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Proteomics. 2016 Jul;16(13):1915-27. doi: 10.1002/pmic.201500310.

In planta chemical cross-linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis.

Zhu X1,2, Yu F3, Yang Z1, Liu S1, Dai C4, Lu X2, Liu C1, Yu W3,5, Li N1,3,6.

Author information

1
Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
2
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.
3
Division of Biomedical Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
4
Proteomics Center, Nanjing Agriculture University, Nanjing, P. R. China.
5
Department of Electronic and Computer Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, P. R. China.
6
HKUST Shenzhen Research Institute, Shenzhen, P. R. China.

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.

KEYWORDS:

Arabidopsis; Exhaustive cross-linked peptides identification tool; In planta chemical cross-linking; Interactomics; Mass spectrometry; Plant proteomics

PMID:
27198063
DOI:
10.1002/pmic.201500310
[Indexed for MEDLINE]

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