基于MS可裂解的交联剂和MeroX软件的交联/质谱分析工作流程，用于研究蛋白质结构和蛋白质-蛋白质相互作用。,Nature Protocols

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基于MS可裂解的交联剂和MeroX软件的交联/质谱分析工作流程，用于研究蛋白质结构和蛋白质-蛋白质相互作用。
Nature Protocols ( IF 13.1 ) Pub Date : 2018-Dec-01 , DOI: 10.1038/s41596-018-0068-8
Claudio Iacobucci , Michael Götze , Christian H. Ihling , Christine Piotrowski , Christian Arlt , Mathias Schäfer , Christoph Hage , Rico Schmidt , Andrea Sinz

化学交联与对所产生的交联产物进行质谱分析相结合是一种新兴技术，旨在从蛋白质和蛋白质复合物中获得有价值的结构信息。我们协议的目标是获得距离限制，以测定蛋白质的结构并研究蛋白质之间的相互作用。我们提出了一种基于蛋白质与MS可裂解试剂进行交联的交联/质谱（MS）集成工作流程，然后进行酶消化，通过强阳离子交换色谱（SCX）和LC富集交联肽/ MS / MS分析。为了充分利用MS可裂解的交联剂的潜力，我们开发了免费版本的MeroX软件的更新版本，用于自动数据分析。市售的本文使用的MS可裂解的交联剂（DSBU和CDI）具有不同的长度，并且与胺以及羟基反应。由于在其MS / MS光谱中形成了两个特征性的26-u双峰，因此与使用经典的不可裂解的交联剂相比，发现的假阳性少得多。该协议在本文中以BSA和整个大肠埃希氏菌核糖体为例，是可靠且广泛适用的，它允许轻松识别交联键，以从纯化的蛋白质和蛋白质复合物中获得空间限制。交联/ MS过程需要2-3天才能完成。不可裂解的交联剂。该协议在本文中以BSA和整个大肠埃希氏菌核糖体为例，是可靠且广泛适用的，它允许轻松识别交联键，以从纯化的蛋白质和蛋白质复合物中获得空间限制。交联/ MS过程需要2-3天才能完成。不可裂解的交联剂。该协议在本文中以BSA和整个大肠埃希氏菌核糖体为例，是可靠且广泛适用的，它允许轻松识别交联键，以从纯化的蛋白质和蛋白质复合物中获得空间限制。交联/ MS过程需要2-3天才能完成。

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A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions.

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.

更新日期：2019-01-26

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