ProteaseGuru: A Tool for Protease Selection in Bottom-Up Proteomics

Bottom-up proteomics is currently the dominant strategy for proteome analysis. It relies critically upon the use of a protease to digest proteins into peptides, which are then identified by liquid chromatography–mass spectrometry (LC-MS). The choice of protease(s) has a substantial impact upon the utility of the bottom-up results obtained. Protease selection determines the nature of the peptides produced, which in turn affects the ability to infer the presence and quantities of the parent proteins and post-translational modifications in the sample. We present here the software tool ProteaseGuru, which provides in silico digestions by candidate proteases, allowing evaluation of their utility for bottom-up proteomic experiments. This information is useful for both studies focused on a single or small number of proteins, and for analysis of entire complex proteomes. ProteaseGuru provides a convenient user interface, valuable peptide information, and data visualizations enabling the comparison of digestion results of different proteases. The information provided includes data tables of theoretical peptide sequences and their biophysical properties, results summaries outlining the numbers of shared and unique peptides per protease, histograms facilitating the comparison of proteome-wide proteolytic data, protein-specific summaries, and sequence coverage maps. Examples are provided of its use to inform analysis of variant-containing proteins in the human proteome, as well as for studies requiring the use of multiple proteomic databases such as a human:mouse xenograft model, and microbiome metaproteomics.自下而上蛋白质组学（bottom-up proteomics）目前是蛋白质组分析的主流研究策略。该技术核心依赖于使用蛋白酶（protease）将蛋白质酶解为肽段，随后通过液相色谱-质谱联用（LC-MS）对肽段进行鉴定。蛋白酶的选择对所得自下而上蛋白质组学结果的实用性具有显著影响：蛋白酶的选择决定了所生成肽段的性质，进而影响对样本中母本蛋白质的存在、丰度以及翻译后修饰（post-translational modifications）的推断能力。本文介绍了一款名为ProteaseGuru的软件工具，该工具可通过候选蛋白酶开展计算机模拟（in silico）酶解，从而评估其在自下而上蛋白质组学实验中的应用价值。该工具的信息不仅适用于以单个或少量蛋白质为研究对象的实验，也可用于完整复杂蛋白质组的分析。ProteaseGuru配备便捷的用户界面，可提供高价值的肽段信息与数据可视化功能，支持不同蛋白酶酶解结果的对比分析。其所提供的信息包括：理论肽段序列及其生物物理特性的数据表、概述每种蛋白酶的共有肽段与独有肽段数量的结果汇总、用于对比全蛋白质组酶解数据的直方图、蛋白质特异性分析摘要以及序列覆盖度图谱。本文还提供了该工具的应用实例，包括用于分析人类蛋白质组中携带变异的蛋白质，以及针对需要使用多套蛋白质组数据库的研究——例如人-小鼠异种移植模型（xenograft model）和微生物组宏蛋白质组学（microbiome metaproteomics）的相关研究。