Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline
Lysine-specific chemical cross-linking of protein complexes and identification of cross-linking sites using LC-MS/MS and the xQuest/xProphet software pipeline
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DOI：
10.1038/nprot.2013.168
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发表时间：
2014-01-01
期刊：
影响因子：
14.8
通讯作者：
Aebersold, Ruedi
中科院分区：
文献类型：
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作者：
Leitner, Alexander;Walzthoeni, Thomas;Aebersold, Ruedi
化学交联与LC-MS/MS （XL-MS）相结合是一种新兴技术，用于获得蛋白质和蛋白质复合物的低分辨率结构（距离）约束。这些约束也可以通过对xml - ms数据的综合建模来表征蛋白质复合物，或者与其他类型的结构信息结合使用，或者单独使用，以建立蛋白质复合物中亚基的空间关系。在这里，我们提出了一个协议，已成功地用于从大量的天然蛋白质和蛋白质复合物生成xml - ms数据。它包括使用二琥珀酰亚基进行交联反应的实验步骤（一种同源双功能，lys氨酸反应性交联试剂），通过肽尺寸排除色谱（SEC；去除较小的非交联肽）对交联肽进行浓缩，串联质谱分析的说明以及通过开源计算软件管道xQuest和xProphet（可从http://proteomics.ethz.ch获得）对质谱数据进行分析。一旦建立，这个健壮的方案应该需要类似的4天来完成，并且通常适用于纯化的蛋白质和蛋白质复合物。
Chemical cross-linking in combination with LC-MS/MS (XL-MS) is an emerging technology to obtain low-resolution structural (distance) restraints of proteins and protein complexes. These restraints can also be used to characterize protein complexes by integrative modeling of the XL-MS data, either in combination with other types of structural information or by themselves, to establish spatial relationships of subunits in protein complexes. Here we present a protocol that has been successfully used to generate XL-MS data from a multitude of native proteins and protein complexes. It includes the experimental steps for performing the cross-linking reaction using disuccinimidyl suberate (a homobifunctional, lysine-reactive cross-linking reagent), the enrichment of cross-linked peptides by peptide size-exclusion chromatography (SEC; to remove smaller, non-cross-linked peptides), instructions for tandem MS analysis and the analysis of MS data via the open-source computational software pipeline xQuest and xProphet (available from http://proteomics.ethz.ch). Once established, this robust protocol should take similar to 4 d to complete, and it is generally applicable to purified proteins and protein complexes.