Quantitative targeted proteomics with a protein internal standrd for analysis of HDL as carrier of biomarkers of cardiovascular disease.

摘要

Mass spectrometric assays have the potential to replace protein immunoassays in many applications. Previous studies have demonstrated the utility of multiple-reaction monitoring mass spectrometry (MRM-MS) for the quantification of proteins in biological samples and many examples of the accuracy of these approaches to quantify spiked analytes have been reported. However, a direct comparison of multiplexed assays using MRM-MS with established immunoassays to measure endogenous proteins has not been reported. We developed two different isotope dilution MRM-MS multiplex assays and validated them for apolipoproteins A-I, C-II, C-III, E, B, and J): one assay used stable isotope labeled peptides (Is_(pep)) and the other used stable isotope labeled apolipoprotein A-I (apoA-I, an abundant HDL protein)(IS_(prot)), as internal standards to control for matrix effects and mass spectrometer performance. Both IS_(prot) and IS_(pep) methods performed equally well against biochemical assays (CV<12%, R=0.7-0.96). We therefore selected the IS_(prot) method, extended it to 45 HDL proteins and applied it to samples from a clinical study of people with cardiovascular disease and people with acute coronary syndrome. The results showed that HDL carries specific modules of proteins and that these modules are differentially altered in acute coronary syndrome and in stable CAD compared to healthy.