Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany.
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University of Leipzig, Germany.
J Lipid Res. 2019 Apr;60(4):900-908. doi: 10.1194/jlr.D084301. Epub 2019 Feb 5.
Apolipoproteins are major structural and functional constituents of lipoprotein particles. As modulators of lipid metabolism, adipose tissue biology, and energy homeostasis, apolipoproteins may serve as biomarkers or potential therapeutic targets for cardiometabolic diseases. Mice are the preferred model to study metabolic disease and CVD, but a comprehensive method to quantify circulating apolipoproteins in mice is lacking. We developed and validated a targeted proteomics assay to quantify eight apolipoproteins in mice via proteotypic signature peptides and corresponding stable isotope-labeled analogs. The LC/MS/MS method requires only a 3 µl sample volume to simultaneously determine mouse apoA-I, apoA-II, apoA-IV, apoB-100, total apoB, apoC-I, apoE, and apoJ concentrations. ApoB-48 concentrations can be calculated by subtracting apoB-100 from total apoB. After we established the analytic performance (sensitivity, linearity, and imprecision) and compared results for selected apolipoproteins against immunoassays, we applied the method to profile apolipoprotein levels in plasma and isolated HDL from normocholesterolemic C57BL/6 mice and from hypercholesterolemic -receptor- and -deficient mice. In conclusion, we present a robust, quantitative LC/MS/MS method for the multiplexed analysis of eight apolipoproteins in mice. This assay can be applied to investigate the effects of genetic manipulation or dietary interventions on apolipoprotein levels in plasma and isolated lipoprotein fractions.
载脂蛋白是脂蛋白颗粒的主要结构和功能成分。作为脂质代谢、脂肪组织生物学和能量稳态的调节剂，载脂蛋白可以作为心血管代谢疾病的生物标志物或潜在治疗靶点。小鼠是研究代谢疾病和 CVD 的首选模型，但缺乏一种全面的方法来定量检测小鼠循环载脂蛋白。我们开发并验证了一种靶向蛋白质组学测定法，通过特征肽和相应的稳定同位素标记类似物来定量测定小鼠中的 8 种载脂蛋白。LC/MS/MS 方法仅需 3µl 样品体积即可同时测定小鼠载脂蛋白 A-I、载脂蛋白 A-II、载脂蛋白 A-IV、载脂蛋白 B-100、总载脂蛋白 B、载脂蛋白 C-I、载脂蛋白 E 和载脂蛋白 J 的浓度。载脂蛋白 B-48 的浓度可以通过从总载脂蛋白 B 中减去载脂蛋白 B-100 来计算。在建立分析性能（灵敏度、线性和精密度）并比较针对选定载脂蛋白的免疫测定结果后，我们将该方法应用于正常胆固醇的 C57BL/6 小鼠和高胆固醇的 -受体-和-缺陷型小鼠的血浆和分离的高密度脂蛋白中载脂蛋白水平的分析。总之，我们提出了一种强大的、定量的 LC/MS/MS 方法，用于在小鼠中同时分析 8 种载脂蛋白。该测定法可用于研究遗传操作或饮食干预对血浆和分离脂蛋白部分中载脂蛋白水平的影响。
