Quantitative Persulfide Site Identification (qPerS-SID) Reveals Protein Targets of H2S Releasing Donors in Mammalian Cells

Abstract

H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents.

Sulfheme formation during homocysteine S-oxygenation by catalase in cancers and neurodegenerative diseases

“…6g). These results suggest that H 2 S inhibits ubiquitously cellular antioxidant enzymes that rapidly consume H 2 O 2 , such as peroxiredoxins and GSH peroxidases, most likely via the formation of Cys persulfides that affect the activity of target proteins69. Regardless, these observations do not provide any information regarding the heme-iron species accountable for SpCAT inhibition in cancer cells and rotenone-treated BE2-M17 cells.…”

N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H2S and Sulfane Sulfur Production

“…Proteins with vicinal thiols (like roGFP2) can rapidly convert hydropersulfide groups to disulfides, but other proteins may acquire the hydropersulfide as a more stable modification. Indeed, protein hydropersulfidation seems to be relatively common as a detectable posttranslational modification (Doka et al, 2016; Gao et al, 2015; Longen et al, 2016; Wedmann et al, 2016). …”

Biological hydropersulfides and related polysulfides – a new concept and perspective in redox biology

“…An advantage of this method compared to others developed previously is that it utilized an alkylation step prior to cellular lysis, thus precluding cell lysis-mediated artifactual oxidation/persulfidation. Further work by Longen and coworkers [29], also using a mass spectrometry-based assay (entitled quantitative PerSulfide Site Identification or qPerS-SID), found that cells treated with H 2 S resulted in the generation of numerous protein hydropersulfides (with significant identification overlap with the above-mentioned work by Ida and coworkers [27]). They also found that H 2 S-mediated protein hydropersulfide formation appeared to exhibit specificity among numerous potential "sulfhydration" sites.…”