title: Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states.
abstract: 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.
authors.affiliation: Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.|National Institute of Biological Sciences, Beijing, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Tan
authors.affiliation: National Institute of Biological Sciences, Beijing, China.|Synthetic and Functional Biomolecules Center, Peking University, Beijing, China.|Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.|Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Mei-Jun
authors.initials: MJ
authors.lastname: Zhang
authors.affiliation: Key Lab of Intelligent Information Processing of Chinese Academy of Sciences, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Liu
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Chengying
authors.initials: C
authors.lastname: Ma
authors.affiliation: Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.|National Institute of Biological Sciences, Beijing, China.
authors.forename: Pan
authors.initials: P
authors.lastname: Zhang
authors.affiliation: Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.|National Institute of Biological Sciences, Beijing, China.
authors.forename: Yue-He
authors.initials: YH
authors.lastname: Ding
authors.affiliation: Key Lab of Intelligent Information Processing of Chinese Academy of Sciences, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Sheng-Bo
authors.initials: SB
authors.lastname: Fan
authors.affiliation: Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.|National Institute of Biological Sciences, Beijing, China.
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Yang
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Xiangke
authors.initials: X
authors.lastname: Li
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Shoucai
authors.initials: S
authors.lastname: Ma
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Liu
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Boya
authors.initials: B
authors.lastname: Feng
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Liu
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Wang
authors.affiliation: Key Lab of Intelligent Information Processing of Chinese Academy of Sciences, Institute of Computing Technology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Si-Min
authors.initials: SM
authors.lastname: He
authors.affiliation: Ministry of Education Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Gao
authors.affiliation: National Institute of Biological Sciences, Beijing, China.
authors.forename: Keqiong
authors.initials: K
authors.lastname: Ye
authors.affiliation: Graduate Program, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.|National Institute of Biological Sciences, Beijing, China.
authors.forename: Meng-Qiu
authors.initials: MQ
authors.lastname: Dong
authors.affiliation: National Institute of Biological Sciences, Beijing, China.|Synthetic and Functional Biomolecules Center, Peking University, Beijing, China.|Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.|Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Lei
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000502:physiology; D029742:Caenorhabditis elegans Proteins / Q000032:analysis / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000502:physiology; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D012270:Ribosomes / Q000737:chemistry
chemical_list: D029742:Caenorhabditis elegans Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins
keywords: 70S ribosome; <i>c. elegans</i>; <i>e. coli</i>; biophysics; cross-linking; exosome; mass spectrometry; protein structure; protein-protein interactions; structural biology