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• W2126156743 abstract "Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities. Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities. Targeted analysis by liquid chromatography-stable isotope dilution-multiple reaction monitoring-MS (LC-SID-MRM-MS) 1The abbreviations used are:LC-SID-MRM-MSLiquid chromatography-stable isotope dilution-multiple reaction monitoring-MSCVcoefficient of variationLOQlimit of quantificationSSPsystem suitability protocolFWHMfull width half maximalRTretention time. 1The abbreviations used are:LC-SID-MRM-MSLiquid chromatography-stable isotope dilution-multiple reaction monitoring-MSCVcoefficient of variationLOQlimit of quantificationSSPsystem suitability protocolFWHMfull width half maximalRTretention time. (also referred to as LC-SID-SRM-MS) has experienced rapid expansion over the last several years for precise relative quantification of peptides in the context of basic biological studies (1Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6940-6945Crossref PubMed Scopus (1542) Google Scholar, 2Barnidge D.R. Dratz E.A. Martin T. Bonilla L.E. Moran L.B. Lindall A. Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards.Anal. Chem. 2003; 75: 445-451Crossref PubMed Scopus (196) Google Scholar, 3Barr J.R. Maggio V.L. Patterson Jr., D.G. Cooper G.R. Henderson L.O. Turner W.E. Smith S.J. Hannon W.H. Needham L.L. Sampson E.J. Isotope-dilution mass spectrometric quantification of specific proteins: model application with apolipoprotein A-1.Clin. Chem. 1996; 42: 1676-1682Crossref PubMed Scopus (321) Google Scholar, 4Kuzyk M.A. Smith D. Yang J. Cross T.J. Jackson A.M. Hardie D.B. Anderson N.L. Borchers C.H. Multiple reaction monitoring-based, multiplexed, absolute quantitation of 45 proteins in human plasma.Mol. Cell. Proteomics. 2009; 8: 1860-1877Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar) and for verification of candidate biomarkers in clinical applications (5Keshishian H. Addona T.A. 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: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 6Abbatiello S.E. Pan Y.X. Zhou M. Wayne A.S. Veenstra T.D. Hunger S.P. Kilberg M.S. Eyler J.R. Richards N.G. Conrads T.P. Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients.J. Proteomics. 2008; 71: 61-70Crossref PubMed Scopus (15) Google Scholar, 7Hoofnagle A.N. Becker J.O. Wener M.H. Heinecke J.W. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry.Clin. Chem. 2008; 54: 1796-1804Crossref PubMed Scopus (242) Google Scholar, 8Keshishian H. Addona T. Burgess M. Mani D. Shi X. Kuhn E. Sabatine M. Gerszten R. Carr S. Quantification of cardiovascular biomarkers in patient plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2009; 8: 2339-2349Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 9Kumar V. Barnidge D.R. Chen L.S. Twentyman J.M. Cradic K.W. Grebe S.K. Singh R.J. Quantification of serum 1–84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry.Clin. Chem. 2010; 56: 306-313Crossref PubMed Scopus (84) Google Scholar, 10Huttenhain R. Soste M. Selevsek N. Rost H. Sethi A. Carapito C. Farrah T. Deutsch E.W. Kusebauch U. Moritz R.L. Nimeus-Malmstrom E. Rinner O. Aebersold R. Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics.Sci. Transl. Med. 2012; 4Crossref PubMed Scopus (208) Google Scholar). As a quantitative proteomics tool, LC-SID-MRM-MS offers numerous benefits. First, the overall precision of analyte quantification, taking into account all sample processing steps from digestion through data acquisition, is often in the range of 5–10% (CV) above the limit of quantification (LOQ) and ≤25% at the LOQ when stable isotope labeled internal standards are employed. The excellent precision is inherent to LC-SID-MRM-MS experimental design and how data are acquired. For example, these assays employ classical SID methodology in which synthetic peptide standards incorporating one or more labeled amino acids (13C, 15N or a combination thereof) are spiked at known amounts into the samples thereby enabling the endogenous peptide concentration to be determined (1Gerber S.A. Rush J. Stemman O. Kirschner M.W. Gygi S.P. Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 6940-6945Crossref PubMed Scopus (1542) Google Scholar, 2Barnidge D.R. Dratz E.A. Martin T. Bonilla L.E. Moran L.B. Lindall A. Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards.Anal. Chem. 2003; 75: 445-451Crossref PubMed Scopus (196) Google Scholar, 3Barr J.R. Maggio V.L. Patterson Jr., D.G. Cooper G.R. Henderson L.O. Turner W.E. Smith S.J. Hannon W.H. Needham L.L. Sampson E.J. Isotope-dilution mass spectrometric quantification of specific proteins: model application with apolipoprotein A-1.Clin. Chem. 1996; 42: 1676-1682Crossref PubMed Scopus (321) Google Scholar). Confidence in detection and quantification of analytes in SID-MRM-MS is based on multiple orthogonal measurements, specifically (1) the labeled internal standard and analyte must co-elute chromatographically, (2) the heavy and light peptides fragment identically (yielding fragment ions that either have identical m/z values or are shifted upward in mass if they contain the label), and (3) the fragment ions have the same relative abundance in both the analyte and internal standard channels. Second, analyte detection sensitivities on the order of ELISA assays (low ng/ml) are achievable when either sample fractionation or stable isotope standards with capture by antipeptide antibodies (SISCAPA) are incorporated into the assay (5Keshishian H. Addona T.A. 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: 2212-2229Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 11Kuhn E. Addona T.A. Keshishian H. Burgess M. Mani D.R. Lee R.T. Sabatine M.S. Gerszten R.E. Carr S.A. Developing multiplexed assays for troponin I and interleukin-33 in plasma by peptide immunoaffinity enrichment and targeted mass spectrometry.Clin. Chem. 2009; 55: 1108-1117Crossref PubMed Scopus (210) Google Scholar, 12Anderson N.L. Anderson N.G. Haines L.R. Hardie D.B. Olafson R.W. Pearson T.W. Mass spectrometric quantitation of peptides and proteins using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA).J. Proteome Res. 2004; 3: 235-244Crossref PubMed Scopus (694) Google Scholar, 13Whiteaker J.R. Zhao L. Anderson L. Paulovich A.G. An automated and multiplexed method for high throughput peptide immunoaffinity enrichment and multiple reaction monitoring mass spectrometry-based quantification of protein biomarkers.Mol. Cell. Proteomics. 2010; 9: 184-196Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). Finally, several hundred analytes can be multiplexed into a single LC-SID-MRM-MS experiment using scheduling software that segregates collection of data from subsets of the analyte peptides into different time periods in an LC-MRM-MS analysis (10Huttenhain R. Soste M. Selevsek N. Rost H. Sethi A. Carapito C. Farrah T. Deutsch E.W. Kusebauch U. Moritz R.L. Nimeus-Malmstrom E. Rinner O. Aebersold R. Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics.Sci. Transl. Med. 2012; 4Crossref PubMed Scopus (208) Google Scholar, 14Stahl-Zeng J. Lange V. Ossola R. Eckhardt K. Aebersold R. Domon B. High sensitivity detection of plasma proteins by multiple reaction monitoring of N-glycosites.Mol. Cell. Proteomics. 2007; 6: 1809-1817Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 15Escher C. Reiter L. MacLean B. Ossola R. Herzog F. Chilton J. MacCoss M.J. Rinner O. Using iRT, a normalized retention time for more targeted measurement of peptides.Proteomics. 2012; 12: 1111-1121Crossref PubMed Scopus (382) Google Scholar). This feature enables implementation of extremely efficient and high throughput quantitative protein assays. Accordingly, these attributes make quantitative LC-SID-MRM-MS workflows attractive for adaptation into a clinical setting (16Hammett-Stabler C.A. Garg U. The evolution of mass spectrometry in the clinical laboratory.Methods Mol. Biol. 2010; 603: 1-7Crossref PubMed Scopus (8) Google Scholar, 17Strathmann F.G. Hoofnagle A.N. Current and future applications of mass spectrometry to the clinical laboratory.Am. J. Clin. Pathol. 2011; 136: 609-616Crossref PubMed Scopus (71) Google Scholar). However, before clinical adoption becomes a reality, robust procedures must be in place to ensure that optimal performance of an LC-SID-MRM-MS instrument platform is maintained throughout the progression of a particular assay (18Percy A.J. Chambers A.G. Smith D.S. Borchers C.H. Standardized Protocols for Quality Control of MRM-based Plasma Proteomic Workflows.J. Proteome Res. 2013; 12: 222-233Crossref PubMed Scopus (60) Google Scholar). Liquid chromatography-stable isotope dilution-multiple reaction monitoring-MS coefficient of variation limit of quantification system suitability protocol full width half maximal retention time. Liquid chromatography-stable isotope dilution-multiple reaction monitoring-MS coefficient of variation limit of quantification system suitability protocol full width half maximal retention time. To date, little work has been presented on the development of standardized protocols that quantitatively assess the “suitability” (19Heller D.N. Guidance for Industry: Mass Spectrometry for Confirmation of the Identity of Animal Drug Residues, C.f.V.M.in: U.S. Department of Health and Human Services, 2003: 1-11Google Scholar) of nanoflow (typically 150–300 nL/min) high performance liquid chromatography (HPLC) systems interfaced to triple quadrupole mass spectrometers during the course of an LC-SID-MRM-MS protein assay. Although promising LC-SID-MRM-MS biomarker verification studies were recently reported for cardiovascular disease (8Keshishian H. Addona T. Burgess M. Mani D. Shi X. Kuhn E. Sabatine M. Gerszten R. Carr S. Quantification of cardiovascular biomarkers in patient plasma by targeted mass spectrometry and stable isotope dilution.Mol. Cell. Proteomics. 2009; 8: 2339-2349Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), cancer (7Hoofnagle A.N. Becker J.O. Wener M.H. Heinecke J.W. Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry.Clin. Chem. 2008; 54: 1796-1804Crossref PubMed Scopus (242) Google Scholar, 10Huttenhain R. Soste M. Selevsek N. Rost H. Sethi A. Carapito C. Farrah T. Deutsch E.W. Kusebauch U. Moritz R.L. Nimeus-Malmstrom E. Rinner O. Aebersold R. Reproducible quantification of cancer-associated proteins in body fluids using targeted proteomics.Sci. Transl. Med. 2012; 4Crossref PubMed Scopus (208) Google Scholar), and other disorders (9Kumar V. Barnidge D.R. Chen L.S. Twentyman J.M. Cradic K.W. Grebe S.K. Singh R.J. Quantification of serum 1–84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry.Clin. Chem. 2010; 56: 306-313Crossref PubMed Scopus (84) Google Scholar), this approach is still in its infancy. Development of quantitative protein/peptide LC-SID-MRM-MS experiments is challenged by many of the same hurdles as those for small molecules and as this technology evolves, other unique issues relevant to separation and quantification of peptides are being realized (20Abbatiello S.E. Mani D.R. Keshishian H. Carr S.A. Automated detection of inaccurate and imprecise transitions in peptide quantification by multiple reaction monitoring mass spectrometry.Clin. Chem. 2010; 56: 291-305Crossref PubMed Scopus (166) Google Scholar). In the most systematic and thorough interlaboratory evaluation of LC-SID-MRM-MS to date, eight laboratories followed a standard operating procedure (SOP) and analyzed identical sample sets to determine the limits of quantification and measures of variation using a set of 10 tryptic peptides generated from seven proteins spiked into human plasma at known concentrations (21Addona 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.L. 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.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (862) Google Scholar). The results from this study detailed the range of reproducibility attainable for individual laboratories performing an identical experimental protocol, based on increasing complexity of the sample workflow (21Addona 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.L. 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.Nat. Biotechnol. 2009; 27: 633-641Crossref PubMed Scopus (862) Google Scholar). Although the focus of this study was on the contributions of sample handling and processing to the intra- and interlaboratory precision (CV) of the measurements, it also uncovered several common issues that contributed to intralaboratory variability. Most notably, HPLC-related problems resulting in peak tailing or random peak shape deformities caused inconsistencies in peak area integration and subsequent quantitative calculations. These performance degradations often went unnoticed by the laboratory that generated the data, and were only discovered when all of the data were evaluated by a small team of expert analysts. In addition, this study confirmed that use of stable isotope labeled peptides as internal standards for each peptide analyte helps to dampen measurement imprecision introduced by HPLC and MS-associated problems, but did not eliminate them, nor did it improve interlaboratory accuracy (22Xia J.Q. Sedransk N. Feng X. Variance Component Analysis of a Multi-Site Study for the Reproducibility of Multiple Reaction Monitoring Measurements of Peptides in Human Plasma.PLoS ONE. 2011; 6: e14590Crossref PubMed Scopus (9) Google Scholar). Recently, measures have been proposed to quantitatively monitor aspects of discovery-based proteomics approaches to better understand technical variability associated with chromatography, dynamic sampling, ion source configuration, signal intensity of MS and MS/MS scans, and peptide identification for data-dependent HPLC-MS/MS acquisitions (23Rudnick 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.L. 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. Witeaker 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: 225-241Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 24Bell A.W. Deutsch E.W. Au C.E. Kearney R.E. Beavis R. Sechi S. Nilsson T. Bergeron J.J. Group H.T.S.W. A HUPO test sample study reveals common problems in mass spectrometry-based proteomics.Nat. Methods. 2009; 6: 423-430Crossref PubMed Scopus (274) Google Scholar, 25Burkhart J.M. Premsler T. Sickmann A. Quality control of nano-LC-MS systems using stable isotope-coded peptides.Proteomics. 2011; 11: 1049-1057Crossref PubMed Scopus (26) Google Scholar, 26Briscoe C.J. Stiles M.R. Hage D.S. System Suitability in bioanalytical LC/MS/MS.J. Pharmaceutical Biomed. Anal. 2007; 44: 484-491Crossref PubMed Scopus (77) Google Scholar). Likewise, there is a critical need for standardized methods to demonstrate that LC-SID-MRM-MS analysis platforms are performing optimally. However, the approaches developed for discovery LC-MS/MS platform evaluation are not directly adaptable to LC-SID-MRM-MS systems, which have their own unique requirements for system performance assessment. In this context, we now describe the development and evaluation of an easy to implement system suitability protocol (SSP) to assess performance metrics of triple quadrupole-based nanoLC-SID-MRM-MS instrument configurations. In this investigation, 11 laboratories comprising 15 individual nanoLC-SID-MRM-MS platforms (from 4 different vendors, including 8 different models of mass spectrometers) participated in development and evaluation of an SSP, including peptide selection and platform-specific LC and MRM-MS optimization. Specific chromatographic and MS metrics including peak width, chromatographic resolution, peak capacity and the variability in peak area and retention time stability were monitored to assess reproducibility of replicate injections of a commercially available peptide mixture generated from trypsin digestion of 6 bovine proteins. Our study demonstrates that the SSP facilitated rapid detection, diagnosis and correction of system problems that were a source of performance degradation in terms of precision (CVs) and sensitivity (limits of detection and quantification, LOD/LOQ). A critical part of the study involved optimization and use of vendor-neutral data analysis tools, including Skyline (27MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2963) Google Scholar), for rapid assessment of reproducibility and data quality in individual laboratories. These tools were then implemented across multiple sites for comparison of variability among different platform configurations. The SSP was also shown to be of value in understanding sources of variation across multiple laboratories performing an identical experimental protocol as well as for tracking an individual instrument over an extended period of time. The effect of a marginally performing LC-SID-MRM-MS system on assay sensitivity, determined by calculating LOQ values for peptide targets, is illustrated and appropriate limits for all system suitability parameters monitored are proposed. An equimolar predigested “Bovine 6 Protein Mix” (P/N PTD/00001/63) was purchased from Bruker-Michrom, Inc. (Auburn, CA). ReproSil-Pur C18-AQ resin (3 μm particle size) was purchased from Dr. Maisch, GmbH (Ammerbuch-Entringen, Germany). Picofrit self-pack columns, 75 μm ID, 10 μm ID tip, were purchased from New Objective (Woburn, MA). The commercial “Bovine 6 Protein Mix” referred to as 6ProteinMix-QC is a trypsin predigested mixture containing beta lactoglobulin, lactoperoxidase, carbonic anhydrase, glutamate dehydrogenase, alpha casein, and serum albumin with each protein at an equimolar amount of 100 pmol per commercial vial (iodoacetic acid was used by the vendor to alkylate cysteine residues). The 6ProteinMix-QC aliquots were centrally prepared at Vanderbilt University by generating stock solutions with concentrations of 1 pmol/μl per protein using an aqueous solution of 30% acetonitrile (v/v) and 0.1% formic acid (v/v) for dissolution. The stock solutions were stored as 10 μl aliquots (at 1 pmol/μl concentration) at −80 °C, and shipped frozen on dry ice to the participating laboratories. As described in detail in the SOP (see supplementary Methods), before analysis individual sites further diluted the stock solution to a working solution of 50 fmol/μl using an aqueous diluent containing 3% acetonitrile (v/v) and 0.1% formic acid (v/v). MRM-MS transition lists were developed that could be applied to all 15 participating triple quadrupole mass spectrometers spanning four different vendors (AB Sciex, ThermoFisher Scientific, Waters and Agilent, for details see below). The selection of MRM transitions for each peptide was performed independently for each platform and aided by the use of Skyline software. Skyline was used to build spectral libraries from data dependent acquisitions and peptide search engine results using the BiblioSpec library builder (28Frewen B. MacCoss M.J. Using BiblioSpec for Creating and Searching Tandem MS Peptide Libraries.Current Protocols in Bioinformatics. John Wiley & Sons, Inc., 2007Google Scholar) so that MRM transitions could be selected based on previously acquired discovery platform data (for AB Sciex and Waters instruments). Alternatively (for Thermo and Agilent instruments), SRM Refinement approaches (29Bereman M.S. MacLean B. Tomazela D.M. Liebler D.C. MacCoss M.J. The development of selected reaction monitoring methods for targeted proteomics via empirical refinement.Proteomics. 2012; 12: 1134-1141Crossref PubMed Scopus (82) Google Scholar) were used on a triple quadrupole MS to determine optimal MRM transitions for each peptide. To select the five best transitions per peptide, spectral libraries for different instrument platform data formats were generated in Skyline, such as an “ABI spectral library” (obtained by data acquisition of the 6 ProteinMix-QC in data dependent IDA mode on a 4000 QTRAP), and a “Waters spectral library” (obtained by data acquisition of the 6 ProteinMix-QC in data dependent mode on a Waters QTOF Premier). For the QTRAP 5500 all transitions were selected to be below m/z 1,250 due to the instrument's upper mass limit of m/z 1250 in Q1 and Q3. For the ThermoFisher Scientific and Agilent platforms, optimal peptides and their corresponding transitions were determined by predicting all tryptic peptides from the 6 ProteinMix-QC and by monitoring all y-ions from y3 to yn-1 on the triple quadrupole TSQ Quantum-Ultra and Agilent 6460 (29Bereman M.S. MacLean B. Tomazela D.M. Liebler D.C. MacCoss M.J. The development of selected reaction monitoring methods for targeted proteomics via empirical refinement.Proteomics. 2012; 12: 1134-1141Crossref PubMed Scopus (82) Google Scholar). The raw data were imported into Skyline and results were refined to select the 5 most abundant transitions for all detectable peptides. From this refined peptide list, 22 peptides that were readily detectable between all MS platforms were selected for the final MS method. One peptide was included with an additional charge state, resulting in a total of 115 transitions. The final list of 22 bovine 6 Protein Mix signature peptides is listed in the SOP (see supplementary Methods). Finally, five different Skyline “Instrument Method templates” were generated, including all specific MRM transition information: (1) Study9S_ABI_MichromMix_template.sky (for 4000 QTRAP instruments), (2) Study9S_ABI5500_MichromMix_template.sky (for QTRAP 5500 instrument), (3) Study9S_Thermo_MichromMix_template.sky, (4) Study9S_Waters_MichromMix_template.sky, and v) Study9S_Agilent_MichromMix_template.sky. All Skyline Method templates were uploaded onto Panorama (panoramaweb.org, additional details below) and further details can be viewed in supplemental Table S1. Skyline Instrument Method templates were distributed to all sites and were used to export a transition list that could be directly imported into the mass spectrometer vendor method set-up (for detailed description see supplementary Methods). Peptide mixtures were separated by on-line reversed phase nanoHPLC systems equipped with autosamplers: two NanoLC-1D Plus systems, seven NanoLC-2D systems and two NanoLC_Ultra systems, one NanoLC_Ultra 1D Plus and one NanoLC_Ultra 2D Plus from Eksigent Technologies (Dublin, CA); one Ultimate 3000 system from Dionex (Sunnyvale, CA); one nanoAquity system (Waters, Milford, MA); and two 1100 series systems (Agilent, Santa Clara, CA). PicoFrit® (New Objective, Woburn," @default.
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• W2126156743 date "2013-09-01" @default.
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• W2126156743 title "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)" @default.
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• W2126156743 doi "https://doi.org/10.1074/mcp.m112.027078" @default.
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