On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins

Apolipoproteins are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). Quantitative measurement of apolipoproteins in serum or plasma by LC-IDMS allows the analysis of numerous proteins in the same sample run. However, the accuracy and precision of the IDMS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight different apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV < 4.4% and inter-assay CV < 8%. In addition, the method was compared with traditional homogeneous digestion coupled IDMS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application.217