Increasing the Depth of Mass-Spectrometry-Based Structural Analysis of Protein Complexes through the Use of Multiple Cross-Linkers

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• 作者：Yue-He Ding ; Sheng-Bo Fan ; Shuang Li ; Bo-Ya Feng ; Ning Gao ; Keqiong Ye ; Si-Min He ; Meng-Qiu Dong
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：April 19, 2016
• 年：2016
• 卷：88
• 期：8
• 页码：4461-4469
• 全文大小：643K
• 年卷期：0
• ISSN：1520-6882

文摘

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS³ and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins. In order to find the cross-linkers that can best complement the performance of BS³ and DSS, we tested seven additional cross-linkers that either have different spacer arm structures or that target different amino acids (BS²G, EGS, AMAS, GMBS, Sulfo-GMBS, EDC, and TFCS). Using BSA, aldolase, the yeast H/ACA protein complex, and E. coli 70S ribosomes, we showed that, in terms of providing structural information not obtained through the use of BS³ and DSS, EGS and Sulfo-GMBS worked better than the other cross-linkers that we tested. EGS generated a large number of cross-links not seen with the other amine-specific cross-linkers, possibly due to its hydrophilic spacer arm. We demonstrate that incorporating the cross-links contributed by the EGS and amine-sulfhydryl cross-linkers greatly increased the accuracy of Rosetta in docking the structure of the yeast H/ACA protein complex. Given the improved depth of useful information it can provide, we suggest that the multilinker CXMS approach should be used routinely when the amount of a sample permits.