Advanced Clinical Biosystems Institute, Heart Institute, Cedars Sinai Medical Center , Los Angeles, California 90048, United States.
Beckman Coulter Life Sciences, Indianapolis, Indiana 46268, United States.
J Proteome Res. 2018 Jan 5;17(1):420-428. doi: 10.1021/acs.jproteome.7b00623. Epub 2017 Nov 10.
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.
用于质谱法蛋白质定量的样品制备需要多个处理步骤，包括变性、还原、烷基化、蛋白酶消化和肽净化。对于分析众多复杂的生物样品，扩展这些程序可能会很繁琐和耗时，因为有许多液体转移步骤和定时反应，其中可能会引入和传播技术差异。我们建立了一个自动化的样品制备工作流程，96 个样品的总处理时间为 5 小时，其中包括 2 小时与胰蛋白酶孵育。肽净化是通过 LC/MS/MS 分析过程中的在线分流来完成的。在针对 6 种血浆生物标志物和β-半乳糖苷酶的靶向选择反应监测（SRM）测定中，5 个血清和 5 个血浆样本的日内和日间循环伏安图（CV）的平均值<20%，持续 5 天。在针对 70 多种蛋白质的高度多重化 SRM 测定中，6 个血浆样本在 3 个不同天重复的 90%的跃迁的总 CV 低于 20%。当工作流程转移到第二个地点时，也得到了类似的结果：93%的肽的 CV<20%。自动化胰蛋白酶消化工作流程可在不到 5 小时内均匀处理样品。在重复、天数、仪器和实验室地点都观察到肽的可重复定量，证明了这种方法的广泛适用性。
