert capillary autosampler (LC Packings, San Francisco) after the flow split. Peptides are reconstituted in 6% acetic acid/0.01% TFA before injection.
Analysis & Quantification.
Target protein (e.g. a phosphorylated protein of A-D above) in a biological sample is quantified using a validated AQUA peptide (as described above). The IAP method is then applied to the complex mixture of peptides derived from proteolytic cleavage of crude cell extracts to which the AQUA peptides have been spiked in.
LC-SRM of the entire sample is then carried out. MS/MS may be performed by using a ThermoFinnigan (San Jose, CA) mass spectrometer (LTQ ion trap or TSQ Quantum triple quadrupole). On the LTQ, parent ions are isolated at 1.6 m/z width, the ion injection time being limited to 100 ms per microscan, with one microscans per peptide, and with an AGC setting of 1 x 105; 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).

Claims

WHAT IS CLAIMED IS:
1. A method for obtaining a phosphorylation profile of protein Kinases that are phosphorylated in Leukemia signaling pathways, said method comprising the step of utilizing one or more isolated antibody that specifically binds a protein Kinase selected from Column A, Rows 210- 291 , of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 210-291 , of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: 210-221 , 223-280, and 281-290), to detect the phosphorylation of one or more of said protein Kinases, thereby obtaining a phosphorylation profile for said Kinases.
2. A method for detecting or quantifying a signaling protein that is tyrosine phosphorylated in Leukemia signaling pathways, said method comprising the step of utilizing one or more of the following reagents to detect or quantify one or more Leukemia-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 :
(i) an isolated phosphorylation site-specific antibody that specifically binds said protein only when phosphorylated at the tyrosine listed in corresponding Column D of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21, 23, 26-30, 32-34, 36-45, 48- 52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182-193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319- 336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440- 474, and 476-480), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine; and/or
(ii) a heavy-isotope labeled peptide (AQUA peptide) for the quantification of said protein, said labeled peptide comprising the phosphorylation site peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21, 23, 26-30, 32-34, 36-45, 48-52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182- 193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319-336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440-474, and 476-480).
3. The method of claim 2, wherein said protein is an adaptor/scaffold protein selected from Column A, Rows 11-59 of Table 1, and wherein
(i) said antibody specifically binds said adaptor/scaffold protein only when phosphorylated at the tyrosine listed in corresponding
Column D, Rows 11-59, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 11-59, of Table 1 (SEQ ID NOs: 10-14, 16, 19-21, 23, 26-30, 32-34, 36-45, 48-52, and 56-58), and (ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 11-59, of Table 1 (SEQ ID NOs: 10-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, and 56-58), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 11-58, of Table 1.
4. The method of claim 2, wherein said protein is an adhesion binding protein selected from Column A, Rows 60-79, of Table 1 , and wherein
(i) said antibody specifically binds said adhesion binding protein only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 60-79, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 60-79, of Table 1 (SEQ ID NOs: 60-78), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 60-79, of Table 1 (SEQ ID NOs: 60-78), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 60-79, of Table 1.
5. The method of claim 2, wherein said protein is a cell cycle regulation protein selected from Column A, Rows 83-87, of Table 1 , and wherein
(i) said antibody specifically binds said cell cycle regulation protein only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 83-87, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 83-87 of Table 1 (SEQ ID NOs: 82-86), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 83-87, of Table 1 (SEQ ID NOs: 82-86), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 83-87, of Table 1.
6. The method of claim 2, wherein said protein is a cell surface protein selected from Column A, Rows 88-94 of Table 1, and wherein
(i) said antibody specifically binds said cell surface protein only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 88-94, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 88-94, of Table 1 (SEQ ID NOs: 87-90, and 93), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 88-94, of Table 1 (SEQ ID NOs: 87-90, and 93), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 88-94, of Table 1.
7. The method of claim 2, wherein said protein is a cytoskeletal protein selected from Column A, Rows 114-170, of Table 1 , and wherein (i) said antibody specifically binds said cytoskeletal protein only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 114-170, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 114-170, of Table 1 (SEQ ID NOs: 113-119, 121-124, 129-151 , 153-160, and 163-169), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 114-170, of Table 1 (SEQ ID NOs: 113-119, 121-124, 129-151 , 153-160, and 163-169), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 114-170, of Table t
8. The method of claim 2, wherein said protein is a G protein selected from Column A, Rows 174-180, of Table 1 , and wherein
(i) said antibody specifically binds said G protein only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 174-180, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 174-180, of Table 1 (SEQ ID NOs: 173-179), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 174-180, of Table 1 (SEQ ID NOs: 173-179), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 174-180, of Table 1.
9. The method of claim 2, wherein said protein is a protein kinase selected from Column A, Rows 210-291 , of Table 1 , and wherein
(i) said antibody specifically binds said protein kinase only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 210-291 , of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: 210-221 , 223-280, and 281-290), and (ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: 210-221 , 223-280, and 281-290), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 210-291, of Table !
10. The method of claim 2, wherein said protein is a lipid kinase selected from Column A, Rows 292-298, of Table 1 , and wherein
(i) said antibody specifically binds said protein kinase only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 292-298, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 292-298, of Table 1 (SEQ ID NOs: 291-297), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 292-298, of Table 1 (SEQ ID NOs: 291-297), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 292-298, of Table 1.
11. The method of claim 2, wherein said protein is a phosphatase selected from Column A, Rows 331-350, of Table 1 , and wherein
(i) said antibody specifically binds said phosphatase only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 331-350, of Table 1, comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 331-350, of Table 1 (SEQ ID NOs: 330-349), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 331-350, of Table 1 (SEQ ID NOs: 330-349), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 331-350, of Table 1.
12. The method of claim 2, wherein said protein is a phosphodiesterase/phospholipase selected from Column A, Rows 351- 359, of Table 1 , and wherein
(i) said antibody specifically binds said phophodiesterase/ phospholipase only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 351-359, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 351-359, of Table 1 (SEQ ID NOs: 350-356, and 358), and
(ii) said labeled peptide comprises the phosphorylation site sequence listed in corresponding Column E, Rows 351-359, of Table 1 (SEQ ID NOs: 350-356, and 358), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 351-359, of Table 1.
13. The method of claim 2, wherein said protein is an receptor protein selected from Column A, Rows 366-386, of Table 1 , and wherein (i) said antibody specifically binds said receptor protein only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 366-386, of Table 1, comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 366-386, of Table 1 (SEQ ID NOs: 365-366, 368-378, and 380-385), and (ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 366-386, of Table 1 (SEQ ID NOs: 365-366, 368-378, and 380-385), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 366- 386, of Table t
14. The method of claim 2, wherein said protein is a transcription factor selected from Column A, Rows 387-425, of Table 1 , and wherein
(i) said antibody specifically binds transcription factor only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 387-425, of Table 1, comprised within the phosphorylation site sequence listed in corresponding Column E, Rows 387-425, of Table 1 (SEQ ID NOs: 386-417, and 419-424), and
(ii) said labeled peptide comprises the phosphorylation site peptide sequence listed in corresponding Column E, Rows 387-425, of Table 1 (SEQ ID NOs: 386-417, and 419-424), comprising the phosphorylated tyrosine listed in corresponding Column D, Rows 387-425 of Table 1.
15. The method of claim 2, wherein said protein is selected from the group consisting of EIF4EBP2, EIF4G2 and EIF4B (Column A, Rows 199, 446, 448 and 460 of Table 1), and wherein
(i) said antibody specifically binds said protein only when phosphorylated at the tyrosine listed in corresponding Column F, Rows
446, 448 and 460, of Table 1 , comprised within the phosphorylation site sequence listed in corresponding Column G, Rows Rows 446, 448 and 460, of Table 1 (SEQ ID NOs: 445, 447 and 459), and
(ii) said labeled peptide comprises the phosphorylation site sequence listed in'corresponding Column G, Rows 446, 448 and 460, of Table 1 (SEQ ID NOs: 445, 447 and 459), comprising the phosphorylated tyrosine listed in corresponding Column F, Rows 446, 448 and 460, of Table 1.
16. An isolated phosphorylation site-specific antibody that specifically binds a human Leukemia-related signaling protein selected from Column A of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48- 52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182-193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319- 336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440- 474, and 476-480), wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine.
17. An isolated phosphorylation site-specific antibody that specifically binds a human Leukemia-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 phosphorylatable peptide sequence listed in corresponding Column E of Table 1 (SEQ ID NOs: 1-7, 9-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48- 52, 56-58, 60-90, 93-119, 121-124, 129-151, 153-160, 163-180, 182-193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319- 336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440- 474, and 476-480), wherein said antibody does not bind said signaling protein when phosphorylated at said tyrosine.
18. A heavy-isotope labeled peptide (AQUA peptide) for the quantification of a human Leukemia-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: 1-1-7, 9-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, 56-58, 60-90, 93-119, 121-124, 129-151 , 153-160, 163-180, 182- 193, 195-197, 199-208, 210-221 , 223-279, 281-294, 296-297, 299-316, 319-336, 339-345, 347-356, 358, 360-366, 368-378, 380-417, 419-438, 440-474, and 476-480), comprising the phosphorylatable tyrosine listed in corresponding Column D, Rows 2-481 , of Table 1.
19. The labeled peptide of claim 18, wherein said phosphorylatable tyrosine is phosphorylated.
20. The labeled peptide of claim 18, wherein said phosphorylatable tyrosine is not phosphorylated.
21. An immortalized cell line producing the antibody of claim 16 or 17.
22. The cell line of claim 21 , wherein said immortalized cell line is a rabbit hybridoma or a mouse hybridoma.
23. The antibody of claim 16, wherein said antibody specifically binds an adaptor/scaffold protein selected from Column A, Rows 11-59, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 11-59, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 11-59, of Table 1 (SEQ ID NOs: 10-14, 16, 19-21 , 23, 26-30, 32-34, 36-45, 48-52, and 56-58), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
24. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of an adaptor/scaffold protein selected from Column A, Rows 11-59, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 11-59, of Table 1 (SEQ ID NOs: 10-14, 16, 19-21, 23, 26- 30, 32-34, 36-45, 48-52, and 56-58), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 11-59, of Table 1.
25. The antibody of claim 16, wherein said antibody specifically binds an adhesion protein selected from Column A, Rows 60-79, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 60-79, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 60-79, of Table 1 (SEQ ID NOs: 60-78), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
26. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of an adhesion protein selected from Column A, Rows 60-79, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 60-79, of Table 1 (SEQ ID NOs: 60-78), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 60- 79, of Table t
27. The antibody of claim 16, wherein said antibody specifically binds a cell cycle regulation protein selected from Column A, Rows 83-87, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 83-87, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 83-87, of Table 1 (SEQ ID NOs: 82-86), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
28. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a cell cycle regulation protein selected from Column A, Rows 83-87, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 83-87, of Table 1 (SEQ ID NOs: 82-86), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 83-87, of Table 1.
29. The antibody of claim 16, wherein said antibody specifically binds a cell surface protein selected from Column A, Rows 88-94, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 88-94, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 88-94, of Table 1 (SEQ ID NOs: 87-90, and 93), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
30. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a cell surface protein selected from Column A, Rows 88-94, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 88-94, of Table 1 (SEQ ID NOs: 87-90, and 93), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 88-94, of Table 1.
31. The antibody of claim 16, wherein said antibody specifically binds a cytoskelatal protein selected from Column A, Rows 114-170, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 114-170, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 114-170 of Table 1 (SEQ ID NOs: 113-119, 121-124, 129-151 , 153-160, and 163- 169), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
32. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of cytoskelatal protein selected from Column A, Rows 114-170, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 114-170, of Table 1 (SEQ ID NOs: 113-119, 121-124, 129-151 , 153- 160, and 163-169), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 114-170, of Table 1.
33. The antibody of claim 16, wherein said antibody specifically binds a G protein selected from Column A, Rows 174-180, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 174-180, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 174-180, of Table 1 (SEQ ID NOs: 173-179), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
34. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a Leukemia-related signaling protein that is a G protein selected from Column A, Rows 174- 180, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 174-180, of Table 1 (SEQ ID NOs: 173-179), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 174-180, of Table 1.
35. The antibody of claim 16, wherein said antibody specifically binds a protein kinase selected from Column A, Rows 210-291, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 210-291 , of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: 210-221, 223-280, and 281-290), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
36. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a protein kinase selected from Column A, Rows 210-291 , said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 210-291 , of Table 1 (SEQ ID NOs: 210-221 , 223-280, and 281-290), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 210-291 , of Table 1.
37. The antibody of claim 16, wherein said antibody specifically binds a lipid kinase selected from Column A, Rows 292-298, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 292-298, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 292-298, of Table 1 (SEQ ID NOs: 291-297), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
38. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a lipid kinase selected from Column A, Rows 292-298, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 292-298, of Table 1 (SEQ ID NOs: 291-297), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 292-298, of Table 1.
39. The antibody of claim 16, wherein said antibody specifically binds a phosphatase selected from Column A, Rows 331-350, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 331-350, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 331-350, of Table 1 (SEQ ID NOs: 330-349), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
40. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a phosphatase selected from Column A, Rows 331-350, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 331-350, of Table 1 (SEQ ID NOs: 330-349), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 331-350, of Table 1.
41. The antibody of claim 16, wherein said antibody specifically binds a phophodiesterase/phospholipase protein selected from Column A, Rows 351-359, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 351-359, of Table 1 , comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 351-359, of Table 1 (SEQ ID NOs: 350-356, and 358), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
42. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a phosphodiesterase/phospholipase protein selected from Column A, Rows 351-359, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E, Rows 351-359, of Table 1 (SEQ ID NOs: 350-356, and 358), which sequence comprises the phosphorylatable tyrosine or serine listed in corresponding Column D, Rows 351-359, of Table 1.
43. The antibody of claim 16, wherein said antibody specifically binds a receptor protein selected from Column A, Rows 366-386, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 366-386, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 366-386, of Table 1 (SEQ ID NOs: 365-366, 368-378, and 380-385), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
44. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a receptor protein selected from Column A, Rows 366-386, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E1 Rows 366-386, of Table 1 (SEQ ID NOs: 365-366, 368-378, and 380-385), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 366-386, of Table 1.
45. The antibody of claim 16, wherein said antibody specifically binds a transcription factor selected from Column A, Rows 387-425, of Table 1 only when phosphorylated at the tyrosine listed in corresponding Column D, Rows 387-425, of Table 1, comprised within the phosphorylatable peptide sequence listed in corresponding Column E, Rows 387-425, of Table 1 (SEQ ID NOs: 386-417, and 419-424), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
46. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a transcription factor selected from Column A, Rows 387-425, said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column E1 Rows 387-425, of Table 1 (SEQ ID NOs: 386-417, and 419- 424), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column D, Rows 387-425, of Table 1.
47. The antibody of claim 16, wherein said antibody specifically binds a protein selected from the group consisting of EIF4EBP2, EIF4G2 and
EIF4B (Column A, Rows 446, 448 and 460 of Table 1) only when phosphorylated at the tyrosine listed in corresponding Column F, Rows 446, 448 and 460 of Table 1), said tyrosine comprised within the phosphorylatable peptide sequence listed in corresponding Column G, Rows 446, 448 and 460, of Table 1 (SEQ ID NOs: 445, 447 and 459), wherein said antibody does not bind said protein when not phosphorylated at said tyrosine.
48. The heavy-isotope labeled peptide (AQUA peptide) of claim 18, wherein said labeled peptide is for the quantification of a protein selected from the group consisting of EIF4EBP2, EIF4G2 and EIF4B (Column A1 Rows 446, 448 and 460 of Table 1), said labeled peptide comprising the phosphorylatable peptide sequence listed in corresponding Column G, Rows 446, 448 and 460, of Table 1 (SEQ ID NOs: 445, 447 and 459), which sequence comprises the phosphorylatable tyrosine listed in corresponding Column F, Rows 446, 448 and 460, of Table 1.
49. An immortalized cell line producing the antibody of any one of claims 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45 and 47.
50. The cell line of claim 49, wherein said immortalized cell line is a rabbit hybridoma or a mouse hybridoma.
51. The heavy-isotope labeled peptide of any one of claims 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48, wherein said phosphorylatable tyrosine is phosphorylated.
52. The heavy-isotope labeled peptide of any one of claims 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48, wherein said phosphorylatable tyrosine is not phosphorylated.
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