Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter

Bo Wei; Patrick Willems; Jingjing Huang; Caiping Tian; Jing Yang; Joris Messens; Frank Van Breusegem

doi:10.3389/fpls.2020.00777

Identification of Sulfenylated Cysteines in Arabidopsis thaliana Proteins Using a Disulfide-Linked Peptide Reporter

Front Plant Sci. 2020 Jul 2:11:777. doi: 10.3389/fpls.2020.00777. eCollection 2020.

Authors

Bo Wei^{1

2

3

4

5}, Patrick Willems^{1

2}, Jingjing Huang^{1

2}, Caiping Tian⁶, Jing Yang⁶, Joris Messens^{3

4

5}, Frank Van Breusegem^{1

2}

Affiliations

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.

Abstract

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.

Keywords: Arabidopsis thaliana; S-sulfenylation (-SOH); YAP1C; affinity purification; cross-linked peptide identification; disulfide; hydrogen peroxide.