Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma. There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma. Biology and clinical medicine are increasingly in need of methods to robustly and reliably measure many tens to hundreds of peptides and proteins in a given sample with high sensitivity, specificity, and reproducibility. Targeted mass spectrometry (MS) methods offer biologists and clinical researchers an ever-increasing suite of experimental approaches and data analysis tools to accomplish this task without the need for immunoassays (1.Grebe S.K. Singh R.J. LC-MS/MS in the clinical laboratory–Where to from here?.Clin. Biochem. Rev. 2011; 32: 5-31PubMed Google Scholar, 2.Picotti P. Aebersold R. 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Skates S.J. Pearson T.W. Paulovich A.G. Carr S.A. Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.Mol. Cell. Proteomics. 2012; 11M111.013854Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Despite numerous reports describing the application of LC-MRM-MS for quantification of target peptides, questions remain about the sensitivity, specificity, reproducibility, quantitative precision, and accuracy of the measurements as well as the transferability of the methods and assays across laboratories. These questions are driven, in part, by the lack of methodological detail or rigorous analytical validation of targeted MS measurements in many published studies, preventing readers from understanding how well the assays work or to be able to implement the described assays in their own laboratories (10.Carr S.A. Abbatiello S.E. Ackermann B.L. Borchers C. Domon B. Deutsch E.W. Grant R.P. Hoofnagle A.N. Hüttenhain R. Koomen J.M. Liebler D.C. Liu T. MacLean B. Mani D.R. Mansfield E. Neubert H. Paulovich A.G. Reiter L. Vitek O. Aebersold R. Anderson L. Bethem R. Blonder J. Boja E. Botelho J. Boyne M. Bradshaw R.A. Burlingame A.L. Chan D. Keshishian H. Kuhn E. Kinsinger C. Lee J.S. Lee S.W. Moritz R. Oses-Prieto J. Rifai N. Ritchie J. Rodriguez H. Srinivas P.R. Townsend R.R. Van Eyk J. Whiteley G. Wiita A. Weintraub S. Targeted peptide measurements in biology and medicine: Best practices for mass spectrometry-based assay development using a fit-for-purpose approach.Mol. Cell. Proteomics. 2014; 13: 907-917Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Since 2005, the Clinical Proteomics Technology Assessment for Cancer (CPTAC) network of the National Cancer Institute has had, as one area of focus, the evaluation, refinement, and application of LC-MRM-MS methodology for peptide-based verification of proteins and their modifications in biofluids and tissue. Our efforts have focused on making these assays more precise, accurate, reproducible, and transferable between different laboratories, expertise levels, and LC-MS instrument platforms with the goal of widespread adoption initially by the proteomics community but ultimately also by the clinical laboratory and biology communities. Previously, we demonstrated the reproducibility and transferability of peptide-based MRM assays across eight laboratories (5.Addona T.A. Abbatiello S.E. Schilling B. Skates S.J. Mani D.R. Bunk D.M. Spiegelman C.H. Zimmerman L.J. Ham A.J. Keshishian H. Hall S.C. Allen S. Blackman R.K. Borchers C.H. Buck C. Cardasis H.L. Cusack M.P. Dodder N.G. Gibson B.W. Held J.M. Hiltke T. Jackson A. Johansen E.B. Kinsinger C.R. Li J. Mesri M. Neubert T.A. Niles R.K. Pulsipher T.C. Ransohoff D. Rodriguez H. Rudnick P.A. Smith D. Tabb D.L. Tegeler T.J. Variyath A.M. Vega-Montoto L.J. Wahlander A. Waldemarson S. Wang M. Whiteaker J.R. Zhao L. Anderson N.L. Fisher S.J. Liebler D.C. Paulovich A.G. Regnier F.E. Tempst P. Carr S.A. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma.Nature Biotech. 2009; 27: 633-641Crossref PubMed Scopus (862) Google Scholar) by measuring levels of 10 signature peptides representing seven proteins that were spiked across a defined concentration range (1–500 fmol/μl) into neat human plasma. The study was performed in three phases whereby each phase introduced additional sources of variability in sample preparation and instrumental analyses. In the final phase, which included all sources of variability, including proteolytic digestion, the median interlaboratory CV of the eight peptides consistently detected was ≤20% across the concentration range tested. This study demonstrated the implementation of a targeted, quantitative, and multiplexed LC-MRM-MS assay across multiple laboratories to reproducibly measure a small number of proteins present at moderate to high abundance (≥2–6 μg/ml in plasma) yielding CVs in an acceptable range for biomarker verification studies (10.Carr S.A. Abbatiello S.E. Ackermann B.L. Borchers C. Domon B. Deutsch E.W. Grant R.P. Hoofnagle A.N. Hüttenhain R. Koomen J.M. Liebler D.C. Liu T. MacLean B. Mani D.R. Mansfield E. Neubert H. Paulovich A.G. Reiter L. Vitek O. Aebersold R. Anderson L. Bethem R. Blonder J. Boja E. Botelho J. Boyne M. Bradshaw R.A. Burlingame A.L. Chan D. Keshishian H. Kuhn E. Kinsinger C. Lee J.S. Lee S.W. Moritz R. Oses-Prieto J. Rifai N. Ritchie J. Rodriguez H. Srinivas P.R. Townsend R.R. Van Eyk J. Whiteley G. Wiita A. Weintraub S. Targeted peptide measurements in biology and medicine: Best practices for mass spectrometry-based assay development using a fit-for-purpose approach.Mol. Cell. Proteomics. 2014; 13: 907-917Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 27.Paulovich A.G. Whiteaker J.R. Hoofnagle A.N. Wang P. The interface between biomarker discovery and clinical validation: The tar pit of the protein biomarker pipeline.Proteome Clin. Appl. 2008; 2: 1386-1402Crossref PubMed Scopus (171) Google Scholar, 32.Rifai N. Gillette M.A. Carr S.A. Protein biomarker discovery and validation: The long and uncertain path to clinical utility.Nature Biotech. 2006; 24: 971-983Crossref PubMed Scopus (1367) Google Scholar). Here, we significantly expand upon our previous work, detailing critical steps in the assay development phase essential for successful development of highly multiplexed MRM assays, including the use of an SSP (33.Abbatiello S.E. Mani D.R. Schilling B. Maclean B. Zimmerman L.J. Feng X. Cusack M.P. Sedransk N. Hall S.C. Addona T. Allen S. Dodder N.G. Ghosh M. Held J.M. Hedrick V. Inerowicz H.D. Jackson A. Keshishian H. Kim J.W. Lyssand J.S. Riley C.P. Rudnick P. Sadowski P. Shaddox K. Smith D. Tomazela D. Wahlander A. Waldemarson S. Whitwell C.A. You J. Zhang S. Kinsinger C.R. Mesri M. Rodriguez H. Borchers C.H. Buck C. Fisher S.J. Gibson B.W. Liebler D. Maccoss M. Neubert T.A. Paulovich A. Regnier F. Skates S.J. Tempst P. Wang M. Carr S.A. Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS).Mol. Cell. Proteomics. 2013; 12: 2623-2639Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) to monitor LC-MRM-MS performance during assay development to detect and correct problems early. We also highlight key advances in hardware and software that we have incorporated into the current design that became available since our initial study. The present study utilized eight different LC-MS instrument configurations in 11 separate laboratories on a total of 14 individual systems to target and quantitatively measure >100 peptides from a total of 34 proteins, including 27 that are cancer relevant (Table I). Similar considerations on a smaller scale have been recently discussed using protein and peptide standards as part of quality control for large quantitative studies (34.Gallien S. Bourmaud A. Domon B. A simple protocol to routinely Assess the uniformity of proteomics analyses.J. Proteome Res. 2014; 13: 2688-2695Crossref PubMed Scopus (22) Google Scholar). In our study, sensitivity for proteins in plasma was increased into the low- to subnanogram/ml level by one-step immunoaffinity depletion as well as gradient optimization to maximize the chromatographic resolution in the sample matrix. Use of heavy-labeled protein internal standards added to samples prior to processing greatly improved the accuracy of protein-level quantification. Intra- and interlaboratory reproducibility sufficient for most biological studies as well as for candidate protein biomarker verification was achieved. Overall, this study demonstrates that highly multiplexed MRM-MS based assays can, with appropriate attention to experimental design, analytical validation, and suitable quality control measures, be implemented by multiple laboratories to provide sensitive, specific, reproducible, and quantitative measurements of proteins and peptides of clinical and biological interest in complex biological matrices, specifically plasma.Table IList of 34 proteins for which MRM-MS assays were developed and the heavy-isotope labeled internal standard (SIS) peptides used.aMRM-MS assays were constructed and run for a total of 365 peptides: 125 peptides were unlabeled and derived from synthesis as well as digestion of the light versions of all of the proteins shown. Another 125 peptides were chemically synthesized heavy-labeled versions, and another 115 peptides derived by digestion from the 27 uniformly heavy-labeled proteins. Peptides annotated with an asterisk (96 of 115) were detected at all sites used throughout the data analysisSwProtProtein descriptionSynthetic internal standard 13C/N15 peptidesbChemically synthesized 13C/N15-labeled peptides used as internal standards; labeled amino acids are indicated in bold. These peptides are labeled on the C-terminal Lys as K8 (13C6, 15N2) or C-terminal Arg as R10 (13C6, 15N4) unless otherwise noted. Exceptions include peptides from ferritin, protein S100-A2, protein S100-B, and myelin basic protein that were labeled with K6 (13C6) Lys or R6 (13C6) Arg. Protein S100-A1 was labeled at an internal Leu, L6 (13C6). Prostate-specific antigen and pancreatic trypsin inhibitor were labeled at an internal Val, V5 (13C5).E coli expressed L/H proteincTwenty-seven proteins expressed in E. coli in both natural isotope abundance and uniformly N15-labeled (U-N15) forms. A total of 115 U15N-labeled peptides derived from the heavy labeled proteins were monitored.P09972Fructose-bisphosphate aldolase CALQASALNAWR*, AEVNGLAAQGK*, ELSDIALR*, TPSALAILENANVLAR*, QVLFSADDR*+/+P04083Annexin A1ALYEAGER*, GTDVNVFNTILTTR*, GVDEATIIDILTK, AAYLQETGKPLDETLK*, TPAQFDADELR*+/+P09525Annexin A4VLVSLSAGGR*, GLGTDEDAIISVLAYR, DEGNYLDDALVR*, GLGTDDNTLIR, GAGTDEGCLIEILASR*+/+P20073Annexin A7LYQAGEGR*, SEIDLVQIK*, GAGTDDSTLVR*, EFSGYVESGLK*, GFGTDEQAIVDVVANR+/+Q53G71Calreticulin variant (Fragment)QIDNPDYK*, GLQTSQDAR, FYALSASFEPFSNK*, EQFLDGDGWTSR*, GQTLVVQFTVK+/+O00299Chloride intracellular channel protein 1LHIVQVVCK*, YLSNAYAR*, IEEFLEAVLCPPR*, GVTFNVTTVDTK*, GFTIPEAFR*+/+P15311EzrinIQVWHAEHR*, EDEVEEWQHR*, SGYLSSER*, IALLEEAR*, SQEQLAAELAEYTAK*+/+Q16658FascinYLTAEAFGFK*, FLIVAHDDGR*, YLAPSGPSGTLK*, VTGTLDANR*, LSCFAQTVSPAEK*+/+P02792Ferritin light chainLGGPEAGLGEYLFER (R6), KPAEDEWGK (K6)+/+P39748Flap endonuclease 1HLTASEAK, WSEPNEEELIK*, SIEEIVR*, QLQQAQAAGAEQEVEK*, LIADVAPSAIR*+/+P09211Glutathione S-transferase PFQDGDLTLYQSNTILR*, YISLIYTNYEAGK, EEVVTVETWQEGSLK*, PPYTVVYFPVR, ASCLYGQLPK*+/+Q04760Lactoylglutathione lyaseFSLYFLAYEDK*, SLDFYTR*, IAWALSR*, GFGHIGIAVPDVYSACK*, FEELGVK*+/+P62993Growth factor receptor-bound protein 2FNSLNELVDYHR*, FGNDVQHFK*, NYVTPVNR*, ATADDELSFK*, ESESAPGDFSLSVK*+/+P04792Heat shock protein beta-1LFDQAFGLPR*, AQLGGPEAAK*, VSLDVNHFAPDELTVK*, DGVVEITGK*, QLSSGVSEIR*+/+Q14116Interleukin-18EDELGDR, SDIIFFQR*, TIFIISMYK*, ISTLSCENK*, GMAVTISVK*+/+P09382Galectin-1DGGAWGTEQR*, DSNNLCLHFNPR*, FNAHGDANTIVCNSK*, SFVLNLGK*, LPDGYEFK*+/+O00151PDZ and LIM domain protein 1CGTGIVGVFVK, GCTDNLTLTVAR*, VAASIGNAQK*, VWSPLVTEEGK*, DFEQPLAISR*+/+P32119Peroxiredoxin-2GLFIIDGK*, TDEGIAYR*, LSEDYGVLK*, ATAVVDGAFK*+/+Q13162Peroxiredoxin-4DYGVYLEDSGHTLR*, LVQAFQYTDK*, IPLLSDLTHQISK*, QITLNDLPVGR*+/+P23297Protein S100-A1ELLQTELSGFLDAQK (L6)+/+P29034Protein S100-A2ELPSFVGEK* (K6), YSCQEGDK (K6)+/+P04271Protein S100-BAMVALIDVFHQYSGR* (R6), ELINNELSHFLEEIK (K6)+/+P54727UV excision repair protein RAD23 homBEQVIAALR*, IDIDPEETVK*, ILNDDTALK*+/+O76070Gamma-synucleinEQANAVSEAVVSSVNTVATK, EGVVGAVEK*, ENVVQSVTSVAEK*, TVEEAENIAVTSGVVR*+/+P09493Tropomyosin alpha-1 chainHIAEDADR, LVIIESDLER*, SIDDLEDELYAQK*, QLEDELVSLQK*+/+O00762Ubiquitin-conjugating enzyme E2 CYLQETYSK*, WSALYDVR*, LSLEFPSGYPYNAPTVK*, DPAATSVAAAR*, GISAFPESDNLFK*+/+P63279SUMO-conjugating enzyme UBC9DHPFGFVAVPTK*, GTPWEGGLFK*, DDYPSSPPK*, DWRPAITIK+/+P00974Pancreatic trypsin inhibitorAGLCQTFVYGGCR (V5)+/−dCommercially available unlabeled protein.P41160LeptinINDISHTQSVSAK+/−eCommercially available unlabeled proteins used as controls (5).P68082MyoglobinLFTGHPETLEK+/−eCommercially available unlabeled prot