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Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

2013; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês

10.1016/j.jmoldx.2013.03.002

1943-7811

Todd Hembrough, Sheeno Thyparambil, Wei-Li Liao, Marlene Darfler, Joe Abdo, Kathleen Bengali, Stephen M. Hewitt, Richard A. Bender, David B. Krizman, Jon Burrows,

Monoclonal and Polyclonal Antibodies Research

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R2 > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies. One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R2 > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies. The aberrant cellular biochemistry that drives tumor growth and cancer progression is mediated largely by sets of proteins organized into complex cell-signaling networks. In the context of cancer, these proteins may be described as oncoproteins because they are part of biochemical pathways that control malignant transformation and permit unregulated cell growth.1Nomura D.K. Dix M.M. Cravatt B.F. Activity-based protein profiling for biochemical pathway discovery in cancer.Nat Rev Cancer. 2010; 10: 630-638Crossref PubMed Scopus (256) Google Scholar, 2Nagaraj N.S. Singh O.V. Merchant N.B. Proteomics: a strategy to understand the novel targets in protein misfolding and cancer therapy.Expert Rev Proteomics. 2010; 7: 613-623Crossref PubMed Scopus (18) Google Scholar Individual members of these sets of proteins have been the targets of targeted therapies that have been developed since the 1990s. A notable example is the use of a humanized monoclonal antibody, trastuzumab, for treatment of patients with HER2-positive breast cancer. HER2 is a member of the human epidermal growth factor receptor (EGFR) family of transmembrane tyrosine kinase receptors, along with HER3 and HER4.3Hudis C.A. Trastuzumab: mechanism of action and use in clinical practice.N Engl J Med. 2007; 357: 39-51Crossref PubMed Scopus (1895) Google Scholar HER family members can drive tumor growth through receptor homodimerization (HER2/HER2), heterodimerization (formation of HER2/HER3 dimers), and receptor transactivation (c-Met activation of EGFR).4Dulak A.M. Gubish C.T. Stabile L.P. Henry C. Siegfried J.M. HGF-independent potentiation of EGFR action by c-Met.Oncogene. 2011; 30: 3625-3635Crossref PubMed Scopus (93) Google Scholar In each case, these signaling pathways help to promote the proliferation and survival of cancer cells.3Hudis C.A. Trastuzumab: mechanism of action and use in clinical practice.N Engl J Med. 2007; 357: 39-51Crossref PubMed Scopus (1895) Google Scholar, 5Earp H.S. Dawson T.L. Li X. Yu H. 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Her4 and Her2/neu tyrosine kinase domains dimerize and activate in a reconstituted in vitro system.J Biol Chem. 2010; 285: 7035-7044Crossref PubMed Scopus (52) Google Scholar Although specifically targeting HER2 inhibits tumor growth and reduces the risk of metastasis, trastuzumab resistance is a common therapeutic occurrence in advanced cancers. In most cases, resistance is driven by alterations in the expression of alternate signaling pathways, not through changes in the drug target, although secondary mutations in EGFR have been described.10Yun C.H. Mengwasser K.E. Toms A.V. Woo M.S. Greulich H. Wong K.K. Meyerson M. Eck M.J. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.Proc Natl Acad Sci U S A. 2008; 105: 2070-2075Crossref PubMed Scopus (1558) Google Scholar Diagnostic testing is essential for identifying the subset of patients with cancer who are best suited for targeted therapy. In the instance of trastuzumab, treatment is restricted to those patients who are HER2+, as defined by either high immunohistochemical (IHC) staining and/or high copy numbers of the HER2 gene, assessed using fluorescence in situ hybridization. These methods have been useful surrogates for measuring HER2 protein expression and were instrumental in defining a population of patients with breast cancer who respond to trastuzumab treatment.3Hudis C.A. Trastuzumab: mechanism of action and use in clinical practice.N Engl J Med. 2007; 357: 39-51Crossref PubMed Scopus (1895) Google Scholar, 11Cuadros M. Villegas R. Systematic review of HER2 breast cancer testing.Appl Immunohistochem Mol Morphol. 2009; 17: 1-7Crossref PubMed Scopus (80) Google Scholar, 12Mass R.D. Press M.F. Anderson S. Cobleigh M.A. Vogel C.L. Dybdal N. Leiberman G. Slamon D.J. Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab.Clin Breast Cancer. 2005; 6: 240-246Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 13Paik S. Bryant J. Tan-Chiu E. Romond E. Hiller W. Park K. Brown A. Yothers G. Anderson S. Smith R. Wickerham D.L. Wolmark N. Real-world performance of HER2 testing: National Surgical Adjuvant Breast and Bowel Project experience.J Natl Cancer Inst. 2002; 94: 852-854Crossref PubMed Scopus (439) Google Scholar Current diagnostic methods do not directly quantify the protein targets nor can they allow for simultaneous multiplex analysis of other proteins involved in tumor growth and drug resistance. Methods using RNA analysis to assess or predict protein expression are not considered universally reliable because they do not directly quantify the expression of the proteins.14Chen G. Gharib T.G. Huang C.C. Taylor J.M. Misek D.E. Kardia S.L. Giordano T.J. Iannettoni M.D. Orringer M.B. Hanash S.M. Beer D.G. Discordant protein and mRNA expression in lung adenocarcinomas.Mol Cell Proteomics. 2002; 1: 304-313Crossref PubMed Scopus (783) Google Scholar Indeed, a shortcoming in the clinic is the lack of availability of diagnostic platforms and methods that can generate quantitative and reproducible diagnostic information about multiple functional proteins and produce valuable information about which proteins should be targeted for therapeutic intervention. More important, these diagnostic methods must be applicable to formalin-fixed, paraffin-embedded (FFPE) tissue to be seamlessly integrated into clinical practice. Mass spectrometry (MS) has been used in clinical assays, such as for the assessment of multiple inborn errors of metabolism15Wilcken B. Wiley V. Hammond J. Carpenter K. Screening newborns for inborn errors of metabolism by tandem mass spectrometry.N Engl J Med. 2003; 348: 2304-2312Crossref PubMed Scopus (540) Google Scholar and vitamin D levels.16Singh R.J. Quantitation of 25-OH-vitamin D (25OHD) using liquid tandem mass spectrometry (LC-MS-MS).Methods Mol Biol. 2010; 603: 509-517Crossref PubMed Scopus (37) Google Scholar The ability to accurately measure target analytes from small amounts of biological samples separates MS from most other diagnostic methods. More recently, MS has begun to be used for quantitative analysis of proteins in biological samples,17Ye X. Blonder J. Veenstra T.D. Targeted proteomics for validation of biomarkers in clinical samples.Brief Funct Genomic Proteomic. 2009; 8: 126-135Crossref PubMed Scopus (33) Google Scholar and application of MS to patient-derived FFPE tissue can be expected to have a profound impact on patient stratification and targeted cancer therapeutics.18Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nat Chem Biol. 2005; 1: 252-262Crossref PubMed Scopus (1316) Google Scholar, 19Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. et al.Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nat Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (862) Google Scholar, 20Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5581) Google Scholar, 21Nilsson T. Mann M. Aebersold R. Yates 3rd, J.R. Bairoch A. Bergeron J.J. Mass spectrometry in high-throughput proteomics: ready for the big time.Nat Methods. 2010; 7: 681-685Crossref PubMed Scopus (387) Google Scholar, 22Rudnick P.A. Clauser K.R. Kilpatrick L.E. Tchekhovskoi D.V. Neta P. Blonder N. Billheimer D.D. Blackman R.K. Bunk D.M. Cardasis H.L. Ham A.J. Jaffe J.D. Kinsinger C.R. Mesri M. Neubert T.A. Schilling B. Tabb D.L. Tegeler T.J. Vega-Montoto L. Variyath A.M. Wang M. Wang P. Whiteaker J.R. Zimmerman L.J. Carr S.A. Fisher S.J. Gibson B.W. Paulovich A.G. Regnier F.E. Rodriguez H. Spiegelman C. Tempst P. Liebler D.C. Stein S.E. Performance metrics for liquid chromatography-tandem mass spectrometry systems in proteomics analyses.Mol Cell Proteomics. 2010; 9: 225-241Crossref PubMed Scopus (152) Google Scholar, 23Gallien S. Duriez E. Domon B. Selected reaction monitoring applied to proteomics.J Mass Spectrom. 2011; 46: 298-312Crossref PubMed Scopus (236) Google Scholar The Liquid Tissue-selected reaction monitoring (SRM) work flow (Figure 1) is a proteomic method by which microdissected FFPE tumor tissue is subjected to Liquid Tissue processing to reverse formalin cross-links. This is followed by trypsinization to completely solubilize all of the protein in the sample. This tryptic peptide mixture is then subjected to SRM analysis using stable isotope-labeled control peptides for accurate quantitation. SRM methods have long been used to quantitate low-abundance protein targets in plasma,24Keshishian H. Addona T. Burgess M. Kuhn E. Carr S.A. Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.Mol Cell Proteomics. 2007; 6: 2212-2229Crossref PubMed Scopus (576) Google Scholar but application of these techniques to FFPE tissue samples has, until recently, been hindered by incomplete solubilization of samples.25Bateman N.W. Sun M. Bhargava R. Hood B.L. Darfler M.M. Kovatich A.J. Hooke J.A. Krizman D.B. Conrads T.P. Differential proteomic analysis of late-stage and recurrent breast cancer from formalin-fixed paraffin-embedded tissues.J Proteome Res. 2011; 10: 1323-1332Crossref PubMed Scopus (36) Google Scholar, 26Cheung W. Darfler M.M. Alvarez H. Hood B.L. Conrads T.P. Habbe N. Krizman D.B. Mollenhauer J. Feldmann G. Maitra A. Application of a global proteomic approach to archival precursor lesions: deleted in malignant brain tumors 1 and tissue transglutaminase 2 are upregulated in pancreatic cancer precursors.Pancreatology. 2008; 8: 608-616Abstract Full Text PDF PubMed Scopus (43) Google Scholar, 27DeSouza L.V. Krakovska O. Darfler M.M. Krizman D.B. Romaschin A.D. Colgan T.J. Siu K.W. mTRAQ-based quantification of potential endometrial carcinoma biomarkers from archived formalin-fixed paraffin-embedded tissues.Proteomics. 2010; 10: 3108-3116Crossref PubMed Scopus (54) Google Scholar, 28Güzel C. Ursem N.T. Dekker L.J. Derkx P. Joore J. van Dijk E. Ligtvoet G. Steegers E.A. Luider T.M. Multiple reaction monitoring assay for pre-eclampsia related calcyclin peptides in formalin fixed paraffin embedded placenta.J Proteome Res. 2011; 10: 3274-3282Crossref PubMed Scopus (28) Google Scholar, 29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar, 30Hood B.L. Darfler M.M. Guiel T.G. Furusato B. Lucas D.A. Ringeisen B.R. Sesterhenn I.A. Conrads T.P. Veenstra T.D. Krizman D.B. Proteomic analysis of formalin-fixed prostate cancer tissue.Mol Cell Proteomics. 2005; 4: 1741-1753Crossref PubMed Scopus (236) Google Scholar, 31Nishimura T. Nomura M. Tojo H. Hamasaki H. Fukuda T. Fujii K. Mikami S. Bando Y. Kato H. Proteomic analysis of laser-microdissected paraffin-embedded tissues: (2) MRM assay for stage-related proteins upon non-metastatic lung adenocarcinoma.J Proteomics. 2010; 73: 1100-1110Crossref PubMed Scopus (58) Google Scholar, 32Patel V. Hood B.L. Molinolo A.A. Lee N.H. Conrads T.P. Braisted J.C. Krizman D.B. Veenstra T.D. Gutkind J.S. Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression.Clin Cancer Res. 2008; 14: 1002-1014Crossref PubMed Scopus (164) Google Scholar, 33Prieto D.A. Hood B.L. Darfler M.M. Guiel T.G. Lucas D.A. Conrads T.P. Veenstra T.D. Krizman D.B. Liquid Tissue: proteomic profiling of formalin-fixed tissues.Biotechniques. 2005; : 32-35Crossref PubMed Google Scholar We have recently described the development and clinical performance of a Liquid Tissue-SRM assay for EGFR in FFPE patient tissues.29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar By using this quantitative EGFR-SRM assay, we assessed EGFR expression in FFPE tumor tissues from a cohort of gefitinib-treated patients with non-small cell lung cancer. Although each of these tumors was positive for EGFR by IHC, by using quantitative SRM, we demonstrated a wide range of EGFR expression in these tissues. Although the focus of the Liquid Tissue-SRM assay development has been on FFPE tissue, recent reports have shown that these MS-based methods can also be performed with equal precision on frozen tissue.12Mass R.D. Press M.F. Anderson S. Cobleigh M.A. Vogel C.L. Dybdal N. Leiberman G. Slamon D.J. Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with trastuzumab.Clin Breast Cancer. 2005; 6: 240-246Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 34Sprung R.W. Martinez M.A. Carpenter K.L. Ham A.J. Washington M.K. Arteaga C. Sanders M. Liebler D.C. Precision of multiple reaction monitoring mass spectrometry analysis of formalin-fixed, paraffin-embedded tissue.J Proteome Res. 2012; 11: 3498-3505Crossref PubMed Scopus (49) Google Scholar This report describes the development of a multiplexed Liquid Tissue-SRM assay to accurately measure the expression of a series of oncological targets: the insulin-like growth factor-1 receptor (IGF-1R and IGF-1R-SRM), HER2 (HER2-SRM), and HER3 (HER3-SRM). We also include the assay development results of EGFR-SRM to place them in the context of this larger multiplexed set.29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar Each of the assays is capable of detecting and measuring attomole (ie, 10−18 mol) amounts of these specific peptides directly in FFPE patient tumor tissue. We extend the assay development to assess the performance of these assays (ie, HER2-SRM, EGFR-SRM, and HER3-SRM) in FFPE patient breast tumor tissues, including patients who had been treated previously with trastuzumab. This study demonstrates the broad, dynamic range of SRM MS, when compared with HER2 IHC, underlining the gaps in using only IHC to assess protein expression levels. Taken together, Liquid Tissue-SRM provides a powerful, quantitative, and multiplexed approach for analysis of critical oncological proteins in FFPE tumor tissues. Ten human cancer cell lines were used: A431 (skin), HCC-827 (lung), MDA-MB-231 (breast), MCF7 (breast), HT29 (colon), Colo205 (colon), T47D (breast), SKBR-3 (breast), PC3 (prostate), and ZR75-30 (breast). A431 and MDA-MB-231 lines were maintained in Dulbecco's modified Eagle's medium; T47D, ZR75-30, and HCC827 cells were maintained in RPMI 1640 medium; MCF7 cells were maintained in Dulbecco's modified Eagle's medium-F12 medium; SKBR-3 and HT29 cells were maintained in McCoy's 5A medium; and PC3 cells were maintained in HAM's-F12 medium. All media were supplemented with 10% fetal bovine serum and antibiotics. Each cell line was grown in sufficient quantity so that cells could be fixed in formalin and embedded in paraffin for SRM analysis, as well as preparing fresh lysates in parallel for analysis by immunoassay [ie, either electrochemiluminescence (ECL) or enzyme-linked immunosorbent assay (ELISA)]. For SRM analysis, the cultured cell lines were prepared by pelleting the cell suspensions, overlaying the pellet with 10% neutral-buffered formalin, and allowing the cells to fix for 18 to 24 hours at 4°C. The 10% formalin was removed, and the pellet was washed with water and then transferred into 70% ethanol. Embedding in paraffin and dividing into sections cells prepared on microscope slides were done using standard histological methods. Tumor tissue was obtained from patients with infiltrating ductal carcinoma of the breast. Ten specimens were obtained from MDR Global (Windber, PA), with no patient identifiers, and collected under preapproved local guidelines. An additional 19 specimens were obtained from the Ontario Tumor Bank (Ontario Institute for Cancer Research, Toronto, ON, CA) under strict Institutional Review Board guidelines through the University of Toronto Health System (Toronto, ON, Canada). Tissue sections (10 μm thick) from each breast cancer tissue block were cut onto Director microdissection slides and deparaffinized and stained with hematoxylin to prepare for microdissection, as previously described.29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar Likewise, blocks containing FFPE cultured cells were divided into sections (10 μm) and placed onto Director slides, prepared for dissection similarly, and microdissected as previously described.29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar A cumulative area of 12 mm2 containing approximately 45,000 malignant cells was microdissected from each FFPE breast cancer specimen. For the FFPE tissue-cultured cell lines, an area of 12 mm2 was also microdissected. For the IGF-1R and HER2 experiments correlating SRM to immunoassay, protein lysates of fresh, unfixed cells were prepared for analysis by ECL signal-based sandwich immunoassay and FFPE cells were prepared for SRM analysis. Protein lysates were prepared from fresh, unfixed cells from the tissue culture cell lines using the manufacturer-provided cell lysis buffer and protocol (Meso Scale Discovery, Rockville, MD). For the IGF-1R experiments, MDA-MB-231, HCC-827, A431, and MCF7 cells were used. For the IGF-1R ECL assay, a standard curve was generated with purified recombinant IGF-1R protein to measure IGF-1R in cell lysates. The IGF-1R standards were assayed in triplicate with good reproducibility [coefficient of variation (CV) range, 2.4% to 7.7%] across serial dilutions that ranged in concentration from 25,000 pg/mL (625 pg) to 1560 pg/mL (39 pg) in assay dilution buffer. A linear standard curve (R2 = 0.9926) was generated to measure IGF-1R protein in detergent lysates of non-formalin-fixed, tissue culture cells. For the HER2 experiments, the MCF7, T47D, HT29, SKBR-3, and ZR75-30 cell lines were used. ECL immunoassays were performed on 10 μg of fresh, unfixed cell lysate in triplicate and analyzed on a SECTOR Imager 2400 instrument (Meso Scale Discovery) to collect ECL data. The EGFR experiments correlating SRM to immunoassay have been previously published29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar and are presented herein to place these results in context with the other protein biomarkers as a multiplexed set. As previously described, the MCF7, HT29, MDA-MB-23, and A431 cell lines were used in this study. Briefly, protein lysates of fresh, unfixed cells for each cell line were prepared for analysis by ELISA using the manufacturer-provided cell lysis buffer and protocol, according to manufacturer's recommendations (Millipore, Billerica, MA). The process of developing SRM assays for IGF-1R (IGF-1R-SRM), HER2 (HER2-SRM), and HER3 (HER3-SRM) was similar to the EGFR-SRM assay, as previously described.29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar Each SRM assay was developed in a stepwise manner. Purified recombinant proteins were obtained and digested with trypsin to produce a set of tryptic peptides. Subsequently, these peptides were analyzed on an Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) equipped with a nanoAcquityLC system (Waters, Milford, MA) or on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific) equipped with a nanoAcquityLC to evaluate all tryptic peptides to identify candidate SRM peptides. Liquid Tissue lysates from the FFPE preparations derived from the cell lines expressing these proteins were analyzed on the TSQ Vantage system. Software programs Pinpoint version 1.1 and Xcalibur version 2.1 (Thermo Scientific) were used to identify optimal tryptic peptides based on reproducible peak heights, retention times, chromatographic ion intensities, and transition ion ratios that were distinctive and reproducible. Methionine and cysteine-containing peptides were excluded to avoid oxidation entities. Peptides with glycosylation motifs were also excluded. The choice of unique peptides for each of the corresponding biomarkers was verified by performing peptide sequence searches using the BLASTP function of the BLAST search engine (http://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed December 12, 2012). Unlabeled (native) and isotopically labeled (internal standard) versions of each peptide were synthesized to develop and perform each assay (Thermo Scientific). Candidate peptides for the IGF-1R-SRM assay were obtained by trypsin digestion mapping of recombinant human IGF-1R protein. They were composed of an equal mixture of a recombinant N-terminus fragment (Met1-ASN932; Sigma, St. Louis, MO) and a recombinant C-terminus fragment (Arg960-Cys1367; Invitrogen, Carlsbad, CA). For IGF-1R, a single unique peptide (single amino acid abbreviation: GNLLINIR), which gave the most reproducible detection in both trypsin-digested recombinant IGF-1R and a Liquid Tissue lysate prepared from FFPE A431 cells, was chosen. Peptide GNLLINIR spans residues 358 to 365 of the IGF-1R extracellular domain. Unlabeled (GNLLINIR) and isotopically labeled [GNL(13C15N)LINIR] versions of this peptide were synthesized. SRM transitions are listed in Table 1.Table 1SRM Assay Parameters for the Four Assays DescribedAssayPrecursor ion (m/z)∗Data in parentheses are the charge.Product ion 1 (m/z)†Data in parentheses are fragment, charge, and collision energy.Product ion 2 (m/z)†Data in parentheses are fragment, charge, and collision energy.Product ion 3 (m/z)†Data in parentheses are fragment, charge, and collision energy.EGFR IPLENLQIIR604.872 (2)756.472885.515998.599(y6, 1, 24)(y7, 1, 24)(y8, 1, 24) IPLENL(HeavyL)QIIR608.380 (2)763.489892.5321005.616(y6, 1, 24)(y7, 1, 24)(y8, 1, 24)HER2 ELVSEFR483.748 (2)538.261625.294724.362(y4, 1, 17)(y5, 1, 17)(y6, 1, 18) ELVSEFSR(HeavyR)488.752 (2)548.27635.302734.37(y4, 1, 17)(y5, 1, 17)(y6, 1, 18)HER3 LAEVPDLLEK563.821 (2)714.403813.471942.514(y6, 1, 17)(y7, 1, 16)(y8, 1, 15) LAEVPDLLEK(HeavyK)567.828 (2)722.417821.485950.528(y6, 1, 17)(y7, 1, 16)(y8, 1, 15)IGF-1R GNLLINIR456.784 (2)515.33628.41741.5(y4, 1, 19)(y5, 1, 19)(y6, 1, 19) GNLL(HeavyL)INIR460.293 (2)515.33635.43748.51(y4, 1, 19)(y5, 1, 19)(y6, 1, 19)∗ Data in parentheses are the charge.† Data in parentheses are fragment, charge, and collision energy. Open table in a new tab Candidate peptides for HER2 were obtained by trypsin digestion mapping of full-length, recombinant human HER2 protein (Origene, Rockville, MD). For HER2, a single unique peptide (ELVSEFSR), which gave the most reproducible detection with the highest intensity in both trypsin-digested recombinant HER2 and a Liquid Tissue lysate prepared from FFPE SKBR-3 cells, was chosen. Peptide ELVSEFSR spans residues 971 to 978 of the HER2 cytoplasmic domain. Unlabeled (ELVSEFSR) and isotopically labeled [ELVSEFS (13C15N)R] versions of this peptide were synthesized to develop and perform the assay (Thermo Scientific). SRM transitions used for quantification of the HER2 native peptide are listed in Table 1. As previously described, a single peptide unique to EGFR was chosen, which spans residues 98 to 108 of the EGFR extracellular domain.29Hembrough T. Thyparambil S. Liao W.L. Darfler M.M. Abdo J. Bengali K.M. Taylor P. Tong J. Lara-Guerra H. Waddell T.K. Moran M.F. Tsao M.S. Krizman D.B. Burrows J. Selected reaction monitoring (SRM) analysis of epidermal growth factor receptor (EGFR) in formalin fixed tumor tissue.Clin Proteomics. 2012; 9: 5Crossref PubMed Scopus (53) Google Scholar Briefly, unlabeled (IPLENLQIIR) and isotopically labeled [IPLEN(13C15N)LQIIR] versions of this peptide were synthesized to develop and perform the assay (Thermo Scientific). SRM transitions used for quantification of the EGFR peptides are listed in Table 1. Candidate peptides for HER3 were obtained by trypsin digestion mapping of full-length, recombinant human HER3 protein (Origene).