Large-scale interlaboratory study to develop, analytically validate and apply highly multiplexed, quantitative peptide assays to measure cancer-relevant proteins in plasma

Susan E. Abbatiello, Birgit Schilling, D. R. Mani, Lisa J. Zimmerman, Steven C. Hall, Brendan MacLean, Matthew Albertolle, Simon Allen, Michael Burgess, Michael P. Cusack, Mousumi Gosh, Victoria Hedrick, Jason M. Held, H. Dorota Inerowicz, Angela Jackson, Hasmik Keshishian, Christopher R. Kinsinger, John Lyssand, Lee Makowski, Mehdi MesriHenry Rodriguez, Paul Rudnick, Pawel Sadowski, Nell Sedransk, Kent Shaddox, Stephen J. Skates, Eric Kuhn, Derek Smith, Jeffery R. Whiteaker, Corbin Whitwell, Shucha Zhang, Christoph H. Borchers, Susan J. Fisher, Bradford W. Gibson, Daniel C. Liebler, Michael J. MacCoss, Thomas A. Neubert, Amanda G. Paulovich, Fred E. Regnier, Paul Tempst, Steven A. Carr

Research output: Contribution to journalArticlepeer-review

136 Scopus citations


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 intraand 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.

Original languageEnglish
Pages (from-to)2357-2374
Number of pages18
JournalMolecular and Cellular Proteomics
Issue number9
StatePublished - Sep 1 2015


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