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

AN Hoofnagle, JO Becker, MN Oda… - Clinical …, 2012 - academic.oup.com
AN Hoofnagle, JO Becker, MN Oda, G Cavigiolio, P Mayer, T Vaisar
Clinical chemistry, 2012academic.oup.com
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 …
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.
Oxford University Press