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Clin Chem. 2012 Apr;58(4):777-81. doi: 10.1373/clinchem.2011.173856. Epub 2012 Feb 3.

Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixtures.

Author information

1
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195-7110, USA. ahoof@u.washington.edu

Abstract

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.

PMID:
22307200
PMCID:
PMC3665768
DOI:
10.1373/clinchem.2011.173856
[Indexed for MEDLINE]
Free PMC Article

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