Department of Biomedicine, Aarhus University, Høegh-Guldbergsgade 10, 8000 Aarhus C, Denmark.
Central Institute for Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany.
Anal Chem. 2021 Apr 6;93(13):5596-5605. doi: 10.1021/acs.analchem.1c00310. Epub 2021 Mar 17.
Site-specific proteolytic processing is an important, irreversible post-translational protein modification with implications in many diseases. Enrichment of protein N-terminal peptides followed by mass spectrometry-based identification and quantification enables proteome-wide characterization of proteolytic processes and protease substrates but is challenged by the lack of specific annotation tools. A common problem is, for example, ambiguous matches of identified peptides to multiple protein entries in the databases used for identification. We developed MaxQuant Advanced N-termini Interpreter (MANTI), a standalone Perl software with an optional graphical user interface that validates and annotates N-terminal peptides identified by database searches with the popular MaxQuant software package by integrating information from multiple data sources. MANTI utilizes diverse annotation information in a multistep decision process to assign a conservative preferred protein entry for each N-terminal peptide, enabling automated classification according to the likely origin and determines significant changes in N-terminal peptide abundance. Auxiliary R scripts included in the software package summarize and visualize key aspects of the data. To showcase the utility of MANTI, we generated two large-scale TAILS N-terminome data sets from two different animal models of chemically and genetically induced kidney disease, puromycin adenonucleoside-treated rats (PAN), and heterozygous Wilms Tumor protein 1 mice (WT1). MANTI enabled rapid validation and autonomous annotation of >10 000 identified terminal peptides, revealing novel proteolytic proteoforms in 905 and 644 proteins, respectively. Quantitative analysis indicated that proteolytic activities with similar sequence specificity are involved in the pathogenesis of kidney injury and proteinuria in both models, whereas coagulation processes and complement activation were specifically induced after chemical injury.
位点特异性蛋白水解加工是一种重要的、不可逆的翻译后蛋白修饰，与许多疾病都有关联。通过富集蛋白 N 端肽段，然后基于质谱的鉴定和定量，可以实现对蛋白水解过程和蛋白酶底物的全蛋白质组学特征分析，但这受到缺乏特异性注释工具的挑战。一个常见的问题是，例如，鉴定出的肽段与用于鉴定的数据库中多个蛋白条目之间存在模糊匹配。我们开发了 MaxQuant Advanced N-termini Interpreter（MANTI），这是一个独立的 Perl 软件，带有可选的图形用户界面，可以通过整合来自多个数据源的信息，对流行的 MaxQuant 软件包进行数据库搜索鉴定的 N 端肽进行验证和注释。MANTI 在一个多步骤决策过程中利用多种注释信息，为每个 N 端肽分配一个保守的首选蛋白条目，从而能够根据可能的来源自动分类，并确定 N 端肽丰度的显著变化。软件包中包含的辅助 R 脚本可对数据的关键方面进行总结和可视化。为了展示 MANTI 的实用性，我们从两种不同的化学和基因诱导的肾脏疾病动物模型（嘌呤霉素腺嘌呤核苷处理的大鼠（PAN）和杂合性 Wilms 瘤蛋白 1 小鼠（WT1））中生成了两个大规模的 TAILS N 端蛋白质组数据集。MANTI 能够快速验证和自主注释超过 10000 个鉴定出的末端肽，分别在 905 种和 644 种蛋白质中揭示了新的蛋白水解蛋白形式。定量分析表明，具有相似序列特异性的蛋白水解活性参与了两种模型中的肾脏损伤和蛋白尿的发病机制，而化学损伤后特异性诱导了凝血过程和补体激活。
