A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions
A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions
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DOI：
10.1038/s41596-018-0068-8
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发表时间：
2018-12-01
期刊：
影响因子：
14.8
通讯作者：
Sinz, Andrea
中科院分区：
文献类型：
--
作者：
Iacobucci, Claudio;Goetze, Michael;Sinz, Andrea
化学交联与所产生的交联产物的质谱分析相结合是一项新兴技术，旨在从蛋白质和蛋白质复合物中获取有价值的结构信息。我们协议的目标是获得蛋白质结构测定的距离约束并研究蛋白质-蛋白质相互作用。我们提出了一种用于交联/质谱 (MS) 的集成工作流程，该工作流程基于使用 MS 可裂解试剂进行蛋白质交联，然后进行酶消化、通过强阳离子交换色谱 (SCX) 富集交联肽以及 LC/MS/MS 分析。为了充分发挥 MS 可裂解交联剂的潜力，我们开发了免费的 MeroX 软件的更新版本，用于自动数据分析。本文使用的市售 MS 可裂解交联剂（DSBU 和 CDI）具有不同的长度并与胺和羟基反应。由于在 MS/MS 谱图中形成了两个特征性 26-u 双峰，因此与使用经典的不可裂解交联剂相比，发现的误报要少得多。本文以 BSA 和整个大肠杆菌核糖体为例的方案是稳健且广泛适用的，并且它允许轻松识别交联，以从纯化的蛋白质和蛋白质复合物中导出空间约束。交联/MS 程序需要 2-3 天才能完成。
Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2-3 days to complete.