AUTHOR=Wei Bo , Willems Patrick , Huang Jingjing , Tian Caiping , Yang Jing , Messens Joris , Van Breusegem Frank 

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

JOURNAL=Frontiers in Plant Science

VOLUME=Volume 11 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2020.00777

DOI=10.3389/fpls.2020.00777

ISSN=1664-462X

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 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.