Ischemia in Tumors Induces Early and Sustained Phosphorylation Changes in Stress Kinase Pathways but Does Not Affect Global Protein Levels

Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis. Protein abundance and phosphorylation convey important information about pathway activity and molecular pathophysiology in diseases including cancer, providing biological insight, informing drug and diagnostic development, and guiding therapeutic intervention. Analyzed tissues are usually collected without tight regulation or documentation of ischemic time. To evaluate the impact of ischemia, we collected human ovarian tumor and breast cancer xenograft tissue without vascular interruption and performed quantitative proteomics and phosphoproteomics after defined ischemic intervals. Although the global expressed proteome and most of the >25,000 quantified phosphosites were unchanged after 60 min, rapid phosphorylation changes were observed in up to 24% of the phosphoproteome, representing activation of critical cancer pathways related to stress response, transcriptional regulation, and cell death. Both pan-tumor and tissue-specific changes were observed. The demonstrated impact of pre-analytical tissue ischemia on tumor biology mandates caution in interpreting stress-pathway activation in such samples and motivates reexamination of collection protocols for phosphoprotein analysis. Genomic analysis of thousands of tumor samples from multiple cancer types is currently being carried out by consortia such as the Cancer Genome Atlas (TCGA) 1The abbreviations used are: used are: TCGA, the Cancer Genome Atlas; iTRAQ, isobaric tags for relative and absolute quantification; PDX, patient-derived xenograft; RPPA, reverse-phase protein array; pTyr, phospho-tyrosine; GO BP, Gene Ontology Biological Process; EGFR, epidermal growth factor receptor. 1The abbreviations used are: used are: TCGA, the Cancer Genome Atlas; iTRAQ, isobaric tags for relative and absolute quantification; PDX, patient-derived xenograft; RPPA, reverse-phase protein array; pTyr, phospho-tyrosine; GO BP, Gene Ontology Biological Process; EGFR, epidermal growth factor receptor. and the International Cancer Genome Consortium. 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Borresen-Dale A.L. Mills G.B. A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers.Clin. Proteomics. 2010; 6: 129-151Crossref PubMed Scopus (177) Google Scholar) has been incorporated into the analysis pipeline for TCGA samples. RPPA is a highly parallelized, chip-based dot-blot approach employing very well-characterized anti-protein and anti-phosphopeptide antibodies. The power of proteomics in the context of TCGA has recently been highlighted by the ability of RPPA to identify new molecular taxonomies in breast cancer subtypes not discernible via genomics (1.Cancer Genome Atlas Network Comprehensive molecular portraits of human breast tumours.Nature. 2012; 490: 61-70Crossref PubMed Scopus (8301) Google Scholar). A limitation of RPPA is that only around 200 antibodies are currently demonstrated to work well in this methodology. We hypothesized that concurrent and complementary evaluation of the functional proteomes (including posttranslational modifications) of tumors will improve our ability to diagnose, treat, and prevent cancers by enabling a better understanding of the molecular basis of these diseases, especially when integrated and analyzed together with the comprehensive genomic characterization information. Recent advances in the preparation of samples and their quantitative analysis by means of mass-spectrometry-based proteomics and phosphoproteomics now yield datasets covering nearly half of the predicted proteome and identifying many thousands of phosphopeptides (10.Wisniewski J.R. Dus K. Mann M. Proteomic workflow for analysis of archival formalin-fixed and paraffin-embedded clinical samples to a depth of 10 000 proteins.Proteomics Clin. Appl. 2013; 7: 225-233Crossref PubMed Scopus (119) Google Scholar, 11.Mertins P. Udeshi N.D. Clauser K.R. Mani D.R. Patel J. Ong S.E. Jaffe J.D. 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Methods. 2013; 10: 634-637Crossref PubMed Scopus (432) Google Scholar). These advances have sparked efforts utilizing state-of-the-art proteomics to characterize the proteomes and define changes in the posttranslational modification landscape of tumor samples. The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium is therefore characterizing the proteomes of large numbers of breast, colon, and ovarian tumor samples that have been genomically characterized as part of the TCGA program. A concern faced by the Clinical Proteomic Tumor Analysis Consortium program is that the samples provided by TCGA for proteomic analysis were not obtained with proteomic studies in mind. Importantly, the total duration of ischemia prior to sample freezing is generally unknown in TCGA samples but frequently spans tens of minutes to over an hour. The total ischemia duration comprises a period of warm ischemia corresponding to the time from blood vessel ligation to surgical excision and then cold ischemia corresponding to the delay time to freezing post-excision (usually a mix of transportation time to pathology and pathological analyses). Until now, the effect of cold ischemia on the stability of proteins and phosphosites has primarily been studied only for selected candidate proteins and phosphosites for which high-quality antibodies were available. A study employing AQUA, a quantitative immunofluorescence technique, found slightly increased expression of hypoxia inducible factor but no changes in protein abundance in the four breast cancer biomarker proteins ER, PR, HER2, and Ki67 over a time span of up to 7 h, though signal reduction occurred in a subset of samples at longer intervals of up to 48 h of cold ischemia (15.Neumeister V.M. Anagnostou V. Siddiqui S. England A.M. Zarrella E.R. Vassilakopoulou M. Parisi F. Kluger Y. Hicks D.G. Rimm D.L. Quantitative assessment of effect of preanalytic cold ischemic time on protein expression in breast cancer tissues.J. Natl. Cancer Inst. 2012; 104: 1815-1824Crossref PubMed Scopus (91) Google Scholar). In another study employing immunohistochemistry, the breast cancer biomarker proteins ER, PR, and Her2 were found to be stable for up to 2 h at room temperature (16.Yildiz-Aktas I.Z. Dabbs D.J. Bhargava R. The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma.Mod. Pathol. 2012; 25: 1098-1105Crossref PubMed Scopus (106) Google Scholar). Evidence from RPPA studies evaluating the time course of changes induced by cold ischemia has shown that even when the total protein levels and levels of many phosphoproteins are constant, the phosphorylation stoichiometry of specific proteins can change, in some cases significantly (9.Hennessy B.T. Lu Y. Gonzalez-Angulo A.M. Carey M.S. Myhre S. Ju Z. Davies M.A. Liu W. Coombes K. Meric-Bernstam F. Bedrosian I. McGahren M. Agarwal R. Zhang F. Overgaard J. Alsner J. Neve R.M. Kuo W.L. Gray J.W. Borresen-Dale A.L. Mills G.B. A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers.Clin. Proteomics. 2010; 6: 129-151Crossref PubMed Scopus (177) Google Scholar, 17.Espina V. Edmiston K.H. Heiby M. Pierobon M. Sciro M. Merritt B. Banks S. Deng J. VanMeter A.J. Geho D.H. Pastore L. Sennesh J. Petricoin 3rd, E.F. Liotta L.A. A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.Mol. Cell. Proteomics. 2008; 7: 1998-2018Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). For example, Espina et al. used 55 phosphosite-specific antibodies in RPPA format to show that in various tissues, including breast, ovary, colon, and endometrium, cold ischemia can cause up to a 2-fold variation in phosphorylation abundance and that these processes can be augmented by phosphatase inhibitors and reduced by kinase inhibitors (17.Espina V. Edmiston K.H. Heiby M. Pierobon M. Sciro M. Merritt B. Banks S. Deng J. VanMeter A.J. Geho D.H. Pastore L. Sennesh J. Petricoin 3rd, E.F. Liotta L.A. A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.Mol. Cell. Proteomics. 2008; 7: 1998-2018Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) or heat stabilization (18.Ahmed M.M. Gardiner K.J. Preserving protein profiles in tissue samples: differing outcomes with and without heat stabilization.J. Neurosci. Methods. 2011; 196: 99-106Crossref PubMed Scopus (32) Google Scholar). However, because of clinical sample handling limitations, the earliest time points evaluated in this study occurred between 4 and 40 min after tissue resection with an average time to cryopreservation of 19.3 min (17.Espina V. Edmiston K.H. Heiby M. Pierobon M. Sciro M. Merritt B. Banks S. Deng J. VanMeter A.J. Geho D.H. Pastore L. Sennesh J. Petricoin 3rd, E.F. Liotta L.A. A portrait of tissue phosphoprotein stability in the clinical tissue procurement process.Mol. Cell. Proteomics. 2008; 7: 1998-2018Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The duration of blood vessel ligation, a common procedure in nearly all surgeries, was not noted, and so the actual total ischemia times (warm ischemia plus cold ischemia) might have been longer. Hennessy et al. analyzed the effects of cold ischemia in breast cancer tumor samples using RPPA and found that 21 of 82 proteins and phosphoproteins demonstrated time-dependent instability at room temperature between 6 and 24 h of cold ischemia but exhibited few effects at earlier time points (9.Hennessy B.T. Lu Y. Gonzalez-Angulo A.M. Carey M.S. Myhre S. Ju Z. Davies M.A. Liu W. Coombes K. Meric-Bernstam F. Bedrosian I. McGahren M. Agarwal R. Zhang F. Overgaard J. Alsner J. Neve R.M. Kuo W.L. Gray J.W. Borresen-Dale A.L. Mills G.B. A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers.Clin. Proteomics. 2010; 6: 129-151Crossref PubMed Scopus (177) Google Scholar). Gundish et al. used both RPPA and LC-MS/MS to analyze a time course of cold ischemia in normal liver tissue from mouse and rat (34.Gündisch S. Grundner-Culemann K. Wolff C. Schott C. Reischauer B. Machatti M. Groelz D. Schaab C. Tebbe A. Becker K.F. Delayed times to tissue fixation result in unpredictable global phosphoproteome changes.J. Proteome Res. 2013; 12: 4424-4434Crossref PubMed Scopus (25) Google Scholar). No significant changes were observed in the phosphoproteome after up to 60 min of cold ischemia, in agreement with the findings of Hennessy et al. In the LC-MS/MS study by Gundisch et al., approximately 1700 phosphosites were quantified, but no statistically significant alterations of individual phosphosites after over 60 min of cold ischemia were found. A few other unbiased mass-spectrometry-based proteomic studies of cold ischemia have been reported. These studies identified a small number of proteins that changed in abundance (primarily as a result of degradation, presumably via proteolysis) after long (> 3 h) periods of cold ischemia (19.Gündisch S. Hauck S. Sarioglu H. Schott C. Viertler C. Kap M. Schuster T. Reischauer B. Rosenberg R. Verhoef C. Mischinger H.J. Riegman P. Zatloukal K. Becker K.F. Variability of protein and phosphoprotein levels in clinical tissue specimens during the preanalytical phase.J. Proteome Res. 2012; 11: 5748-5762Crossref PubMed Scopus (46) Google Scholar, 20.Li J. Kil C. Considine K. Smarkucki B. Stankewich M.C. Balgley B. Vortmeyer A.O. Intrinsic indicators for specimen degradation.Lab. Invest. 2013; 93: 242-253Crossref PubMed Scopus (7) Google Scholar). For example, Li et al., using two-dimensional fluorescence difference gel electrophoresis in conjunction with mass spectrometry, identified 26 proteins that changed over 48 h of cold ischemia time, chiefly as a result of degradation (20.Li J. Kil C. Considine K. Smarkucki B. Stankewich M.C. Balgley B. Vortmeyer A.O. Intrinsic indicators for specimen degradation.Lab. Invest. 2013; 93: 242-253Crossref PubMed Scopus (7) Google Scholar). The sample collection and analysis approaches used in most of these studies did not permit assessment of changes within the one-hour interval investigated in our study. Before embarking on analyses of posttranslational modifications in TCGA-profiled tumor samples, we wanted to better understand the potential effects of ischemia on the proteome and phosphoproteome of tumor tissue. Here we describe our time-course studies of patient-derived xenografts of human breast cancer tumors and patient-derived ovarian cancer tumors. Tumor tissues were excised prior to vascular ligation in order to accurately define the ischemia time, and they were prepared centrally using a cryopulverization method that maintained the tissue in a frozen state. Samples were analyzed using a common sample-processing workflow for global, quantitative proteome and phosphoproteome analysis using iTRAQ chemical mass tag labeling for quantification (11.Mertins P. Udeshi N.D. Clauser K.R. Mani D.R. Patel J. Ong S.E. Jaffe J.D. Carr S.A. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics.Mol. Cell. Proteomics. 2012; 11Abstract Full Text Full Text PDF Scopus (138) Google Scholar, 21.Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar) and state-of-the-art high-performance, multidimensional LC-MS/MS on high-performance mass spectrometers for data collection. After obtaining consent to Institutional Review Board–approved protocols, we collected tissue from high-grade serous ovarian carcinoma tumors from four patients. Immediately after resection, the tumors were dissected into four contiguous and adjacent strips, each no larger than 10 mm × 3 mm × 3 mm. Tumor strips were placed into cryovials and frozen in liquid nitrogen at specified time points. All human tissues for these experiments were processed in compliance with NIH regulations and institutional guidelines approved by the Institutional Review Board at Washington University. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis, MO. Patient-derived xenograft (PDX) tumors from established basal (WHIM6) and luminal (WHIM20) breast cancer subtypes were raised subcutaneously in 8-week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (Jackson Labs, Bar Harbor, ME) as previously described (22.Li S. Shen D. Shao J. Crowder R. Liu W. Prat A. He X. Liu S. Hoog J. Lu C. Ding L. Griffith O.L. Miller C. Larson D. Fulton R.S. Harrison M. Mooney T. McMichael J.F. Luo J. Tao Y. Goncalves R. Schlosberg C. Hiken J.F. Saied L. Sanchez C. Giuntoli T. Bumb C. Cooper C. Kitchens R.T. Lin A. Phommaly C. Davies S.R. Zhang J. Kavuri M.S. McEachern D. Dong Y.Y. Ma C. Pluard T. Naughton M. Bose R. Suresh R. McDowell R. Michel L. Aft R. Gillanders W. DeSchryver K. Wilson R.K. Wang S. Mills G.B. Gonzalez-Angulo A. Edwards J.R. Maher C. Perou C.M. Mardis E.R. Ellis M.J. Endocrine therapy resistant ESR1 variants revealed by genomic characterization of breast cancer derived xenografts.Cell Reports. 2013; 4: 1116-1130Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, 23.Ding L. Ellis M.J. Li S. Larson D.E. Chen K. Wallis J.W. Harris C.C. McLellan M.D. Fulton R.S. Fulton L.L. Abbott R.M. Hoog J. Dooling D.J. Koboldt D.C. Schmidt H. Kalicki J. Zhang Q. Chen L. Lin L. Wendl M.C. McMichael J.F. Magrini V.J. Cook L. McGrath S.D. Vickery T.L. Appelbaum E. Deschryver K. Davies S. Guintoli T. Lin L. Crowder R. Tao Y. Snider J.E. Smith S.M. Dukes A.F. Sanderson G.E. Pohl C.S. Delehaunty K.D. Fronick C.C. Pape K.A. Reed J.S. Robinson J.S. Hodges J.S. Schierding W. Dees N.D. Shen D. Locke D.P. Wiechert M.E. Eldred J.M. Peck J.B. Oberkfell B.J. Lolofie J.T. Du F. Hawkins A.E. O'Laughlin M.D. Bernard K.E. Cunningham M. Elliott G. Mason M.D. Thompson Jr., D.M. Ivanovich J.L. Goodfellow P.J. Perou C.M. Weinstock G.M. Aft R. Watson M. Ley T.J. Wilson R.K. Mardis E.R. Genome remodelling in a basal-like breast cancer metastasis and xenograft.Nature. 2010; 464: 999-1005Crossref PubMed Scopus (989) Google Scholar). Tumors from each animal were harvested by surgical excision at ∼1.5 cm3, rapidly divided into four pieces, and snap-frozen by immersion in a liquid nitrogen bath at times 0 (∼30 s), 5, 30, and 60 min post-excision. Proteins extracted from tumor tissues (∼50-mg tissue weight for ovarian and ∼100-mg tissue weight for breast cancer xenograft) were reduced, alkylated, and subjected to enzyme digestion. Peptides were labeled with 4-plex iTRAQ reagents and separated using an off-line high pH (7.5 or 10) reversed-phase column. Fractions were collected and concatenated (14.Mertins P. Qiao J.W. Patel J. Udeshi N.D. Clauser K.R. Mani D.R. Burgess M.W. Gillette M.A. Jaffe J.D. Carr S.A. Integrated proteomic analysis of post-translational modifications by serial enrichment.Nat. Methods. 2013; 10: 634-637Crossref PubMed Scopus (432) Google Scholar, 24.Wang Y. Yang F. Gritsenko M.A. Wang Y. Clauss T. Liu T. Shen Y. Monroe M.E. Lopez-Ferrer D. Reno T. Moore R.J. Klemke R.L. Camp 2nd, D.G. Smith R.D. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells.Proteomics. 2011; 11: 2019-2026Crossref PubMed Scopus (397) Google Scholar) into 24 fractions, and 5% of each fraction was analyzed via LC-MS/MS for quantitative global proteomics measurement. The remainder (95%) of each of the 24 fractions was further concatenated into 12 fractions, and phosphopeptides were enriched using immobilized metal (Fe3+) affinity chromatography prior to LC-MS/MS analysis. Peptides from ovarian cancer samples were analyzed using a nanoLC system coupled to an LTQ-Orbitrap-Velos MS (Thermo Scientific) at Pacific Northwest National Laboratories. Peptides from breast cancer PDX samples were analyzed using a nanoLC system coupled to a Q Exactive MS (Thermo Scientific) at the Broad Institute. All mass spectrometry data were analyzed using the Spectrum Mill software package, v4.1 beta (Agilent Technologies, Santa Clara, CA). Peptide identifications and quantification information were further combined into protein and phosphosite tables. Statistical analysis of cold-ischemia regulated proteins and phosphosites via kinetic modeling and moderated F-test was performed using the R software environment (version 2.15.2). Protein expression or phosphorylation was measured via RPPA as previously described (9.Hennessy B.T. Lu Y. Gonzalez-Angulo A.M. Carey M.S. Myhre S. Ju Z. Davies M.A. Liu W. Coombes K. Meric-Bernstam F. Bedrosian I. McGahren M. Agarwal R. Zhang F. Overgaard J. Alsner J. Neve R.M. Kuo W.L. Gray J.W. Borresen-Dale A.L. Mills G.B. A technical assessment of the utility of reverse phase protein arrays for the study of the functional proteome in non-microdissected human breast cancers.Clin. Proteomics. 2010; 6: 129-151Crossref PubMed Scopus (177) Google Scholar). More detailed experimental methods are presented in the supplemental "Materials and Methods" section. In this study we analyzed the effect of delayed freezing on protein and phosphoprotein stability in tumor samples derived from four ovarian cancer patients and two breast cancer PDXs (one basal and one luminal) (22.Li S. Shen D. Shao J. Crowder R. Liu W. Prat A. He X. Liu S. Hoog J. Lu C. Ding L. Griffith O.L. Miller C. Larson D. Fulton R.S. Harrison M. Mooney T. McMichael J.F. Luo J. Tao Y. Goncalves R. Schlosberg C. Hiken J.F. Saied L. Sanchez C. Giuntoli T. Bumb C. Cooper C. Kitchens R.T. Lin A. Phommaly C. Davies S.R. Zhang J. Kavuri M.S. McEachern D. Dong Y.Y. Ma C. Pluard T. Naughton M. Bose R. Suresh R. McDowell R. Michel L. Aft R. Gillanders W. DeSchryver K. Wilson R.K. Wang S. Mills G.B. Gonzalez-Angulo A. Edwards J.R. Maher C. Perou C.M. Mardis E.R. Ellis M.J. Endocrine therapy resistant ESR1 variants revealed by genomic characterization of breast cancer derived xenografts.Cell Reports. 2013; 4: 1116-1130Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). Blood vessels were not ligated prior to tumor excision, which eliminated effects of warm ischemia. Individual tumor samples were cut into four sections, which were subsequently stored at room temperature for predefined time intervals (Fig. 1A). Upon partitioning, the first section was frozen immediately with a time span of less than 1 min from excision to freezing in liquid nitrogen. Later time-point sections were left at room temperature and frozen after 5, 30, or 60 min in order to investigate the kinetics of changes in the proteome and phosphoproteome caused by delayed freezing. The ovarian tumors were obtained from four individual cancer patients with high-grade serous ovarian carcinoma, and the four time-point samples per patient were derived from four contiguous, adjacent portions of the same tumor. Although the use of surgically excised tumors was desirable because it recapitulated a typical biospecimen collection scenario, it was recognized that intratumor heterogeneity (e.g. genetic differences or differences in non-tumor elements) could result in proteomic variations not related to ischemic time. To address this concern, as well as to extend our analysis to another tumor type, we carried out the same ischemia time-course study in xenografts from two distinct molecular phenotypes of breast cancer, ER+ (luminal) and ER− (basal-like). Tumor xenografts have less cellular infiltration and therefore are less heterogeneous than tumors excised from patients. Additionally, because the total tumor material was not limiting for the breast cancer xenograft samples, we pooled 5 to 10 xenografts (derived from the same luminal and basal-like tumors) at each time point to dampen the effects of biological variability. We generated three individual pools for the basal and the luminal breast cancer xenograft samples which can be considered as biological replicates, as each individually grown xenograft tumor was present only in one pooled sample. For analysis of the proteome and phosphoproteome, frozen tumor samples were cryofractured, and proteins were extracted with an 8 m urea buffer and digested into peptides using trypsin (Fig. 1B). For relative quantification across the four ischemia time points of each tumor, peptide samples were chemically labeled with iTRAQ reagents and subsequently combined into a single sample. iTRAQ labeling covalently attaches isotope-coded isobaric mass tags to primary amine groups at the N termini of peptides and lysine side chains. Upon fragmentation in the mass spectrometer, reporter ions at defined masses are released, and the ratios of these reporter ions to one another enables relative quantification (21.Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3680) Google Scholar). After iTRAQ labeling, peptide samples were fractionated using basic reverse-phase liquid chromatography to reduce the overall sample complexity per fraction and thus increase the depth of coverage (24.Wang Y. Yang F. Gritsenko M.A. Wang Y. Clauss T. Liu T. Shen Y. Monroe M.E. Lopez-Ferrer D. Reno T. Moore R.J. Klemke R.L. Camp 2nd, D.G. Smith R.D. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells.Proteomics. 2011; 11: 2019-2026Crossref PubMed Scopus (397) Google Scholar). Basic reverse-phase liquid chromatography fractions were combined in a noncontiguous manner into 24 proteome and 12 phosphoproteome fractions, with 5% of the total material contributing to the proteome analysis and 95% to the phosphoproteome analysis. Phosphopeptides were further enriched using immobilized metal affinity chromatography. Our quantitative LC-MS/MS platform was extensively tested within the Clinical Proteomic Tumor Analysis Consortium program to ensure the reproducibility of peptide identification and quantific