Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

Background: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid-throughput analysis for three blood-based proteomes: naïve plasma, plasma depleted of the 14 most abundant proteins, and dried blood. Methods: Optimal conditions for sample preparation and DIA-MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high-throughput or deep profile analysis with mid-throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of an 8-point dilution curve. Result: Using our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Naive plasma samples had the highest rates of identifications where a lower limit of detection or quantitation could be determined. Combining the analysis of both high-throughput plasma fractions exceed the number of reliably identified proteins for individual biofluids in the mid-throughput workflows. Conclusion: The workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.研究背景：精准的发现型检测工作流程是在多种血液基质中鉴定真实循环蛋白质生物标志物的关键。最大化不同生物流体间蛋白质组学工作流程的共性，可简化研究方法并提升重现性。我们开发了一套可适配高通量及中通量分析需求的工作流程，适用于三类血液来源的蛋白质组样本：天然血浆（naïve plasma）、去除14种丰度最高蛋白的血浆，以及干血样。 研究方法：首先在血浆中确立样本制备与数据非依赖采集-质谱（DIA-MS）分析的最优条件，随后将其自动化并适配至去除丰度蛋白的血浆及全血样本。本质谱工作流程可进行调整，以支持灵敏的高通量或深度覆盖分析，同时兼容中通量分析模式。分析性能通过3天内重复完成的5套完整工作流程，以及8点稀释曲线的线性分析进行评估。 研究结果：采用本高通量工作流程时，血浆、去除丰度蛋白的血浆及全血中分别有74%、93%、87%的肽段的批间变异系数（CV, Coefficient of Variation）小于30%。而采用中通量工作流程时，上述三类样本中分别有67%、90%、78%的肽段满足CV<30%的标准。天然血浆（naïve plasma）样本的蛋白鉴定率最高，可确定其检测下限或定量下限。将两类高通量血浆样本的分析结果合并后，可靠鉴定的蛋白数量超过了中通量工作流程中单种生物流体的可靠鉴定蛋白数。 研究结论：本研究所确立的工作流程可实现对宽动态范围蛋白的可靠检测。我们预计，将该标准化工作流程大规模应用，将助力候选标志物向临床应用的转化。