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Mol Cell Proteomics DOI:10.1074/mcp.M111.013854

Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma.

Publication TypeJournal Article
Year of Publication2012
AuthorsKuhn, E, Whiteaker, JR, Mani, DR, Jackson, AM, Zhao, L, Pope, ME, Smith, D, Rivera, KD, N Anderson, L, Skates, SJ, Pearson, TW, Paulovich, AG, Carr, SA
JournalMol Cell Proteomics
Date Published2012 Jun
KeywordsAnimals, Automation, Laboratory, Blood Proteins, Chromatography, Affinity, Granulocyte Colony-Stimulating Factor, Humans, Immunoprecipitation, Limit of Detection, Peptide Fragments, Rabbits, Reproducibility of Results, Tandem Mass Spectrometry

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 μl 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


Alternate JournalMol. Cell Proteomics
PubMed ID22199228
PubMed Central IDPMC3433918
Grant ListU24 CA160034 / CA / NCI NIH HHS / United States
R01 HL096738 / HL / NHLBI NIH HHS / United States
HHSN268201000033C / / PHS HHS / United States
HHSN268201000033C / HL / NHLBI NIH HHS / United States
U24 CA126476 / CA / NCI NIH HHS / United States
R01HL096738-02 / HL / NHLBI NIH HHS / United States
1U24CA160034 / CA / NCI NIH HHS / United States
1U24 CA126476-02 / CA / NCI NIH HHS / United States