Fig. 4: Cyanide induces posttranslational protein modifications.

a–c, Proteome-wide and site-specific changes in S-cyanylation in HepG2 cells treated with 1 µM KCN (a), HepG2 cells treated with 10 mM glycine (Gly; b) and mouse liver tissue lysates treated with 10 mM Gly (c; n = 5 per group, biological replicates). d, Gene Ontology (GO) term enrichment (biological process) of the proteins whose S-cyanylation is significantly increased in HepG2 cells treated with Gly and cyanide releasers and decreased in cells cultured in glycine/serine-free medium. Using DAVID for enrichment, the outcomes were visualized through REVIGO. Significant GO terms passed the Benjamini-adjusted P-value threshold of 0.01. Circle dimensions denote the protein count within specific GO terms, while colour gradients communicate the degree of significance. e, GO term enrichment analysis (cellular localization) of cyanylated proteins. f, Zn²⁺-catalysed transformation of cyanylated peptides to light and heavy tetrazole, used to increase specificity of detected modifications. g, Venn diagram comparing the proteins found to contain ¹³C¹⁵N (heavy cyano) cyanylation with the endogenously cyanylated proteins (light cyano) in liver tissue lysates treated with ¹³C¹⁵N-labelled Gly (n = 5, biological replicates). h,i, Annotated MS/MS spectrum of peptides from Rab GDP dissociation inhibitor beta (UniProt accession: Q61598) displaying two cysteine sites—C203 labelled with light tetrazole (blue L) and C202 labelled with heavy tetrazole (red H; h)—and corresponding quantification of extracted ion chromatogram (XIC) area (n = 5 per group, biological replicates; i). j, Proposed scheme of protein S-cyanylation. After reaction with reactive oxygen species (ROS), thiols (RSH) become oxidized to either sulfenic acid (RSOH) or disulfides (RSSR), which could be both intramolecular and intermolecular disulfides. Both ROSH and RSSR could react with HCN leading to protein cyanylation (RSCN). When SH groups are hyperoxidized, they are no longer reactive to cyanide. k, Left, Enzymatic activity of GAPDH pre-incubated with H₂O₂ (10 µM), KCN (10 µM) or H₂O₂/KCN (at least n = 6 per group, biological replicates). Right, Detection of high-pH-induced peptide bond cleavage at cyanylation sites of GAPDH that was treated with a different combination of KCN, H₂O₂ or diamide (Dm; SDS–PAGE analysis). l, Left, Enzymatic activity of GPDH pre-incubated with H₂O₂ (10 µM), KCN (10 µM) or H₂O₂/KCN (n = 3 per group, biological replicates). Right, Detection of high-pH-induced peptide bond cleavage at cyanylation sites of GPDH that was treated with a different combination of KCN, H₂O₂ or diamide (Dm; SDS–PAGE analysis). Data in i, k and l are expressed as the mean ± s.e.m. Data in k and l were analysed with a two-way ANOVA followed by Bonferroni’s multiple-comparisons test. *P < 0.05 and **P < 0.01 indicate significant differences.

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