Unit of Cardiovascular and Renal Research, Department of Molecular Medicine, University of Southern Denmark, 5000 Odense C, Denmark.
Department of Clinical Biochemistry, Odense University Hospital, 5000 Odense C, Denmark.
J Proteome Res. 2023 May 5;22(5):1385-1393. doi: 10.1021/acs.jproteome.2c00189. Epub 2022 Jun 14.
Atherosclerotic cardiovascular disease is the leading cause of death worldwide. For decades, mouse modeling of atherosclerosis has been the mainstay for preclinical testing of genetic and pharmacological intervention. Mouse models of atherosclerosis depend on supraphysiological levels of circulating cholesterol carried in lipoprotein particles. Lipoprotein particles vary in atherogenicity, and it is critical to monitor lipoprotein levels during preclinical interventions in mice. Unfortunately, the small plasma volumes typically harvested during preclinical experiments limit analyses to measuring total cholesterol and triglyceride levels. Here we developed a high-throughput, low-cost targeted multiple reaction monitoring (MRM) stable isotope dilution (SID) mass spectrometry assay for simultaneous relative quantification of nine apolipoproteins using a few microliters of mouse plasma. We applied the MRM assay to investigate the plasma apolipoproteome of two atherosclerosis models: the widely used ApoE knockout model and the emerging recombinant adeno-associated virus-mediated hepatic Pcsk9 overexpression model. By applying the assay on size-exclusion chromatography-separated plasma pools, we provide in-depth characterization of apolipoprotein distribution across lipoprotein species in these models, and finally, we use the assay to quantify apolipoprotein deposition in mouse atherosclerotic plaques. Taken together, we report development and application of an MRM assay that can be adopted by fellow researchers to monitor the mouse plasma apolipoproteome during preclinical investigations.
动脉粥样硬化性心血管疾病是全球范围内的主要死亡原因。几十年来，动脉粥样硬化的小鼠模型一直是遗传和药物干预的临床前测试的主要方法。动脉粥样硬化的小鼠模型依赖于脂蛋白颗粒中载脂蛋白颗粒的超生理水平循环胆固醇。脂蛋白颗粒的致动脉粥样硬化性不同，在小鼠的临床前干预期间监测脂蛋白水平至关重要。不幸的是，临床前实验中通常采集的小血浆量限制了分析只能测量总胆固醇和甘油三酯水平。在这里，我们开发了一种高通量、低成本的靶向多重反应监测（MRM）稳定同位素稀释（SID）质谱测定法，使用几微升小鼠血浆即可同时相对定量 9 种载脂蛋白。我们应用 MRM 测定法研究了两种动脉粥样硬化模型的血浆载脂蛋白组：广泛使用的 ApoE 敲除模型和新兴的重组腺相关病毒介导的肝 Pcsk9 过表达模型。通过应用该测定法对尺寸排阻色谱分离的血浆池进行分析，我们深入描述了这些模型中载脂蛋白在脂蛋白种类中的分布情况，最后，我们使用该测定法定量了小鼠动脉粥样硬化斑块中的载脂蛋白沉积。总之，我们报告了一种 MRM 测定法的开发和应用，该测定法可以被同行研究人员采用，以在临床前研究中监测小鼠血浆载脂蛋白组。
