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

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

Affiliations

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

Bo Wei et al. Front Plant Sci..

Abstract

In proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known asS-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identifiedS-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 theS-sulfenylated amino acid residues within a protein remained enigmatic. By using the same transgenic YAP1C probe, we present here a technological advancement to identifyin situ sulfenylated cysteine sites inArabidopsis thaliana cells under control condition and oxidative stress. Briefly, the total extract of transgenic YAP1CA. 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.

Copyright © 2020 Wei, Willems, Huang, Tian, Yang, Messens and Van Breusegem.

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Figures

FIGURE 1
FIGURE 1
Enrichment strategies for YAP1C cross-linked (CL) peptide identification. Trypsin digestion under non-reducing conditions results in YAP1C C598SEIWDR CL peptides containing the redox-active Cys598. Next to a non-enriched proteomic shotgun (sample A), CL peptide enrichment was tested with anti-C598SEIWDR polyclonal antibodies (sample B). In addition, YAP1C-interacting protein complexes were purified on IgG-Sepharose beads, followed by an on-bead trypsin digestion (sample D). The obtained peptides were further enriched for C598SEIWDR CL peptides on beads coupled to the anti-C598SEIWDR antibody (sample C). IAM, iodoacetamide, indicated by purple dots; LC-MS/MS, liquid chromatography-tandem mass spectrometry; ProtG, two IgG-binding domains of protein G; SBP, streptavidin-binding peptide.
FIGURE 2
FIGURE 2
Gene set and protein domain enrichment of YAP1C C598SEIWDR CL proteins and sites.(A) Heatmap of the 1,132 C598SEIWDR CL peptides (FDR ≤ 1%; ≥2 PSMs) showing the number of PSMs per replicate in cells untreated (left) and treated with H2O2 for 30 min (right). The overlap of C598SEIWDR CL peptides between conditions is shown in a Venn diagram.(B) Gene set enrichment of the 570 proteins matched uniquely by the C598SEIWDR CL peptides with the DAVID tool (Huang et al., 2009). The assessed gene sets were KEGG pathways, gene ontology (GO) terms for biological process (GO-BP), molecular function (GO-MF), and cellular component (GO-CC, not plotted). All results are presented in Supplementary Dataset S3A.(C) Enrichment of 1,132 sulfenylated cysteines in PROSITE profiles (see section “Materials and Methods”). Enrichment was plotted as a black line (-log10 FDR, topx-axis) and the number of CL sites as a bar chart (bottomx-axis). All results are reported in Supplementary Dataset S3B.
FIGURE 3
FIGURE 3
Characteristic properties and fragmentation ions of C598SEIWDR CL peptides.(A) Mass distribution for non-redundant linear (left) and C598SEIWDR CL peptides (right). A 892-Da difference is indicated between the median peptide masses of both distributions.(B) Peptide precursor charge distribution for PSMs to linear and C598SEIWDR CL peptides.(C) Annotated spectrum of C#SEIWDR CL (blue fragment ions) to HMIEDDC#TDNGIPLPNVTSK (red fragment ions).(D) Parsing of 6,971 PSMs of the YAP1C cross-links (FDR ≤ 1%) containing at least five C598SEIWDR b, y, or precursor ions (withinm/z 0.01) for diagnostic ions. To this end, the occurrence of a peak was counted at am/z 0.001 interval (irrespective of its intensity) to identify consistent fragment ions. Ten characteristic C598SEIWDR CL peptide ions were displayed in the peptide fragmentation scheme and table, with y2* and y3* indicating an ammonia neutral loss (-NH3). A triplet ion resulting from disulfide fragmentation patterns is highlighted.(E) Occurrence of triplet ion peaks characteristic of disulfide cleavage in 6,971 PSMs of C598SEIWDR CL peptides (FDR ≤ 1%, ≥5 fragment ions). The expected C598SEIWDR precursor mass is indicated in black (m/z 908.3931) and is flanked by Cys dehydroalanine (DHA, blue;m/z 874.4054) and Cys persulfide (green;m/z 970.3652). Brown peaks correspond to the MH+ precursor minus two hydrogens corresponding to an intact disulfide bond. MH+, C598SEIWDR single-charged peptide precursor; MH++S, Cys persulfide precursor; MH+-SH2, Cys DHA precursor.
FIGURE 4
FIGURE 4
S-sulfenylated sites identified by C598SEIWDR CL peptides corresponding to previously identified redox-sensitive cysteines.(A) Annotated MS/MS spectrum of C#SEIWDR (blue) linked to VAVGAPDVLGDC#PFSQR (red, Cys20 of DHAR2). The y1 fragment ion (corresponding to the C-terminal Arg) (orange) is shared by both peptides.(B) DHAR2 structure (PDB ID: 5LOL) (Bodra et al., 2017) is shown. YAP1C disulfide CL Cys6 and Cys20 are highlighted in red sticks, glutathione in the active site in brown, and the 96 residues covered by linear peptides in blue. The structure was generated using Chimera version 1.12 (Pettersen et al., 2004).(C) Venn diagram displaying the overlap of 1,132S-sulfenylated sites (≥ 2PSMs) identified in this study (black) with previously reportedArabidopsis S-sulfenylated sites (blue) (Huang et al., 2019),S-nitrosylated sites (pink) (Fares et al., 2011; Puyaubert et al., 2014; Hu et al., 2015) and reversibly oxidized cysteine sites (green) (Liu et al., 2014, 2015).(D) Annotated MS/MS spectrum of C#SEIWDR (blue) linked to DLKPSNLLLNANC#DLK (red; Cys181 of MPK 4).(E) Annotated MS/MS spectrum of C#SEIWDR (blue) linked to VAVGAPDVLGDCPFSQR (red). For all annotated MS/MS spectra, the C598SEIWDR fragment ions are colored in dark blue. Persulfide and cysteine-to-dehydroalanine fragment ions are in green and light blue, respectively. Fragment ions ofArabidopsis peptides linked with sulfenylated cysteines are in red.
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References

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