Sample preparation for protein quantification by mass spectrometry requires multiple processing steps including denaturation, reduction, alkylation, protease digestion, and peptide cleanup. Scaling these procedures for the analysis of numerous complex biological samples can be tedious and time-consuming, as there are many liquid transfer steps and timed reactions where technical variations can be introduced and propagated. We established an automated sample preparation workflow with a total processing time for 96 samples of 5 h, including a 2 h incubation with trypsin. Peptide cleanup is accomplished by online diversion during the LC/MS/MS analysis. In a selected reaction monitoring (SRM) assay targeting 6 plasma biomarkers and spiked β-galactosidase, mean intraday and interday cyclic voltammograms (CVs) for 5 serum and 5 plasma samples over 5 days were <20%. In a highly multiplexed SRM assay targeting more than 70 proteins, 90% of the transitions from 6 plasma samples repeated on 3 separate days had total CVs below 20%. Similar results were obtained when the workflow was transferred to a second site: 93% of peptides had CVs below 20%. An automated trypsin digestion workflow yields uniformly processed samples in less than 5 h. Reproducible quantification of peptides was observed across replicates, days, instruments, and laboratory sites, demonstrating the broad applicability of this approach.

中文翻译：

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高度可重复的自动化蛋白质组学样品制备工作流程，用于定量质谱

用于通过质谱定量蛋白质的样品制备需要多个处理步骤，包括变性，还原，烷基化，蛋白酶消化和肽纯化。扩展这些程序以分析众多复杂的生物样品可能既乏味又耗时，因为存在许多液体转移步骤和定时反应，可以引入和传播技术变化。我们建立了一个自动样品制备工作流程，总处理时间为5小时的96个样品，其中包括与胰蛋白酶孵育2小时的时间。在LC / MS / MS分析过程中，通过在线转移可以完成肽的纯化。在针对6种血浆生物标志物和加标的β-半乳糖苷酶的选择性反应监测（SRM）分析中，5个血清和5个血浆样品在5天内的平均日间和日间循环伏安图（CV）<20％。在针对70多种蛋白质的高度多重SRM分析中，在3天中重复进行的6个血浆样品的90％转换的总CV低于20％。当将工作流程转移到第二个位置时，获得了相似的结果：93％的肽的CV低于20％。自动化的胰蛋白酶消化工作流程可在不到5小时的时间内产生均匀处理的样品。在重复，天数，仪器和实验室位置均观察到了可重复的多肽定量，这证明了该方法的广泛适用性。当将工作流程转移到第二个位置时，获得了相似的结果：93％的肽的CV低于20％。自动化的胰蛋白酶消化工作流程可在不到5小时的时间内产生均匀处理的样品。在重复，天数，仪器和实验室位置均观察到了可重复的多肽定量，这证明了该方法的广泛适用性。当将工作流程转移到第二个位置时，获得了相似的结果：93％的肽的CV低于20％。自动化的胰蛋白酶消化工作流程可在不到5小时的时间内产生均匀处理的样品。在重复，天数，仪器和实验室位置均观察到了可重复的多肽定量，这证明了该方法的广泛适用性。