Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Abbatiello SE

PROVIDER: S-EPMC4563721 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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

Abbatiello Susan E SE Schilling Birgit B Mani D R DR Zimmerman Lisa J LJ Hall Steven C SC MacLean Brendan B Albertolle Matthew M Allen Simon S Burgess Michael M Cusack Michael P MP Gosh Mousumi M Hedrick Victoria V Held Jason M JM Inerowicz H Dorota HD Jackson Angela A Keshishian Hasmik H Kinsinger Christopher R CR Lyssand John J Makowski Lee L Mesri Mehdi M Rodriguez Henry H Rudnick Paul P Sadowski Pawel P Sedransk Nell N Shaddox Kent K Skates Stephen J SJ Kuhn Eric E Smith Derek D Whiteaker Jeffery R JR Whitwell Corbin C Zhang Shucha S Borchers Christoph H CH Fisher Susan J SJ Gibson Bradford W BW Liebler Daniel C DC MacCoss Michael J MJ Neubert Thomas A TA Paulovich Amanda G AG Regnier Fred E FE Tempst Paul P Carr Steven A SA

Molecular & cellular proteomics : MCP 20150218 9

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 whi ...[more]

PMID: 25693799

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Dataset Information

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

Publications

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

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