The Fifth People Hospital and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Fudan University, Shanghai, 200433, China.
Anal Chem. 2021 Jan 26;93(3):1578-1585. doi: 10.1021/acs.analchem.0c03985. Epub 2020 Dec 29.
Fast, robust, and high-throughput mass spectrometry-based serum proteomic pipelines have great potential to yield information for biomarker discovery and daily clinical practice. Here, we developed a simple and rapid sample preparation (RSP) workflow by reducing the classical pretreatment time from overnight to less than 1.5 h in an ordinary system. In HeLa cell lysates and serum samples, the number of proteins and tryptic peptides generated using the RSP was comparable to that generated using conventional methods. For fast scanning of the serum proteome, the RSP-supported pipeline could complete a test in less than 2 h with 30 min of LC-MS/MS analysis. Nearly 390 proteins spanning 8 magnitudes of abundance range were identified with high reproducibility, containing over 90 cancer-associated proteins and over 50 FDA-approved biomarkers. For fast assay development, eight candidate biomarker peptides for cardiovascular disease (CVD) were quantified by MRM with high accuracy (CV% <10). After a simple highly abundant protein removal, a deep serum proteome of over 1400 proteins was reached. By analyzing the depleted serum in DIA acquisition mode, over 700 proteins were quantified. The differentially expressed proteins could help us unambiguously distinguish the serum samples from healthy people and patients with pancreatic cancer (PC). Potential biomarkers for PC were also found. The new RSP method, which is rapid and simple, meets the demands of both deep mining and fast analysis of serum proteins. We believe that it will be widely used in serum protein studies and accelerate the transformation from biomarker discovery to clinical application.
基于快速、稳健和高通量的质谱血清蛋白质组学分析方法具有为生物标志物发现和日常临床实践提供信息的巨大潜力。在这里，我们通过将经典的预处理时间从一整夜减少到不到 1.5 小时，在普通系统中开发了一种简单而快速的样品制备 (RSP) 工作流程。在 HeLa 细胞裂解物和血清样本中，使用 RSP 生成的蛋白质和胰蛋白酶肽数量与使用传统方法生成的数量相当。为了快速扫描血清蛋白质组，RSP 支持的工作流程可以在不到 2 小时的时间内完成测试，其中包括 30 分钟的 LC-MS/MS 分析。使用 RSP 支持的工作流程可以以高重复性鉴定近 390 种跨越 8 个丰度范围的蛋白质，其中包含超过 90 种癌症相关蛋白质和超过 50 种 FDA 批准的生物标志物。为了快速开发检测方法，通过 MRM 对 8 种候选心血管疾病 (CVD) 生物标志物肽进行了高精度定量 (CV%<10)。在简单地去除高丰度蛋白质后，可达到超过 1400 种蛋白质的深度血清蛋白质组。通过在 DIA 采集模式下分析耗尽的血清，可以定量超过 700 种蛋白质。差异表达的蛋白质可以帮助我们明确区分健康人和胰腺癌 (PC) 患者的血清样本。还发现了用于 PC 的潜在生物标志物。这种新的 RSP 方法快速而简单，满足了血清蛋白质的深度挖掘和快速分析的需求。我们相信它将在血清蛋白质研究中得到广泛应用，并加速从生物标志物发现到临床应用的转化。
