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In planta chemical cross‐linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis
Author(s) -
Zhu Xinliang,
Yu Fengchao,
Yang Zhu,
Liu Shichang,
Dai Chen,
Lu Xiaoyun,
Liu Chenyu,
Yu Weichuan,
Li Ning
Publication year - 2016
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201500310
Subject(s) - computational biology , proteomics , eukaryote , mass spectrometry , chemistry , arabidopsis , arabidopsis thaliana , protein–protein interaction , in vivo , biochemistry , biology , genetics , genome , chromatography , gene , mutant
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.