Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma

被引：139

作者：

Kuhn, Eric ^{[1
]}

Whiteaker, Jeffrey R. ^{[2
]}

Mani, D. R. ^{[1
]}

Jackson, Angela M. ^{[3
]}

Zhao, Lei ^{[2
]}

Pope, Matthew E.

Smith, Derek ^{[3
]}

Rivera, Keith D. ^{[5
]}

Anderson, N. Leigh ^{[6
]}

Skates, Steven J. ^{[7
]}

Pearson, Terry W. ^{[4
]}

Paulovich, Amanda G. ^{[2
]}

Carr, Steven A. ^{[1
]}

机构：

[1] Broad Inst MIT & Harvard, Cambridge, MA USA

[2] Fred Hutchinson Canc Res Ctr, Seattle, WA 98104 USA

[3] Univ Victoria, Genome British Columbia Prote Core Facil, Victoria, BC, Canada

[4] Univ Victoria, Dept Biochem & Microbiol, Victoria, BC, Canada

[5] Cold Spring Harbor Lab, Cold Spring Harbor, NY 11724 USA

[6] Plasma Proteome Inst, Washington, DC USA

[7] Massachusetts Gen Hosp, Ctr Biostat, Boston, MA 02114 USA

来源：

MOLECULAR & CELLULAR PROTEOMICS | 2012年 / 11卷 / 06期

基金：

美国国家卫生研究院;

关键词：

BIOMARKER DISCOVERY; QUANTIFICATION; ANTIBODIES; SERUM; PIPELINE; ASSAYS; IMMUNOASSAYS; QUANTITATION; VALIDATION; VERIFICATION;

D O I：

10.1074/mcp.M111.013854

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The inability to quantify large numbers of proteins in tissues and biofluids with high precision, sensitivity, and throughput is a major bottleneck in biomarker studies. We previously demonstrated that coupling immunoaffinity enrichment using anti-peptide antibodies (SISCAPA) to multiple reaction monitoring mass spectrometry (MRM-MS) produces Immunoprecipitation MRM-MS (immuno-MRM-MS) assays that can be multiplexed to quantify proteins in plasma with high sensitivity, specificity, and precision. Here we report the first systematic evaluation of the interlaboratory performance of multiplexed (8-plex) immuno-MRM-MS in three independent labs. A staged study was carried out in which the effect of each processing and analysis step on assay coefficient of variance, limit of detection, limit of quantification, and recovery was evaluated. Limits of detection were at or below 1 ng/ml for the assayed proteins in 30 mu l of plasma. Assay reproducibility was acceptable for verification studies, with median intra-and interlaboratory coefficients of variance above the limit of quantification of 11% and <14%, respectively, for the entire immuno-MRM-MS assay process, including enzymatic digestion of plasma. Trypsin digestion and its requisite sample handling contributed the most to assay variability and reduced the recovery of target peptides from digested proteins. Using a stable isotope-labeled protein as an internal standard instead of stable isotope-labeled pep-tides to account for losses in the digestion process nearly doubled assay accuracy for this while improving assay precision 5%. Our results demonstrate that multiplexed immuno-MRM-MS can be made reproducible across independent laboratories and has the potential to be adopted widely for assaying proteins in matrices as complex as plasma. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.013854, 1-14, 2012.

引用

页数：14

共 40 条

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