Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Réunion Océan Indien (DéTROI), Plateforme CYROI, Saint-Denis de La Réunion, France. Electronic address: mailto:
L'Institut du Thorax, INSERM, CNRS, University of Nantes, Nantes, France.
J Lipid Res. 2020 Jul;61(7):1128-1139. doi: 10.1194/jlr.D120000835. Epub 2020 May 13.
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.
载脂蛋白控制脂蛋白代谢，是代谢和心血管疾病有前途的生物标志物。与免疫测定不同，MS 能够定量和表型分析多种载脂蛋白。因此，在这里，我们旨在开发一种 LC-MS/MS 测定法，该方法可以同时定量测定 18 种人类载脂蛋白 [A-I、A-II、A-IV、A-V、B48、B100、C-I、C-II、C-III、C-IV、C-IV、D、E、F、H、J、L1、M 和 (a)]，并确定人血浆和血清样品中的 apoE、apoL1 和 apo(a)表型。通过优化的程序将血浆和血清载脂蛋白用胰蛋白酶消化，并通过 LC-MS/MS 提取和分析肽。该方法按照标准指南在添加已知肽量的样品中进行验证。将 LC-MS/MS 结果与其他技术获得的结果进行比较，还评估了重现性、稀释效应和稳定性。成功地选择了肽标记物用于靶向载脂蛋白定量和表型分析。经过优化，该测定法的线性、定量下限、准确度（偏差：-14.8%至 12.1%）、日内变异系数（CVs：1.5-14.2%）和日间重复性（CVs：4.1-14.3%）均得到验证。Bland-Altman 图表明 LC-MS/MS 与其他技术之间没有显著的统计学差异。在五个重复实验中，LC-MS/MS 结果具有重现性（CVs：1.8-13.7%），并且我们发现血浆和血清样品之间存在明显差异。这里开发的 LC-MS/MS 测定法快速，仅需要小的采样量，且成本合理，因此适用于广泛的载脂蛋白代谢研究。我们还强调了该测定法如何在实验室中实施。
