Publication Information
Wei et al., 2020
Abstract
Front Plant Sci. 2020 Jul 2;11:777. doi: 10.3389/fpls.2020.00777. eCollection
2020.
Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using
a Disulfide-Linked Peptide Reporter.
Wei B(1)(2)(3)(4)(5), Willems P(1)(2), Huang J(1)(2), Tian C(6), Yang J(6),
Messens J(3)(4)(5), Van Breusegem F(1)(2).
Author information:
(1)Department of Plant Biotechnology and Bioinformatics, Ghent University,
Ghent, Belgium.
(2)VIB Center for Plant Systems Biology, Ghent, Belgium.
(3)VIB-VUB Center for Structural Biology, Brussels, Belgium.
(4)Brussels Center for Redox Biology, Brussels, Belgium.
(5)Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
(6)State Key Laboratory of Proteomics, Beijing Proteome Research Center,
National Center for Protein Sciences, Beijing Institute of Lifeomics, Beijing,
China.
In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to
form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine
oxidation events can steer diverse cellular processes by altering protein
interactions, trafficking, conformation, and function. Previously, we had
identified S-sulfenylated proteins by using a tagged proteinaceous probe based
on the yeast AP-1-like (Yap1) transcription factor that specifically reacts with
sulfenic acids and traps them through a mixed disulfide bond. However, the
identity of the S-sulfenylated amino acid residues within a protein remained
enigmatic. By using the same transgenic YAP1C probe, we present here a
technological advancement to identify in situ sulfenylated cysteine sites in
Arabidopsis thaliana cells under control condition and oxidative stress.
Briefly, the total extract of transgenic YAP1C A. thaliana cells was initially
purified on IgG-Sepharose beads, followed by a tryptic digest. Then, the mixed
disulfide-linked peptides were further enriched at the peptide level on an
anti-YAP1C-derived peptide (C598SEIWDR) antibody. Subsequent mass spectrometry
analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating
sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these
YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines
(S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The
presented methodology provides a noninvasive approach to identify sulfenylated
cysteines in any species that can be genetically modified.
Copyright © 2020 Wei, Willems, Huang, Tian, Yang, Messens and Van Breusegem.
DOI: 10.3389/fpls.2020.00777
PMCID: PMC7343964
PMID: 32714340