Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids
Standardized Workflow for Precise Mid- and High-Throughput Proteomics of Blood Biofluids
复制标题
DOI：
10.1093/clinchem/hvab202
复制
发表时间：
2021-10-23
影响因子：
9.3
通讯作者：
Van Eyk, Jennifer E.
中科院分区：
文献类型：
--
作者：
Mc Ardle, Angela;Binek, Aleksandra;Van Eyk, Jennifer E.
准确的发现测定工作流程对于鉴定不同血液基质中的真实循环蛋白质生物标志物至关重要。最大化不同生物流体之间蛋白质组学工作流程的共性简化了方法并增加了再现性的可能性。我们开发了一个工作流程，可以适应3个血液蛋白质组：幼稚血浆，耗尽血浆和干血。方法在血浆中建立最佳的样品制备和数据独立采集-质谱分析条件，然后对贫化血浆和干血进行自动化分析。对质谱工作流程进行了修改，以促进灵敏的高通量分析或更深入的中通量分析。通过肽和蛋白质对6点或7点稀释曲线的线性响应以及每种生物流体在3个不同日期每天5次消化重复的相对肽和蛋白质强度的再现性来评价分析性能。结果使用高通量工作流程，74%（血浆）、93%（贫化）和87%（干血）显示日间CV
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 can accommodate 3 blood-based proteomes: naive plasma, depleted plasma and dried blood. Methods Optimal conditions for sample preparation and data independent acquisition-mass spectrometry analysis were established in plasma then automated for depleted plasma and dried blood. The mass spectrometry workflow was modified to facilitate sensitive high-throughput analysis or deeper profiling with mid-throughput analysis. Analytical performance was evaluated by the linear response of peptides and proteins to a 6- or 7-point dilution curve and the reproducibility of the relative peptide and protein intensity for 5 digestion replicates per day on 3 different days for each biofluid. Results Using the high-throughput workflow, 74% (plasma), 93% (depleted), and 87% (dried blood) displayed an inter-day CV