Journal
CLINICAL CHEMISTRY
Volume 58, Issue 4, Pages 777-781Publisher
AMER ASSOC CLINICAL CHEMISTRY
DOI: 10.1373/clinchem.2011.173856
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Funding
- University of Washington (UW) Clinical Mass Spectrometry Facility and UW Proteomics Resource [UWPR95794]
- UW Nutrition and Obesity Research Center [NIH P30DK035816]
- UW Diabetes and Endocrinology Research Center [NIH P30DK017047]
- Waters
- Bruker-Daltonics
- NIH [HL77268, HL089504]
- Tobacco-Related Disease Research Program of California [18KT-0021]
- American Heart Association [0830231N]
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BACKGROUND: Mass spectrometric assays could potentially replace protein immunoassays in many applications. Previous studies have demonstrated the utility of liquid chromatography-multiple-reaction monitoring-mass spectrometry(LC-MRM/MS) for the quantification of proteins in biological samples, and many examples of the accuracy of these approaches to quantify supplemented analytes have been reported. However, a direct comparison of multiplexed assays that use LC-MRM/MS with established immunoassays to measure endogenous proteins has not been reported. METHODS: We purified HDL from the plasma of 30 human donors and used label-free shotgun proteomics approaches to analyze each sample. We then developed 2 different isotope-dilution LC-MRM/MS 6-plex assays (for apoliporoteins A-I, C-II, C-III, E, B, and J): 1 assay used stable isotope-labeled peptides and the other used stable isotope-labeled apolipoprotein A-I (an abundant HDL protein) as an internal standard to control for matrix effects and mass spectrometer performance. The shotgun and LC-MRM/MS assays were then compared with commercially available immunoassays for each of the 6 analytes. RESULTS: Relative quantification by shotgun proteomics approaches correlated poorly with the 6 protein immunoassays. In contrast, the isotope dilution LC-MRM/MS approaches showed correlations with immunoassays of r = 0.61-0.96. The LC-MRM/MS approaches had acceptable reproducibility (<13% CV) and linearity (r >= 0.99). Strikingly, a single protein internal standard applied to all proteins performed as well as multiple protein-specific peptide internal standards. CONCLUSIONS: Because peak area ratios measured in multiplexed LC-MRM/MS assays correlate well with immunochemical measurements and have acceptable operating characteristics, we propose that LC-MRM/MS could be used to replace immunoassays in a variety of settings.
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