From the Protein Center, and Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
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ABSTRACT |
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Tyrosine phosphorylation is the least abundant post-translational modification (PTM)1 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 (12). This situation presents a major challenge to develop and implement adequate tyrosine phosphoproteome analysis tools. Mass spectrometry, already the standard method to identify proteins (13), has also gained popularity for phosphoproteome analysis, as practiced either with or without prior gel fractionation of the cellular proteome or subsets thereof (1420). To this end, trace enrichment procedures have been developed to ensure adequate analysis of phosphotyrosine (p-Tyr) containing proteins, including immunoaffinity-based methods (1, 14, 15, 20, 21), chemical modification of the phosphate moiety for subsequent affinity capture (22, 23), and, at least in case of phosphopeptides, IMAC (19, 24, 25). 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 (14, 15, 19), may not always exist within the range achievable for clinical studies. It has been estimated (26) 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 (27). 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) (26). 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 (28), bacteria and bacterial toxins (29), potential biomarkers in human serum (30), and protein expression profiling in human oral cavity tissues (31) or cultured cells (32). 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 (33, 34), detection of auto-Abs in serum from patients with autoimmune disorders (35), screening of human serum for the presence of allergen-specific IgE (36), and expression analysis of a limited number of proteins in esophageal carcinoma and prostate cancer specimens (37).
Despite its proven potential and diversity of many developed applications (38), 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 (39) 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 (40) 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 (41). The Bcr-Abl fusion protein is a constitutively active tyrosine kinase and can be specifically inhibited by the anti-cancer drug STI-571 (Gleevec) (8, 9). 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 (8) or Gefitinib (11).
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EXPERIMENTAL PROCEDURES |
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mAb Development
p-Tyr was coupled to mcKLH using glutaraldehyde as described previously (42). 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 intraperitoneal immunization with 50 µg of antigen emulsified in TiterMax® was repeated. Four weeks later, the last injection of 50 µg of immunogen in PBS was performed intravenously, and after 3 days, the animal was sacrificed, and the spleen was removed and fused with SP2/0-Ag14 myeloma cells (American Type Culture Collection, Manassas, VA). Test bleeds were performed 1 week after each injection and before sacrificing the animal (terminal bleed). Fused cells were grown in hybridoma SFM medium supplemented with 10 mM sodium hypoxanthine, 1.6 µM thymidine, 15% fetal bovine serum (v/v) and 1% hybridoma cloning factor (v/v). Hybridoma SFM medium and HT supplement were from Invitrogen (Carlsbad, CA) and Origen® hybridoma cloning factor from Fisher Scientific (Pittsburg, PA). Hybridomas that secrete anti-p-Tyr Abs were selected by ELISAs and sub-cloned three times by the limited dilution method. PY-KD1 mAb was produced in vitro using a CELLine bioreactor (Integra BioSciences, Chur, Switzerland) and purified on a HiTrapTM protein G column (Amersham Biosciences) according to the manufacturers recommendations.
ELISA
Assays were performed by coating 96-well 4HBX microplates (Dynex Technologies, Chantilly, VA) with 75-ng/well hapten-BSA conjugate in PBS. Conjugated peptides contained either N- or C-terminal cysteine for easy covalent attachment to maleimide-activated BSA. Acetylated p-Tyr was coupled to BSA using EDC. Conjugation reactions were performed as recommended by Pierce. Excess of nonconjugated haptens was removed by dialysis against PBS using dialysis membranes with a molecular weight cut-off 7,000. Prepared conjugates were aliquoted and stored at 20 °C until further use. Coating of microplates was performed overnight at 4 °C. Prepared microplates were washed four times with PBS containing 0.05% Tween® 20 (PBST), blocked with 10 mg/ml BSA in PBST for 1 h at 37 °C and washed four times with PBST again. Samples (sera from immunized mice or hybridoma supernatants) were incubated in microplates for 1 h at 37 °C followed by four washes with PBST. Horseradish peroxidase-conjugated secondary Ab was added to the plates and incubated for 1 h at 37 °C followed by four washes with PBST. Color development was achieved with ImmunoPure® TMB substrate reagent kit according to the manufacturer recommendations. Plates were read at 450 nm in a Bio-Rad 550 microplate reader.
Cell Cultures
Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. RT10+ cells were obtained from Dr. Bayard Clarkson (MSKCC) and grown in Iscoves modified Dulbeccos medium supplemented with 20% (v/v) heat-inactivated fetal bovine serum and a penicillin-streptomycin mixture. They were maintained twice weekly by addition of fresh medium to a dilution of 2 x 105 cells/ml. HeLa cells (American Type Culture Collection) were grown in Dulbeccos modified Eagles medium (high glucose) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and a penicillin-streptomycin mixture. Upon reaching confluence (approximately every 4 days), they were routinely passaged by trypsinization. RT10+ cultures (density 1.21.5 x 106 cells/ml) were treated with Gleevec (1 µM) for 2 h. Subconfluent cultures of HeLa cells were grown in serum-free medium for 24 h followed by EGF treatment (150 ng/ml) for 1 h. STI-571 (Gleevec) was synthesized by the Preparative Chemistry Core Facility (MSKCC); tissue culture grade EGF was purchased from Upstate USA, Inc.
To prepare lysates, cells were collected and washed four times with ice-cold PBS without calcium or magnesium. The extraction buffer typically was Tris-HCl (50 mM, pH 7.3) supplemented with EDTA (1 mM), IGEPAL (1%), NaF (1 mM), Na3VO4 (2 mM) and HaltTM protease inhibitor mixture (1x). Ice-cold extraction buffer was added to cells (1 ml per 107 cells). Proteins were extracted for 15 min on a rocking platform at 4 °C. Cell debris was removed by centrifugation at 15,000 x g for 30 min at 4 °C. Protein concentration of the extract was determined using the Pierce micro BCA reagent kit. When the cell lysate was prepared for subsequent direct labeling with a fluorescent dye, the Tris buffer was substituted by carbonate buffer (200 mM, pH 9.3).
Protein and Ab Labeling
In a typical experiment, 2.6 ml of protein extract (0.2 mg/ml) was labeled with Cy5 fluorescent dye (Amersham Biosciences). The dye (200 nmol) was dissolved in a total volume of protein extract to be labeled and incubated in the dark at room temperature and gentle rocking for 30 min. Separation of unincorporated dye was done by gel-filtration over a Sephadex G-25 column (Amersham Biosciences), equilibrated with PBST. An equal volume of unlabeled protein extract was also applied to a G-25 column to exchange the extraction buffer to PBST. The same labeling/purification procedure was used to attach Cy5 dye to the PY-KD1 mAb.
Biotinylation of the mAb was performed using EZ-LinkTM sulfo-NHS-LC-biotin (Pierce). Two micrograms of purified, anti-p-Tyr IgG1 (clone PY-KD1), in a final volume of 1 ml of PBS, was mixed with 27 µl of 10 mM reagent for 1 h at room temperature on an end-over-end mixer. Excess free biotin was removed by extensive dialysis against PBS using a dialysis membrane with a 10,000 molecular weight cut-off. Biotinylation was determined to be approximately six molecules of biotin per molecule of mAb by using the EZTM biotin quantification kit (Pierce) according to the manufacturers procedure.
Immunoaffinity Capture of p-Tyr-Containing Proteins
Ten milligrams of streptavidin-coated paramagnetic particles (Dynabeads® M-280; Dynal Biotech ASA, Oslo, Norway) were collected, washed with 1 ml of PBS for 10 min, and collected; this cycle was repeated twice. The final pellet was resuspended in the solution of biotinylated PY-KD1 mAb (4 mg mAb in 2 ml of PBS) and incubated for 3 h at room temperature on an end-over-end mixer. The Ab-coupled particles were then washed for 10 min; twice with PBS, 0.1% Tween® 20; twice with PBS, 0.1% Triton® X-100; and twice with PBS. For collection of the beads from large volumes, high-powered cobalt magnetic discs (catalog no. CR30352-75; Edmund Scientific, Tonawanda, NY) were placed underneath 15-ml Falcon tubes during centrifugation in a GS-6KR rotor (Beckman, Fullerton, CA) at 700 x g for 5 min at 4 °C. For smaller volumes, in Eppendorf tubes, a hand-held MPC-S magnet (Dynal Biotech) was used. mAb beads were stored at 4 °C until further use.
RT10+ cells (5 x 108) were washed with ice-cold PBS and resuspended in 50 ml of ice-cold RIPA buffer (50 mM Tris buffer, pH 7.4, 1% IGEPAL, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM PMSF, 1 mM Na3VO4, containing 1x HaltTM protease inhibitor mixture). Cells were lysed for 15 min on a shaking platform at 4 °C, centrifuged at 14,000 x g for 15 min at 4 °C, and the supernatant stored at 80 °C. Lysate (50 ml, 0.5 mg/ml protein) was incubated with 5 mg of mAb beads overnight at 4 °C on an end-over-end mixer. The particles were pelleted as described above, transferred to a 2-ml tube, and washed for 10 min at 4 °C; thrice with PBS; thrice with PBS, 0.1% Tween® 20; and thrice with PBS, 0.1% Triton® X-100. p-Tyr-containing proteins were eluted in 20 µl of 20 mM phenylphosphate (in PBS) for 2 h at 4 °C under slight agitation. The eluate was then mixed with 20 µl of 2x Laemmli buffer, heated at 60 °C for 10 min, and the proteins were separated on a precast 415% Tris-glycine polyacrylamide gel (Bio-Rad) and stained with Coomassie® Brilliant Blue R-250 (Bio-Rad).
Mass Spectrometry
Gel-resolved proteins were digested with trypsin, the mixtures fractionated on a Poros 50 R2 RP micro-tip, and resulting peptide pools individually analyzed by MALDI reflectron TOF (MALDI-reTOF) MS using a Bruker UltraFlex TOF/TOF instrument (Bruker Daltonics, Bremen, Germany), in the presence of three peptide calibrants (6 fmol each; calculated monoisotopic masses of 2,108.155 Da, 1,307.762 Da, and 969.575 Da in the protonated form), as described (43, 44). Spectra were obtained by averaging multiple signals; laser irradiance and number of acquisitions (typically 100150) were operator-adjusted to yield maximal peak deflections derived from the digitizer in real time. The monoisotopic masses were assigned for all prominent peaks after visual inspection, and the low- and high-end internal standards were used for recalibration. Pass/fail criterion for recalibration is correct assignment of an m/z value for the middle calibrant with a mass accuracy equal or better than 15 ppm. After removal of autolytic and keratin-derived tryptic peptides, selected fragment ions (m/z) were taken to search the human segment of a "nonredundant" protein database (NR; 109,000 entries; National Center for Biotechnology Information, Bethesda, MD) utilizing the PeptideSearch algorithm (Matthias Mann, Southern Denmark University, Odense, Denmark; an updated version of this program is currently available as "PepSea" from MDS-Denmark). A molecular mass range double the apparent molecular weight (as estimated from gel electrophoretic relative mobility) was covered, with a mass accuracy restriction better than 40 ppm, and a maximum of one missed cleavage site allowed per peptide. Iterative cycles of database searching and removal of all m/z values that matched a chosen protein sequence were carried out.
To confirm some of the peptide mass fingerprinting (PMF) results, limited MS sequencing of selected peptides was done by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples, using the UltraFlex instrument in "LIFT" mode. Fragment ion spectra were then taken to search the NR database using the MASCOT MS/MS Ion Search program, version 2.0.04 for Windows (Matrix Science Ltd., London, United Kingdom) (45). Any tentative confirmation (Mascot score 30) of a PMF result thus obtained was further verified by comparing the computer-generated fragment ion series of the predicted tryptic peptide with the experimental MS/MS data. No identifications in this study were based on MS/MS data alone.
Immunoprecipitation and Western blotting
Ten micrograms of purified specific Ab was added to 500 µl (0.31.2 mg of protein) of cell lysate and incubated at 4 °C for 3 h with slow rotation on a Dynal sample mixer. Dynabeads® Protein G beads (50 µl) were washed four times with PBST using a Dynal MPC-S magnet. The beads were then added to the cell lysate-Ab mixture and incubated at 4 °C for 1 h with slow rotation. After completion of the incubation, the beads were washed again as described above, suspended in 50 µl of 1x Laemmli sample buffer, and boiled for 5 min. The beads were pulled down with the magnet, and supernatant was used for analysis. The SDS-gel electrophoresis and Western blotting were performed as described elsewhere (46). The concentration of PY-KD1 or protein-specific Abs used in Western blotting was 1 µg/ml. The concentration of secondary Abs conjugated to horseradish peroxidase was 200 ng/ml. The detection of immune complexes was performed with the ECLTM reagent kit (Amersham Biosciences) according to instructions provided by the manufacturer. The films were scanned and respective bands quantified using a Molecular Dynamics Personal Densitometer SI and ImageQuant software (Amersham Pharmacia Biotech).
Microarray Fabrication
Abs for microarray fabrication were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and printed on HydroGelTM coated slides (Perkin Elmer Life Sciences, Boston, MA) using MicroSpot 2500 pins and a BioRobotics MicroGrid II arrayer (Genomic Solutions, Ann Arbor, MI). Printing ink was PBS containing 0.2% gelatin and 0.1% sodium azide. Printing concentration of each Ab was 200 µg/ml. Each Ab was spotted onto the array at least nine times. The spacing between spots was 300 µm. Quality control of Ab deposition was performed using a Cy5-labeled nonspecific Ab. Quality control of retention of Abs was performed using deposition of mouse IgG that was detected at the completion of experiments with Cy5-labeled goat anti-mouse Ab. After completion of a printing cycle, arrays were incubated in the dark at room temperature and 65% relative humidity for at least 48 h. They were washed three times with PBST for 30 min on an orbital shaker. Finally, they were dipped in PBS, centrifuged at 100 x g for 5 min, and left at 37 °C for a few minutes to allow to dry completely. Arrays were stored in a noncondensing atmosphere at 4 °C.
Before an experiment, arrays were allowed to reach room temperature and blocked with a PBST solution containing 10 mg/ml BSA for at least an hour with gentle agitation. Arrays were then dipped in PBS, centrifuged at 100 x g for 5 min, and placed at 37 °C for a few minutes to allow drying.
Detection of Fluorescently Labeled Proteins Using Microarrays
Each microarray was incubated with 100 µl of Cy5-labeled protein (1100 µg/ml) extract for 1 h at 37 °C. Protein extract was supplemented with 1 mg/ml BSA. Upon completion of incubation, arrays were washed four times with PBST for 15 min at room temperature on an orbital shaker. They were dipped in PBS, centrifuged at 100 x g for 5 min, and left at 37 °C for a few minutes to allow drying. Arrays were scanned using a GeneArray microarray scanner (Affymetrix, Santa Clara, CA).
Detection of p-Tyr Proteins Using Microarrays
Each microarray was incubated with 100 µl of unlabeled protein extract (0.001100 µg/ml) for 1 h at 37 °C. Protein extract was always supplemented with 1 mg/ml BSA. Upon completion of incubation, arrays were washed with PBST four times for 15 min at room temperature on an orbital shaker. Cy5-PY-KD1 mAb was diluted in an Ab dilution buffer (PBST with 1 mg/ml BSA) to a final concentration 5 µg/ml, and 100 µl of this solution was incubated with each array for 1 h at 37 °C. Arrays were washed with PBST four times for 15 min at room temperature on an orbital shaker. They were dipped in PBS, centrifuged at 100 x g for 5 min, and left at 37 °C for a few minutes to allow them to dry. Arrays were scanned using a GeneArray microarray scanner.
Microarray Data Analysis
The location of each Ab feature on the array was determined by creating a Gal file with the clone tracking option of MicroGrid II software (Genomic Solutions) and importing it to GenePix Pro 4.0 software (Axon Instruments, Union City, CA). The fluorescence signal from each spot was determined as the average of the pixel intensities within the boundary outlined by the software. The local background was subtracted from the signal at each spot. Corrected signal to local background (signal-to-noise) ratio was then calculated for each spot (arbitrary signal). Spots with a net fluorescence below 150 or with obvious defects were eliminated from analysis. Fold reduction of the arbitrary signal by Gleevec, R(r), was calculated as the ratio of arbitrary signals obtained from extracts of untreated and Gleevec-treated RT10+ cells. Fold induction of the arbitrary signal by EGF, R(i), was assessed as the ratio of arbitrary signals from extracts of EGF-treated and serum-starved, untreated HeLa cells.
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RESULTS |
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Selection and Characterization of Abs for Microarray Fabrication
Commercially available Abs were obtained for 18 of the 32 potential targets (Table II; 118; group "A"); eight of which have been identified in two independent analyses (no. 1, 5, 7, 8, 1013), eight others in this report (no. 24, 6, 9, 1416) and two more by Salomon et al. (no. 17, 18). We realized that some of these proteins, GRB2 for example, were actually not "direct" targets of the Bcr-Abl kinase activity but rather interacted with p-Tyr proteins. Nevertheless, they will likely produce an "indirect" signal on the arrays (see Fig. 2; the captured protein binds a p-Tyr protein, which is in turn recognized by the labeled PY-KD1 mAb) and therefore be useful for comparative analysis, even if the bound phospho-protein(s) is (are) unknown. We then also selected 10 Abs for cognate proteins (no. 1928; group "B") also previously shown to be tyrosine-phosphorylated, albeit not necessarily in human myeloid leukemia cells. Finally, we included seven Abs, recognizing proteins (no. 2935; group "C") that, to the best of our knowledge, have never been shown to contain p-Tyr. All 35 Abs were first characterized in a series of immunocapturing (with PY-KD1 beads) and immunoprecipitation experiments, followed by Western blot analyses using either PY-KD1 mAb or each of the 35 "self" Abs. The results are summarized in Table II.
Twenty-three of the 35 cognate proteins could be captured from RT10+ cell extracts using immobilized PY-KD1 mAb, as indicated by a positive signal in subsequent Western blots with each specific "self" Ab; 14/18 in group A, 7/10 in group B, and 2/7 in group C. It is unlikely that the four group-A proteins (Gads, Shc, Dock180, and Cas-L) not detected in this analysis had been misidentified as Shc was independently identified twice and Gads yielded PMF sequence coverage of 30% (Table II). Rather, the Abs may not have worked in the Western blot. Of the 14 group-A proteins that were confirmed in the bead pull-downs, 10 (c-Abl, p67phox, PI3Kp85, SHIP-1 and 2, SH-PTP2, DOK-1 and 2, Syk, and Gab1) were found to be bona fide p-Tyr proteins, as species of the correct molecular mass were detected in Western blots with PY-KD1 after immunoprecipitation (IP) with "self" Abs. By contrast, three cognate proteins (GRB2, Cbl, RasGap) in the bead pull-downs were detected after similar IPs when probed with "self" Abs but not with PY-KD1, indicating an apparent absence of p-Tyr. SLP-76 could not be immunoprecipitated; i.e. there was no signal in Western blots using "self" Abs.
Of the seven group-B proteins detected in the bead pull-downs, three (PLC-1, Crk-L, and Raf-1) were likely Tyr-phosphorylated; the four others (ZAP-70, IRS-1, ERK-1, and NF
Bp65) were not immunoprecipitated with "self" Abs. Interestingly, Lck, which was not detected in the earlier pull-down with PY-KD1, was also found to be Tyr-phosphorylated after IP with an anti-Lck Ab. Lck is a well-characterized p-Tyr protein in T cells (49), and failure to recover it in our bead pull-down could be due to an inaccessible phosphorylation site. As for members of the "negative control" group (C), presence of both cyclin A and MEK-1 was confirmed.
Evaluation of the Tyr Phosphorylation Ab Microarray Platform
Without the availability of proper calibrants, the method is not really quantitative, yet changes in Tyr phosphorylation state, under carefully controlled conditions, can conceivably be measured. We wanted to test this concept in both a positive and negative model; i.e. up- or down-regulation of global Tyr phosphorylation. As the kinase activity of the oncogenic Bcr-Abl fusion product can be inhibited by the anti-cancer drug STI-571 (Gleevec) (8, 9), RT10+ cells provide a convenient model system to analyze down-regulation. On the other hand, activation of the EGFR, a tyrosine kinase, in serum-starved HeLa cells has often been used to analyze induction of a subset of the tyrosine phosphoproteome (14, 15). Both types of cells were therefore grown in culture and separate aliquots treated with either EGF or Gleevec for 1 or 2 h, respectively. Lysates of both treated and untreated cells were then analyzed by gel electrophoresis and immunoblotting with the PY-KD1 mAb. Optimization experiments suggested that about 10- to 100-fold more protein from the EGF-treated HeLa cells was required to get comparable signals as for RT10+ cells. As expected, all major bands were substantially reduced in protein extract from Gleevec-treated RT10+ cells and enhanced in EGF-treated HeLa cells as compared with the respective untreated controls (Fig. 5). Similar experiments were then carried out using our 35-feature Ab microarrays (Table II) and the anti-p-Tyr mAb for sandwich-type detection.
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DISCUSSION |
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Differences in reactivity of the PY-KD1 mAb and its commonly used commercial counterparts (PY-100, 4G10, and PY-20) were observed in ELISAs, and indicated that PY-KD1 is one of the few if not the only available anti-pTyr mAb right now with negligible affinities for p-Ser, p-Thr, and nonphosphorylated Tyr. In assays typically requiring high Ab titers, as for example in immunohistochemistry or protein arrays, such cross-reactivity of the commercial anti-p-Tyr mAbs may become problematic, all the more so considering that Ser and Thr phosporylation sites and nonphosphorylated Tyr residues in proteins are far more abundant than p-Tyr. This situation may also apply to affinity immunocapture procedures whereby the highest possible quantity of Ab has been immobilized to yield high-ligand density, high-capacity particles, as was the case in a component of this study.
Pilot Ab array experiments suggested the presence of several cognate proteins in RT10+ cells with a different degree of Tyr phosphorylation. Interestingly, the micro-sandwich assay with Cy5-PY-KD1 (for p-Tyr) was 1,000 times more sensitive than the assay with direct Cy5-labeling (for protein) of the extract. Similar limitations in sensitivity of direct fluorescent labeling of proteins have previously been reported (27, 30, 40, 50). To ensure adequate sensitivity of Tyr phosphorylation microarray analyses, we used an excess of detection Ab. At 5 µg/ml Cy5-PY-KD1, detection could be readily achieved using only 10 ng/ml total protein from RT10+ cells. Consistent with Western blotting results (Fig. 5), a substantially higher total protein concentration, 1 µg/ml, was required to generate detectable signals on a p-Tyr chip when using extracts from EGF-treated HeLa cells. Of course, the sensitivity of the p-Tyr microarray platform is entirely dependent on the overall abundance of Tyr-phosphorylated proteins in the sample and therefore largely cell-type specific. Even so, the sensitivities we have observed for p-Tyr detection with our system so far are remarkable by any measure of post-translational modification analysis, and even for protein analysis in general.
Using array incubation volumes of 100 µl, total protein amounts consumed per chip range from 1 to 100 ng depending on cell type. In the case of RT10+ and HeLa cells, lysates of 1 x 107 cells yield typically 12 mg of total protein, in a 1-ml volume, that is then serially diluted 1,000-fold (HeLa extract) or 100,000-fold (RT10+ extract) to a final concentration of 1 µg/ml or 10 ng/ml, respectively. This is the equivalent of a 1-ml extract made from either 10,000 (HeLa) or as little as 100 RT10+ cells; each sufficient for 10 microarray analyses. Conceivably, an even smaller number of cells could be lysed in sub-milliliter volumes to yield identical protein concentrations, but these experiments have yet to be done. It should be noted that low-volume processing of very small numbers of cells could pose specific experimental problems not encountered during larger-scale sample preparation followed by serial dilution. But even when future developments would not match the most optimistic projections, the required number of cells would still be in marked contrast to those in other types of tyrosine phosphoproteome profiling, such as affinity capture in combination with mass spectrometric identification which, in fact, was also used as part of this study. In those studies, both in our laboratory and elsewhere, typically 1 x 108 to 5 x 109 cells have been used (14, 15, 21). Compared with MS-based methods, the Ab array method falls significantly short at this time in terms of protein coverage and mapping of the p-Tyr sites; i.e. only dozens of cognate proteins are being profiled instead of the entire cell population, without any information on possible modification sites. With this in mind, we have tried to at least maximize our chances for getting broader p-Tyr protein coverage by carefully selecting the Abs for printing. Besides sensitivity, the benefits of our Ab array approach are a capacity for i) improved sample throughput, ii) virtually unlimited antigen multiplexing by increasing the number of Ab features deposited on the chip, and iii) multiplexing of the sandwich-based detection by using two or more differentially labeled affinity probes.
Whereas a quantitative Tyr phosphorylation microarray platform would be a welcome addition to the bio-analytical toolbox, such process requires calibration against standards, for example recombinant or purified p-Tyr proteins, as in a conventional ELISA. Unfortunately, those specific standards are not readily available. Therefore, at this time, the p-Tyr microarray platform described herein is only applicable to a comparative analysis of two or more samples rather than exact quantitation of phosphorylation of individual proteins. We also cannot account for the possible presence of multiple, often independently changing p-Tyr sites on individual proteins. In addition, the arbitrary signal for any particular protein is strongly dependent on the properties of the capturing Ab, as well as on variously induced changes (e.g. by phosphorylation itself) in three-dimensional structure that could affect binding to the Ab, making it difficult to compare phosphorylation states of the different cognate proteins captured by the various Abs on the same chip. Instead, this platform is designed to specifically cross-compare each individual member of a set of proteins between two or more samples, whereby corresponding proteins are captured from the different lysates on separate chips but using the exact same Abs under the same conditions.
Evaluation of a 35-feature, p-Tyr microarray platform demonstrated its utility in monitoring both up- and down-regulation of the Tyr phosphoproteome. A ratiometric approach was used to describe changes in Tyr phosphorylation of two protein populations by calculating ratios of arbitrary signals from control and treated samples or vice versa (R(r) for negative regulation or R(i) for positive regulation). Microarray analyses of RT10+ cells treated with Gleevec and HeLa cells treated with EGF indicated that R(r) or R(i) values of 1.8 or more suggest a good probability of changes in Tyr phosphorylation. By contrast, R(r) or R(i) values of 1.5 or less denote the likely absence of such differences. Some proteins had R(r) or R(i) values between 1.5 and 1.8, making it difficult to deduce if their p-Tyr levels had changed, quite likely due to limitations in sensitivity. It is important to understand that, depending on the experimental system and objectives, each study will require its own threshold (R(r) or R(i) value) to qualify as a valid indicator of changes in protein phosphorylation. In certain situations it may be reasonable to apply a more stringent approach by choosing R(r) or R(i) values of 2.0 or more as a "difference threshold."
Tyr phosphorylation array data generated for Bcr-Abl-expressing cells treated with a specific kinase inhibitor was in good agreement with the extensive body of knowledge that exists for this experimental system. Most proteins that had reduced levels (R(r) > 1.8) of phosphorylation after exposure to Gleevec had previously been reported as direct substrates of the Bcr-Abl tyrosine kinase, including PI3K, SHIP-1, SHIP-2, PLC-1, Dok-1, Dok-2, Cbl, Crk-L, Shc, RasGap, and Irs-1 (7, 9, 51), or to be down-regulated by Gleevec in cells transfected with a p210Bcr/Abl-expressing plasmid, including c-Abl, SHIP-2, SH-PTP2, Gab-1, Dok-1, Irs-1, and Erk-1 (19, 51, 52).
In conventional affinity methods for p-Tyr protein enrichment, such as immunocapture or -precipitation, it is generally accepted that not all detected proteins are phosphorylated, as unmodified proteins can bind to and co-precipitate with the phosphoproteins (18). The p-Tyr detection procedure described herein is based on immunoaffinity capture of 35 cognate proteins on a microarray. The platform is therefore also subject to detection of some proteins that are in complex with phosphorylated partners (Fig. 2). GRB2, Gads, and DOCK180, and perhaps also RasGap and Cbl, all identified by MS in this study, are likely examples of such interactions in RT10+ cells. GRB2 and Gads are SH2/SH3 domain-containing adapter molecules interacting with Tyr-phosphorylated motifs of various other proteins (53, 54). DOCK180 has been shown to be part of FAK-Src-p130Cas-DOCK180 signaling complex where all interacting partners but DOCK180 itself are Tyr-phosphorylated (55). In keeping with the MS results, the aforementioned proteins all had R(r) > 1.8 in our array experiments, which may, therefore, equally reflect changes in Tyr phosphorylation of interacting proteins. Cyclin A is also not known to be Tyr-phosphorylated, and was not detected by MS in this study, yet displayed R(r) > 1.8 in microarray experiments. This can be explained by the existence of a complex between cyclin A and Cdk2, which, itself, is Tyr-phosphorylated (56). Finally, ZAP-70, Fyn, Syk, and p67-phox are Tyr-phosphorylated in various cell types, but the apparent reduction of Tyr phosphorylation by Gleevec in RT10+ cells has not been previously reported and may therefore represent a novel finding.
Several proteins that had elevated levels of Tyr phosphorylation (R(r) > 1.8) in HeLa cells after EGF treatment are known to be involved or associated with EGFR signaling, including c-Abl, PI3K, Cbl, Shc, Raf-1, and NFB (57). Participation of SHIP proteins in signal transduction cascades downstream of EGFR is also possible (57). It is further known that a Dok-related protein (Dok-R) that can be phosphorylated at two tyrosines and associated with activation of EGFR shares a high degree of homology with p62dok (Dok-1) (58). Therefore, Dok-R may have been captured on the chip by the anti-Dok-1 Ab and detected as differentially phosphorylated. Furthermore, MEKs can form a complex with Raf-1 and c-Jun with Jnk in response to EGF stimulation (5961). Both Raf-1 and Jnk are Tyr-phosphorylated and may therefore have contributed to the increase in arbitrary signal of MEK-1/2 and c-Jun. The results for cyclin A and Gads in HeLa cells can be similarly interpreted as for the RT10+ cells. As for the three remaining, known p-Tyr proteins (p67-phox, ZAP-70, and SLP-76), they have not been previously reported as putative targets of the EGF/EGFR pathway.
Reviewing the two applications discussed above, it might appear peculiar at first that the majority of the cognate proteins of the 35-feature Ab array were co-regulated for either increase (HeLa plus EGF) or decrease (RT10+ plus Gleevec) of their p-Tyr levels. As pointed out throughout the results section, most of the proteins were bona fide p-Tyr-derivatized or p-Tyr protein interactors in Bcr-Abl-expressing cells. They could therefore have been expected to respond similarly (i.e. become less Tyr-phosphorylated) to an inhibitor of a prominent tyrosine kinase, or respond similarly (i.e. become more Tyr-phosphorylated) to an activator in a different cell type, or to not respond at all, but rather unlikely to respond diametrically under any single set of conditions. On the other hand, changes in Tyr phosphorylation of individual proteins (as measured by arbitrary signal changes on the chip using labeled anti-pTyr mAb) differed significantly between cell types and treatments, also as could have been expected.
In conclusion, we have designed and successfully tested a sandwich Ab microarray platform utilizing a fluorescently labeled p-Tyr-specific detection mAb and describe a generalized, ratiometric approach to analyze data generated by this assay. Our approach offers an advantage of extremely low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with micro-fluidic devices and automation. Future work will focus on development of potential applications of this platform to define signal transduction pathways, for molecular classification of tumors, compound profiling and toxicology studies, and analysis of patients individual sensitivities to tyrosine kinase inhibitors, such as the efficacy of Gleevec (8) or Gefitinib (11) targeted cancer therapy, among many.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The 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.
* Supported by Developmental Funds from National Cancer Institute Grant P30 CA08748. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Published, MCP Papers in Press, September 8, 2004, DOI 10.1074/mcp.M400075-MCP200
To whom correspondence should be addressed: Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Tel.: 212-639-8923; E-mail: p-tempst{at}mskcc.org
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