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

• DOI: 10.1074/mcp.m111.013854
• Publication Date: 2011-12-23T06:04:31Z
• Authors: Eric Kuhn; Jeffrey R. Whiteaker; D.R. Mani; Angela M. Jackson; Lei Zhao; Matthew E. Pope; Derek Smith; Keith D. Rivera; N. Leigh Anderson; Steven J. Skates; Terry W. Pearson; Amanda G. Paulovich; Steven A. Carr
• Keywords: Automation, Laboratory; 0303 health sciences; Reproducibility of Results; Blood Proteins; Chromatography, Affinity; Peptide Fragments; 03 medical and health sciences; Limit of Detection; Tandem Mass Spectrometry; Granulocyte Colony-Stimulating Factor; Animals; Humans; Immunoprecipitation; Rabbits

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