Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states

eLife

Pan Zhang¹  Qiang Li²  Junjie Liu²  Hong-Wei Wang²  Xiangke Li²  Chengying Ma³  Chao Liu⁴  Xiaohui Liu⁵  Shoucai Ma⁵  Dan Tan⁵  Bing Yang⁵  Boya Feng⁶  Sheng-Bo Fan⁶  Meng-Qiu Dong⁷  Li Tao⁷  Xiaoguang Lei⁷  Si-Min He⁸  Mei-Jun Zhang⁸  Keqiong Ye⁸  Ning Gao⁸  Yue-He Ding⁸ 

[1] Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, China;National Institute of Biological Sciences, Beijing, China;Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China;Synthetic and Functional Biomolecules Center, Peking University, Beijing, China;Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China;Key Lab of Intelligent Information Processing of Chinese Academy of Sciences, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China;Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China;National Institute of Biological Sciences, Beijing, China;

关键词: cross-linking;    mass spectrometry;    protein structure;    protein-protein interactions;    70S ribosome;    exosome;   

DOI  :  10.7554/eLife.12509

来源: DOAJ

【 摘 要 】

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

【 授权许可】

Unknown