Abstract
Apolipoproteins govern lipoprotein metabolism and are promising biomarkers of metabolic and cardiovascular diseases. Unlike immunoassays, MS enables the quantification and phenotyping of multiple apolipoproteins. Hence, here, we aimed to develop a LC-MS/MS assay that can simultaneously quantitate 18 human apolipoproteins [A-I, A-II, A-IV, A-V, B48, B100, C-I, C-II, C-III, C-IV, D, E, F, H, J, L1, M, and (a)] and determined apoE, apoL1, and apo(a) phenotypes in human plasma and serum samples. The plasma and serum apolipoproteins were trypsin digested through an optimized procedure and peptides were extracted and analyzed by LC-MS/MS. The method was validated according to standard guidelines in samples spiked with known peptide amounts. The LC-MS/MS results were compared with those obtained with other techniques, and reproducibility, dilution effects, and stabilities were also assessed. Peptide markers were successfully selected for targeted apolipoprotein quantification and phenotyping. After optimization, the assay was validated for linearity, lower limits of quantification, accuracy (biases: -14.8% to 12.1%), intra-assay variability [coefficients of variation (CVs): 1.5-14.2%], and inter-assay repeatability (CVs: 4.1-14.3%). Bland-Altman plots indicated no major statistically significant differences between LC-MS/MS and other techniques. The LC-MS/MS results were reproducible over five repeated experiments (CVs: 1.8-13.7%), and we identified marked differences among the plasma and serum samples. The LC-MS/MS assay developed here is rapid, requires only small sampling volumes, and incurs reasonable costs, thus making it amenable for a wide range of studies of apolipoprotein metabolism. We also highlight how this assay can be implemented in laboratories.
Highlights
Apolipoproteins govern lipoprotein metabolism and are promising biomarkers of metabolic and cardiovascular diseases
Selection of peptide markers In silico investigations led to the identification of numerous peptide candidates per target apolipoprotein
Peptide candidates were primarily detected as doubly charged precursor ions, except for apoA-I
Summary
Apolipoproteins govern lipoprotein metabolism and are promising biomarkers of metabolic and cardiovascular diseases. Here, we aimed to develop a LC-MS/MS assay that can simultaneously quantitate 18 human apolipoproteins [A-I, A-II, A-IV, A-V, B48, B100, C-I, C-II, C-III, C-IV, D, E, F, H, J, L1, M, and (a)] and determined apoE, apoL1, and apo(a) phenotypes in human plasma and serum samples. The plasma and serum apolipoproteins were trypsin digested through an optimized procedure and peptides were extracted and analyzed by LC-MS/MS. Peptide markers were successfully selected for targeted apolipoprotein quantification and phenotyping. The LC-MS/MS results were reproducible over five repeated experiments (CVs: 1.8–13.7%), and we identified marked differences among the plasma and serum samples. ApoB48, apoCs, and apoE have become predominant markers of CVD-related risk in this emerging field [4, 8]. The phenotyping of the major apoE isoforms and other apolipoproteins or apolipoprotein-like proteins [1, 2, 4, 5, 9] is used to diagnose mixed dyslipidemias [10,11,12]
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