RESUMO
Laborious sample pretreatment of biological samples represents the most limiting factor for the translation of targeted proteomics assays from research to clinical routine. An optimized method for the simultaneous quantitation of 12 major apolipoproteins (apos) combining on-line SPE and fast LC-MS/MS analysis in 6.5 min total run time was developed, reducing the manual sample pretreatment time of 3 µL serum or plasma by 60%. Within-run and between-day imprecisions below 10 and 15% (n = 10) and high recovery rates (94-131%) were obtained applying the high-throughput setup. High-quality porcine trypsin was used, which outperformed cost-effective bovine trypsin regarding digestion efficiency. Comparisons with immunoassays and another LC-MS/MS assay demonstrated good correlation (Pearson's R: 0.81-0.98). Further, requirements on sample quality concerning sampling, processing, and long-term storage up to 1 year were investigated revealing significant influences of the applied sampling material and coagulant on quantitation results. Apo profiles of 1339 subjects of the LIFE-Adult-Study were associated with lifestyle and physiological parameters as well as establish parameters of lipid metabolism (e.g., triglycerides, cholesterol). Besides gender effects, most significant impact was seen regarding lipid-lowering medication. In conclusion, this novel highly standardized, high-throughput targeted proteomics assay utilizes a fast, simultaneous analysis of 12 apos from least sample amounts.