通过FASP消化和数据非依赖分析对EPS尿液进行蛋白质组学分析。

Proteomic Profile of EPS-Urine through FASP Digestion and Data-Independent Analysis.

作者信息

Prestagiacomo Licia E, Gabriele Caterina, Morelli Paola, Rota Maria Antonietta, Alba Stefano, Cuda Giovanni, Damiano Rocco, Gaspari Marco

机构信息

Research Centre for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro;

Research Centre for Advanced Biochemistry and Molecular Biology, Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro.

出版信息

J Vis Exp. 2021 May 8(171). doi: 10.3791/62512.

DOI:10.3791/62512

PMID:34028441

Abstract

Filter-aided sample protocol (FASP) is widely used for proteomics sample preparation because it allows to concentrate diluted samples and it is compatible with a wide variety of detergents. Bottom-up proteomics workflows like FASP increasingly rely on LC-MS/MS methods performed in data-independent analysis (DIA) mode, a scanning method that allows deep proteome coverage and low incidence of missing values. In this report, we will provide the details of a workflow that combines a FASP protocol, a double StageTip purification step and LC-MS/MS in DIA mode for urinary proteome mapping. As a model sample, we analyzed expressed prostatic secretions (EPS)-urine, a sample collected after a digital rectal exam (DRE), which is of interest in prostate cancer biomarker discovery studies.

摘要

过滤辅助样品制备方法（FASP）被广泛应用于蛋白质组学样品制备，因为它能够浓缩稀释后的样品，并且与多种去污剂兼容。像FASP这样的自下而上蛋白质组学工作流程越来越依赖于在数据非依赖采集（DIA）模式下执行的液相色谱-串联质谱（LC-MS/MS）方法，这是一种扫描方法，能够实现深度蛋白质组覆盖且缺失值发生率低。在本报告中，我们将详细介绍一种工作流程，该流程结合了FASP方法、双阶段Tip柱纯化步骤以及DIA模式下的LC-MS/MS，用于尿液蛋白质组图谱分析。作为模型样品，我们分析了前列腺按摩液（EPS）-尿液，这是一种在直肠指检（DRE）后采集的样品，在前列腺癌生物标志物发现研究中具有重要意义。