Kuhn, E., Whiteaker, J. R., Mani, D. R., Jackson, A. M., Zhao, L., Pope, M. E., Smith, D., Rivera, K. D., Anderson, N. L., Skates, S. J., Pearson, T. W., Paulovich, A. G., Carr, S. A. (June 2012) Interlaboratory Evaluation of Automated, Multiplexed Peptide Immunoaffinity Enrichment Coupled to Multiple Reaction Monitoring Mass Spectrometry for Quantifying Proteins in Plasma. Molecular & Cellular Proteomics, 11 (6). ISSN 1535-9476
Abstract
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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | biomarker discovery quantification antibodies serum pipeline assays immunoassays quantitation validation verification |
| Subjects: | Investigative techniques and equipment Investigative techniques and equipment > spectroscopy > mass spectrometry Investigative techniques and equipment > spectroscopy |
| CSHL Authors: | |
| Communities: | CSHL labs > Pappin lab |
| Depositing User: | Matt Covey |
| Date: | June 2012 |
| Date Deposited: | 30 Jan 2013 17:03 |
| Last Modified: | 19 Jul 2021 20:44 |
| PMCID: | PMC3433918 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/26983 |
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