A Prototype Antibody Microarray Platform to Monitor Changes in Protein Tyrosine Phosphorylation

Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of Bcr-Abl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs. Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of Bcr-Abl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs. The complex network of all proteins in a living cell that are stably or transiently phosphorylated at tyrosine residues constitutes the tyrosine phosphoproteome. Reversible tyrosine phosphorylation, through the actions of specialized enzymes, kinases, and phosphatases, plays an important role in cell signaling, development, proliferation, and growth control (1Hunter T. The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: Its role in cell growth and disease..Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1998; 353: 583-605Crossref PubMed Scopus (364) Google Scholar, 2Schlessinger J. Cell signaling by receptor tyrosine kinases..Cell. 2000; 103: 211-225Abstract Full Text Full Text PDF PubMed Scopus (3538) Google Scholar, 3Pawson T. Nash P. Assembly of cell regulatory systems through protein interaction domains..Science. 2003; 300: 445-452Crossref PubMed Scopus (1151) Google Scholar). Deregulation has been implicated in disease, most frequently in cancer (4Daley G.Q. Ben-Neriah Y. Implicating the bcr/abl gene in the pathogenesis of Philadelphia chromosome-positive human leukemia..Adv. 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The molecular biology of chronic myeloid leukemia..Blood. 2000; 96: 3343-3356Crossref PubMed Google Scholar). By identifying these targets, diagnostic tools may be developed for some cancers and, importantly, the modification process could be a target for therapeutic intervention (8Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase..Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1621) Google Scholar, 9Kurzrock R. Kantarjian H.M. Bruker B.J. Talpaz M. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics..Ann. Intern. Med. 2003; 138: 819-830Crossref PubMed Scopus (271) Google Scholar, 10Mendelsohn J. Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer..J. Clin. Oncol. 2003; 21: 2787-2799Crossref PubMed Scopus (1185) Google Scholar, 11Blagosklonny M.V. Gefitinib (Iressa) in oncogene-active cancers and therapy for common cancers..Cancer Biol. Ther. 2004; 3 ([epub ahead of print])Crossref PubMed Scopus (11) Google Scholar). Monitoring changes in the tyrosine phosphoproteome that accompany cell growth, differentiation, genetic alteration, disease, or exposure to bioactive substances and drugs represents therefore a fundamental task in contemporary proteomics. Tyrosine phosphorylation is the least abundant post-translational modification (PTM) 1The abbreviations used are: PTM, post-translational modification; Ab, antibody; mAb, monoclonal antibody; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; p-Tyr, phosphotyrosine; p-Ser, phosphoserine; p-Thr, phosphothreonine; PMF, peptide mass fingerprinting; EDC, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride; mcKLH, mariculture keyhole limpet hemocyanin; FPA, forward phase arrays; RPA, reversed phase arrays; MALDI-reTOF, MALDI reflectron TOF; PBST, PBS containing 0.05% Tween® 20; NR, nonredundant; IP, immunoprecipitation. compared with phospho-serine (p-Ser) or -threonine (p-Thr) and is estimated to be less than 0.05% of the total cellular protein phospho-amino acid content (12Hunter T. Sefton B. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine..Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1311-1315Crossref PubMed Scopus (1556) Google Scholar). This situation presents a major challenge to develop and implement adequate tyrosine phosphoproteome analysis tools. Mass spectrometry, already the standard method to identify proteins (13Baldwin M.A. Protein identification by mass spectrometry: Issues to be considered..Mol. Cell. Proteomics. 2004; 3: 1-9Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), has also gained popularity for phosphoproteome analysis, as practiced either with or without prior gel fractionation of the cellular proteome or subsets thereof (14Pandey A. Podtelejnikov A.V. Blagoev B. Bustelo X.R. Mann M. Lodish H.F. Analysis of receptor signaling pathways by mass spectrometry: Identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 179-184Crossref PubMed Scopus (376) Google Scholar, 15Pandey A. Fernandez M.M. Steen H. Blagoev B. Nielsen M.N. Roche S. Mann M. Lodish H.F. 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Several technical and practical problems remain unresolved, however, particularly in the analysis of low-abundance proteins in cell or tissue extracts, which necessitates fairly large amounts of starting material. This may be relatively straightforward in the case of cultured cells but very difficult or simply impossible when dealing with tissues. Clinical samples, such as patient biopsies, are unique and often limited in amount and concentration of the analytes. The number of cells required for mass spectrometric identification, typically 108 to 109 (14Pandey A. Podtelejnikov A.V. Blagoev B. Bustelo X.R. Mann M. Lodish H.F. Analysis of receptor signaling pathways by mass spectrometry: Identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors..Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 179-184Crossref PubMed Scopus (376) Google Scholar, 15Pandey A. Fernandez M.M. Steen H. Blagoev B. Nielsen M.N. Roche S. Mann M. Lodish H.F. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth factor and cytokine receptor signaling pathways..J. Biol. Chem. 2000; 275: 38633-38639Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 19Salomon A.R. Ficarro S.B. Brill L.M. Brinker A. Phung Q.T. Ericson C. Sauer K. Brock A. Horn D.M. Shultz P.G. Peters E.C. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 443-448Crossref PubMed Scopus (265) Google Scholar), may not always exist within the range achievable for clinical studies. It has been estimated (26Liotta L.A. Espina V. Mehta A.I. Calvert V. Rosenblatt K. Geho D. Munson P.J. Young L. Wulfkuhle J. Petricoin E.F. Protein microarrays: Meeting analytical challenges for clinical applications..Cancer Cell. 2003; 3: 317-325Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar) that, whereas a cubic centimeter of tissue contains ∼109 cells, those numbers could be less than 105 in a core needle biopsy or cell aspirate. It is therefore of major importance to develop new tyrosine phosphoproteome analysis tools with more stringent requirements in terms of sensitivity and throughput. This can be satisfied by exploring antibody (Ab) microarray technology that utilizes extremely low sample and reagent volumes. In one of the first comprehensive evaluations of an Ab microarray platform for analysis of proteins in complex mixtures, direct fluorescent labeling of the analytes was used for detection at protein concentrations in the low microgram per milliliter range (27Haab B.B. Dunham M.J. Brown P.O. Protein microarrays for highly parallel detection and quantification of specific proteins and antibodies in complex solutions..Genome Biol. 2001; 2 (research0004.1–0004.13)Crossref PubMed Google Scholar). Since then, several other assays based on protein microarrays have been developed utilizing two major formats: forward phase arrays (FPA) and reversed phase arrays (RPA) (26Liotta L.A. Espina V. Mehta A.I. Calvert V. Rosenblatt K. Geho D. Munson P.J. Young L. Wulfkuhle J. Petricoin E.F. Protein microarrays: Meeting analytical challenges for clinical applications..Cancer Cell. 2003; 3: 317-325Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). FPA assays (typically with multiple Abs printed on the chip to probe a single tissue extract or biological fluid sample at a time) have been applied to detect clinically relevant cytokines (28Moody M.D. Van Arsdell S.W. Murphy K.P. Orencole S.F. Burns C. Array-based ELISAs for high-throughput analysis of human cytokines..BioTechniques. 2001; 31: 186-190Crossref PubMed Scopus (164) Google Scholar), bacteria and bacterial toxins (29Delehanty J.B. Ligler F.C. A microarray immunoassay for simultaneous detection of proteins and bacteria..Anal. Chem. 2002; 74: 5681-5687Crossref PubMed Scopus (284) Google Scholar), potential biomarkers in human serum (30Miller J.C. Zhou H. Kwekel J. Cavallo R. Burke J. Butler E.B. The B.S. Haab B.B. Antibody microarray profiling of human prostate cancer serum: Antibody screening and identification of potential biomarkers..Proteomics. 2003; 3: 56-63Crossref PubMed Scopus (353) Google Scholar), and protein expression profiling in human oral cavity tissues (31Knezevic V. Leethanakul C. Bichsel V.E. Worth J.M. Prabhu V.V. Gutkind J.S. Liotta L.A. Munson P.J. Petricoin E.F. Krizman D.B. Proteomic profiling of the cancer microenvironment by antibody arrays..Proteomics. 2001; 1: 1271-1278Crossref PubMed Scopus (283) Google Scholar) or cultured cells (32Sreekumar A. Nyati M.K. Varambally S. Barrette T.R. Ghosh D. Lawrence T.S. Chinnaiyan A.M. Profiling of cancer cells using protein microarrays: Discovery of novel radiation-regulated proteins..Cancer Res. 2001; 61: 7585-7593PubMed Google Scholar). RPA assays (typically with single antigens or multiple antigen-containing samples printed on the chip for incubation with soluble Abs, either purified or in biological fluids) have been utilized in determination of Ab specificity and cross-reactivity (33Lueking A. Possling A. Huber O. Beveridge A. Horn M. Eickhoff H. Schuchardt J. Lehrach H. Cahill D.J. A nonredundant human protein chip for antibody screening and serum profiling..Mol. Cell. Proteomics. 2003; 2: 1342-1349Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 34Michaud G.A. Salcius M. Zhou F. Bangham R. Bonin J. Guo H. Snyder M. Predki P.F. Schweitzer B.I. Analyzing antibody specificity with whole proteome microarrays..Nat. Biotechnol. 2003; 21: 1509-1512Crossref PubMed Scopus (248) Google Scholar), detection of auto-Abs in serum from patients with autoimmune disorders (35Robinson W.H. DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H.E. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses..Nat. Med. 2002; 8: 295-301Crossref PubMed Scopus (642) Google Scholar), screening of human serum for the presence of allergen-specific IgE (36Fall B.I. Eberlein-Konig B. Behrendt H. Niessner R. Ring J. Weller M.G. Microarrays for the screening of allergen-specific IgE in human serum..Anal. Chem. 2003; 75: 556-562Crossref PubMed Scopus (119) Google Scholar), and expression analysis of a limited number of proteins in esophageal carcinoma and prostate cancer specimens (37Paweletz C.P. Charboneau L. Bichsel V. Simone N.L. Chen T. Gillespie J.W. Emmert-Buck M.R. Roth M.J. Petricoin E.F. Liotta L.A. Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front..Oncogene. 2001; 20: 1089-1981Crossref Scopus (842) Google Scholar). Despite its proven potential and diversity of many developed applications (38Zhu H. Bilgin M. Snyder M. Proteomics..Annu. Rev. Biochem. 2003; 72: 783-812Crossref PubMed Scopus (353) Google Scholar), Ab microarrays have not been widely used in the analysis of the tyrosine phosphoproteome. To this date, only two assay platforms for investigation of PTMs of proteins have been proposed. Grubb and co-workers (39Grubb R.L. Calvert V.S. Wulkuhle J.D. Paweletz C.P. Linehan W.M. Phillips J.L. Chuaqui R. Valasco A. Gillespie J. Emmert-Buck M. Liotta L.A. Petricoin E.F. Signal pathway profiling of prostate cancer using reverse phase protein arrays..Proteomics. 2003; 3: 2142-2146Crossref PubMed Scopus (176) Google Scholar) described an RPA assay for analysis of relative phosphorylation of six cell-signaling proteins in prostate cancer specimens using sequence-specific Abs against p-Tyr-containing peptides. As only small numbers of cells were required, the assay was successfully coupled with laser capture microdissection of clinical specimens. This assay also offers the advantage of parallel analysis of a large number of clinical samples deposited on the same array. The collection of samples must be readily available at the time of array fabrication, therefore making the assay more applicable to screening archived samples from tissue/tumor banks rather than monitoring effectiveness of therapy or compound profiling in drug discovery. Because the assay requires sequence specific Abs, it is also impossible to detect phosphorylation of more than one protein within a single microarray slide. Nielsen and co-workers (40Nielsen U.B. Cardone M.H. Sinskey A.J. MacBeath G. Sorger P.K. Profiling receptor tyrosine kinase activation by using Ab microarrays..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9330-9335Crossref PubMed Scopus (202) Google Scholar) utilized an FPA approach to achieve simultaneous detection of two tyrosine-phosphorylated proteins. They developed a micro-sandwich assay by arraying anti-ErbB1 (epidermal growth factor receptor, EGFR) and anti-ErbB2 Abs and detecting the phosphorylation signal with corresponding anti-[p-Tyr1068]EGFR and anti-[p-Tyr1248]ErbB2 Abs. Increasing the number of arrayed elements in that system will require the use of complicated mixtures of corresponding detection Abs. The high complexity of detection "cocktails" may bring about a much-increased probability of Ab cross-reactivity, higher nonspecific binding and overall background, reduction of signal-to-noise ratios, and an elevated cost of experiments. In the present study, we describe a novel, high-sensitivity, FPA micro-sandwich assay platform, using a labeled p-Tyr-specific Ab and ratiometric data analysis, that is applicable to monitoring changes in the tyrosine phosphoproteome and conducive to a high degree of multiplexing and improved throughput. The reduction of this platform to practice was initially hindered by the absence of one key reagent, namely an anti-p-Tyr monoclonal antibody (mAb) that would meet the following stringent criteria: i) recognition of p-Tyr in all cellular proteins whenever present, ii) no reactivity toward nonphosphorylated tyrosine, iii) no other moieties as part of proteins including phosphorylated serine and threonine are recognized, iv) recognition is independent of the surrounding amino acid sequence, i.e. the recognition epitope is exclusively limited to p-Tyr, and v) a satisfactory performance in microarray-based assays that requires, for example, rather high concentration of mAb. We report herein development of PY-KD1 mAb that satisfies the above criteria and that is much better suited for this technique than the commercially available Abs. To evaluate our assay platform, we have used the tyrosine phosphoproteome of RT10+ and HeLa cells as model systems. RT10+ cells were originally established by transfecting human megakaryoblastic leukemia cells with the Bcr-Abl-expressing plasmid pGD210 (41Berman E. Jhanwar S. McBride M. Strife A. Wisniewski D. Lambek C. Clarkson B. Characterization of two novel sublines established from a human megakaryoblastic leukemia cell line transfected with p210BCR-ABL..Leukemia Res. 2000; 24: 289-297Crossref PubMed Scopus (8) Google Scholar). The Bcr-Abl fusion protein is a constitutively active tyrosine kinase and can be specifically inhibited by the anti-cancer drug STI-571 (Gleevec) (8Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase..Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1621) Google Scholar, 9Kurzrock R. Kantarjian H.M. Bruker B.J. Talpaz M. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics..Ann. Intern. Med. 2003; 138: 819-830Crossref PubMed Scopus (271) Google Scholar). To select the majority of Abs for the microarray fabrication, we first performed MALDI-TOF mass spectrometric identifications of p-Tyr-containing proteins in RT10+ cells after prior immunocapture on magnetic particles bearing immobilized PY-KD1 mAb. Fabricated Ab microarrays were evaluated by assessing changes in the tyrosine phosphoproteome of i) RT10+ cells after treatment with Gleevec and also of ii) HeLa cells after treatment with epidermal growth factor (EGF). Analyses can, in principle, be carried out using 103 to 105 cells, or less, depending on the cell or tissue type. Our studies confirmed the identification of a number of Bcr-Abl and EGFR targets, and associated proteins that had been previously reported. The sandwich Ab microarray assay described herein may hold particular promise for molecular classification of tumors and for compound profiling in development of novel target-cancer drugs similar to Gleevec (8Schindler T. Bornmann W. Pellicena P. Miller W.T. Clarkson B. Kuriyan J. Structural mechanism for STI-571 inhibition of abelson tyrosine kinase..Science. 2000; 289: 1938-1942Crossref PubMed Scopus (1621) Google Scholar) or Gefitinib (11Blagosklonny M.V. Gefitinib (Iressa) in oncogene-active cancers and therapy for common cancers..Cancer Biol. Ther. 2004; 3 ([epub ahead of print])Crossref PubMed Scopus (11) Google Scholar). Anti-p-Tyr Abs were purchased from the following vendors: 4G10 from Upstate USA, Inc. (Charlottesville, VA), PY-20 from BD Transduction Laboratories (Los Angeles, CA), and PY-100 from Cell Signaling Technology (Beverly, MA). Secondary Abs and streptavidin conjugated to horseradish peroxidase, mariculture keyhole limpet hemocyanin (mcKLH), maleimide-activated BSA, D-Salt™ dextran columns, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), Imject® conjugation buffers, ImmunoPure® TMB substrate kit, Halt™ protease inhibitor mixture, micro BCA™ protein assay reagent kit, and Slide-A-Lyzer® dialysis cassettes were obtained from Pierce (Rockford, IL). Recombinant Yersinia protein tyrosine phosphatase was obtained from Calbiochem (San Diego, CA). IgG-free, protease-free BSA was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Acetylated p-Tyr was custom synthesized by AnaSpec, Inc. (San Jose, CA) and all peptides by the Microchemistry Core Facility (MSKCC, New York, NY). Protein molecular weight markers were purchased from Amersham Biosciences (Piscataway, NJ). Ready Gel® polyacrylamide precast gels, polyoxyethylene sorbitan monolaurate (Tween® 20), and PVDF membranes were obtained from Bio-Rad Laboratories (Hercules, CA). Other reagents and supplies were obtained from Sigma (St. Louis, MO). p-Tyr was coupled to mcKLH using glutaraldehyde as described previously (42Frackelton R.A. Posner M. Kannan B. Mermelstein F. Generation of monoclonal antibodies against phosphotyrosine and their use for affinity purification of phosphotyrosine-containing proteins..Methods Enzymol. 1991; 201: 79-92Crossref PubMed Scopus (34) Google Scholar). Excess of nonconjugated p-Tyr was removed by passing the reaction mixture through a cross-linked dextran gel filtration column equilibrated with a purification buffer containing mcKLH stabilizers (Pierce). Prepared conjugate was aliquoted and stored frozen until used. Immunization of animals, fusion, and purification of the mAb was performed by the Monoclonal Antibody Core Facility (MSKCC), and all animal work was done under the protocol approved by the IACUC (Institutional Animal Care and Use Committee). Briefly, 50 μg of the antigen was emulsified in 500 μl of 50% TiterMax® adjuvant and used for the first intraperitoneal immunization of female BALB/c mice. Three weeks later, mice were injected intraperitoneally with 50 μg of the same conjugate emulsified in TiterMax®; after an additional 3 weeks, the intraper