PT  - JOURNAL ARTICLE
AU  - Bo Wei
AU  - Patrick Willems
AU  - Jingjing Huang
AU  - Caiping Tian
AU  - Jing Yang
AU  - Joris Messens
AU  - Frank Van Breusegem
TI  - Identification of sulfenylated cysteines in <em>Arabidopsis thaliana</em> proteins using a disulfide-linked peptide reporter
AID  - 10.1101/2020.03.25.989145
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.03.25.989145
4099  - http://biorxiv.org/content/early/2020/03/25/2020.03.25.989145.short
4100  - http://biorxiv.org/content/early/2020/03/25/2020.03.25.989145.full
AB  - 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 remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. 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.