Clinical Protein Science & Imaging, Department of Biomedical Engineering, Lund University , BMC D13, Lund SE-221 84, Sweden.
Department of Drug Metabolism, H Lundbeck & Co AS , Copenhagen, DK-2500, Denmark.
J Proteome Res. 2017 Sep 1;16(9):3242-3254. doi: 10.1021/acs.jproteome.7b00235. Epub 2017 Aug 8.
A multiple reaction monitoring (MRM) assay was developed for precise quantitation of 87 plasma proteins including the three isoforms of apolipoprotein E (APOE) associated with cardiovascular diseases using nanoscale liquid chromatography separation and stable isotope dilution strategy. The analytical performance of the assay was evaluated and we found an average technical variation of 4.7% in 4-5 orders of magnitude dynamic range (≈0.2 mg/L to 4.5 g/L) from whole plasma digest. Here, we report a complete workflow, including sample processing adapted to 96-well plate format and normalization strategy for large-scale studies. To further investigate the MS-based quantitation the amount of six selected proteins was measured by routinely used clinical chemistry assays as well and the two methods showed excellent correlation with high significance (p-value < 10e-5) for the six proteins, in addition for the cardiovascular predictor factor, APOB: APOA1 ratio (r = 0.969, p-value < 10e-5). Moreover, we utilized the developed assay for screening of biobank samples from patients with myocardial infarction and performed the comparative analysis of patient groups with STEMI (ST- segment elevation myocardial infarction), NSTEMI (non ST- segment elevation myocardial infarction) and type-2 AMI (type-2 myocardial infarction) patients.
建立了一种多重反应监测（MRM）分析方法，用于使用纳升液相色谱分离和稳定同位素稀释策略，对与心血管疾病相关的 87 种血浆蛋白（包括三种载脂蛋白 E（APOE）同工型）进行精确定量。评估了分析方法的性能，我们发现整个血浆消化物在 4-5 个数量级的动态范围内（约 0.2 mg/L 至 4.5 g/L）的平均技术变异为 4.7%。在这里，我们报告了一个完整的工作流程，包括适应 96 孔板格式的样品处理和用于大规模研究的归一化策略。为了进一步研究基于 MS 的定量，还通过常规使用的临床化学分析方法测量了六个选定蛋白的量，这两种方法显示出出色的相关性，对于六个蛋白的相关性具有高度显著性（p 值 < 10e-5），对于心血管预测因子 APOB:APOA1 比值也是如此（r = 0.969，p 值 < 10e-5）。此外，我们利用开发的分析方法筛选了心肌梗死患者的生物库样本，并对 ST 段抬高型心肌梗死（STEMI）、非 ST 段抬高型心肌梗死（NSTEMI）和 2 型 AMI（2 型心肌梗死）患者的患者组进行了比较分析。
