title: In planta chemical cross-linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis.
abstract: Site-specific chemical cross-linking in combination with mass spectrometry analysis has emerged as a powerful proteomic approach for studying the three-dimensional structure of protein complexes and in mapping protein-protein interactions (PPIs). Building on the success of MS analysis of in vitro cross-linked proteins, which has been widely used to investigate specific interactions of bait proteins and their targets in various organisms, we report a workflow for in vivo chemical cross-linking and MS analysis in a multicellular eukaryote. This approach optimizes the in vivo protein cross-linking conditions in Arabidopsis thaliana, establishes a MudPIT procedure for the enrichment of cross-linked peptides, and develops an integrated software program, exhaustive cross-linked peptides identification tool (ECL), to identify the MS spectra of in planta chemical cross-linked peptides. In total, two pairs of in vivo cross-linked peptides of high confidence have been identified from two independent biological replicates. This work demarks the beginning of an alternative proteomic approach in the study of in vivo protein tertiary structure and PPIs in multicellular eukaryotes.
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.|Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi Province, P. R. China.
authors.forename: Xinliang
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
authors.forename: Fengchao
authors.initials: F
authors.lastname: Yu
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
authors.forename: Shichang
authors.initials: S
authors.lastname: Liu
authors.affiliation: Proteomics Center, Nanjing Agriculture University, Nanjing, P. R. China.
authors.forename: Chen
authors.initials: C
authors.lastname: Dai
authors.affiliation: Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi Province, P. R. China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Lu
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
authors.forename: Chenyu
authors.initials: C
authors.lastname: Liu
authors.affiliation: Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.|Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
authors.forename: Weichuan
authors.initials: W
authors.lastname: Yu
authors.affiliation: Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.|Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.|HKUST Shenzhen Research Institute, Shenzhen, P. R. China.
authors.forename: Ning
authors.initials: N
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000032:analysis / Q000378:metabolism; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D012984:Software; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029681:Arabidopsis Proteins; D003432:Cross-Linking Reagents; D010455:Peptides
keywords: Arabidopsis; Exhaustive cross-linked peptides identification tool; In planta chemical cross-linking; Interactomics; Mass spectrometry; Plant proteomics