Dayon Loïc, Núñez Galindo Antonio, Cominetti Ornella, Corthésy John, Kussmann Martin
Nestlé Institute of Health Sciences SA, EPFL Innovation Park, Bâtiment H, 1015, Lausanne, Switzerland.
Methods Mol Biol. 2017;1619:433-449. doi: 10.1007/978-1-4939-7057-5_30.
With recent technological developments, protein biomarker discoveries directly from blood have regained interest due to elevated feasibility. Mass spectrometry (MS)-based proteomics can now characterize human plasma proteomes to a greater extent than has ever been possible before. Such deep proteome coverage comes, however, with important limitations in terms of analysis time which is a critical factor in the case of clinical studies. As a consequence, compromises still need to be made to balance the proteome coverage with realistic analysis time frame in clinical research. The analysis of a sufficient number of samples is compulsory to empower statistically robust candidate biomarker findings. We have, therefore, recently developed a scalable automated proteomic pipeline (ASAP) to enable the proteomic analysis of large numbers of plasma and cerebrospinal fluid (CSF) samples, from dozens to a thousand of samples, with the latter number being currently processed in 15 weeks. A distinct characteristic of ASAP relies on the possibility to prepare samples in a highly automated way, mostly using 96-well plates. We describe herein a sample preparation procedure for human plasma that includes internal standard spiking, abundant protein removal, buffer exchange, reduction, alkylation, tryptic digestion, isobaric labeling, pooling, and sample purification. Other key elements of the pipeline (i.e., study design, sample tracking, liquid chromatography (LC) tandem MS (MS/MS), data processing, and data analysis) are also highlighted.
随着近期技术的发展，由于可行性提高，直接从血液中发现蛋白质生物标志物重新引起了人们的关注。基于质谱（MS）的蛋白质组学如今能够比以往任何时候都更全面地表征人类血浆蛋白质组。然而，如此深入的蛋白质组覆盖在分析时间方面存在重要限制，而分析时间在临床研究中是一个关键因素。因此，在临床研究中仍需做出妥协，以在蛋白质组覆盖范围与实际分析时间框架之间取得平衡。分析足够数量的样本对于获得具有统计学稳健性的候选生物标志物发现至关重要。因此，我们最近开发了一种可扩展的自动化蛋白质组学流程（ASAP），以实现对大量血浆和脑脊液（CSF）样本的蛋白质组分析，样本数量从几十到一千个不等，目前处理一千个样本需要15周时间。ASAP的一个显著特点是能够以高度自动化的方式制备样本，主要使用96孔板。本文描述了一种用于人类血浆的样本制备程序，包括内标加样、去除丰富蛋白质、缓冲液交换、还原、烷基化、胰蛋白酶消化、等压标记、合并和样本纯化。还强调了该流程的其他关键要素（即研究设计、样本跟踪、液相色谱（LC）串联质谱（MS/MS）、数据处理和数据分析）。
