Plasma is a potentially rich source of protein biomarkers for disease progression and drug response. Large multi-center studies are often carried out to increase the number of samples analyzed in a given study. This may increase the chances of variation in blood processing and handling, leading to altered proteomic results. This study evaluates the impact of blood processing variation on LC–MS/MS proteomic analysis of plasma. Initially two batches of patient plasma samples (120 and 204 samples, respectively) were analyzed using LC–MS/MS shotgun proteomics. Follow-up experiments were designed and carried out on healthy donor blood in order to examine the effects of different centrifugation conditions, length of delay until first centrifugation, storage temperature and anticoagulant type on results from shotgun proteomics. Variable levels of intracellular proteins were observed in subsets of patient plasma samples from the initial batches analyzed. This observation correlated strongly with the site of collection, implicating variability in blood processing procedures. Results from the healthy donor blood analysis did not demonstrate a significant impact of centrifugation conditions to plasma proteome variation. The time delay until first centrifugation had a major impact on variability, while storage temperature and anticoagulant showed less pronounced but still significant effects. The intracellular proteins associated with study site effect in patient plasma samples were significantly altered by delayed processing also. Variable blood processing procedures contribute significantly to plasma proteomic variation and may give rise to increased intracellular proteins in plasma. Accounting for these effects can be important both at study design and data analysis stages. This understanding will be valuable to incorporate in the planning of protein-based biomarker discovery efforts in the future.

中文翻译：

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可变的血液处理程序会导致血浆蛋白质组变异

血浆是疾病进展和药物反应的蛋白质生物标志物的潜在丰富来源。通常会进行大型的多中心研究，以增加给定研究中分析的样本数量。这可能会增加血液处理和处理变化的机会，从而导致蛋白质组学结果发生变化。这项研究评估了血液处理变化对血浆LC-MS / MS蛋白质组学分析的影响。最初使用LC-MS / MS shot弹枪蛋白质组学分析了两批患者血浆样品（分别为120和204个样品）。设计并在健康的献血者血液上进行后续实验，以检查不同离心条件，首次离心的延迟时间，储存温度和抗凝剂类型对shot弹枪蛋白质组学结果的影响。在最初分析的患者血浆样本子集中观察到可变水平的细胞内蛋白质。该观察结果与采集部位密切相关，暗示血液处理程序的可变性。健康献血者血液分析的结果并未显示出离心条件对血浆蛋白质组变异的显着影响。直到首次离心的时间延迟对变异性有重大影响，而储存温度和抗凝剂的作用虽然不太明显，但仍然很明显。患者血浆样品中与研究部位效应相关的细胞内蛋白也因延迟处理而发生了显着变化。可变的血液处理程序会极大地影响血浆蛋白质组学变异，并可能导致血浆中细胞内蛋白质增加。在研究设计和数据分析阶段，考虑到这些影响都可能很重要。这种理解对于将来在基于蛋白质的生物标志物发现工作的规划中将是有价值的。