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

• DOI: 10.7554/elife.12509
• Publication Date: 2016-03-03T12:30:04Z
• Authors: Dan Tan; Qiang Li; Mei-Jun Zhang; Chao Liu; Chengying Ma; Pan Zhang; Yue-He Ding; Sheng-Bo Fan; Li Tao; Bing Yang; Xiangke Li; Shoucai Ma; Junjie Liu; Boya Feng; Xiaohui Liu; Hong-Wei Wang; Si-Min He; Ning Gao; Keqiong Ye; Meng-Qiu Dong; Xiaoguang Lei
• Keywords: 0301 basic medicine; QH301-705.5; Protein Conformation; Science; protein-protein interactions; 03 medical and health sciences; Protein Interaction Mapping; Escherichia coli; exosome; Animals; Protein Interaction Maps; protein structure; Biology (General); Caenorhabditis elegans; Caenorhabditis elegans Proteins; mass spectrometry; Escherichia coli Proteins; Q; R; Biophysics and Structural Biology; Cross-Linking Reagents; Medicine; 70S ribosome; Ribosomes; cross-linking

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.