Cysteine redoxome landscape in mouse brown adipose tissue under acute cold exposure

Figures

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  Figure 1. Cysteine redoxome profile in mouse brown adipose tissue under RT and cold conditions (A) Differential alkylation-based bioswitch labeling workflow: (1) Protein extraction from brown adipose tissues, (2) alkylation of native thiol groups (represented as S–H) on cysteines using n-ethylmaleimide (NEM, represented in a blue circle), (3) reduction of reversibly oxidized post-translational modifications (represented as S-X) on cysteines with tris(2-carboxyethyl) phosphine (TCEP), (4) labeling of nascent thiol groups (represented as S-H) with biotin-peac5-maleimide (BPM; represented in a red circle), (5) trypsin digestion of proteins to generate labeled and unlabeled peptides, (6) high pH reverse-phase peptide fractionation to desalt proteins and remove impurities, and (7) LC-MS analysis of peptides to identify total cysteine residues in the sample. (B) Number of all peptides, cysteine containing peptide and cysteine containing protein in three replicates. (C) Percentage (%) of all quantified reduced or oxidized cysteine residues in three replicates. (D) Subcellular distribution of oxidized and reduced cysteine residues in BAT in three replicates. (E) Volcano plot depicting changes in…

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  Figure 2. Identifying dynamic cysteines along with GO terms, KEGG enrichment analysis, and functional prediction (A) Venn diagram depicting overlap and exclusivity of reproducible oxidized and reduced cysteine residues validated in triplicate experiments under RT and Cold as shown in Figure S1. Cysteine residues in Regions L, M, and N are denoted as the cold-reactive cysteine residues. Region L – reproducible cysteine residues found reduced under RT and oxidized under cold; Region M – reproducible cysteine residues found reduced & oxidized under RT and oxidized under cold; Region N – reproducible cysteine residues found reduced under RT and reduced & oxidized under cold. (B) Sequence motif analyses of dynamic cysteine residues, examining proximal positions (± seven positions) relative to the cysteine sites. Horizontal red lines indicate significance at a p-value of 0.05. (C) Heatmaps illustrating amino acid sequences surrounding dynamic cysteine sites (B). The heatmaps display position weight matrix (PWM) values, reflecting amino acid frequencies proximal to the cysteine and categorized by amino acid properties. (D–F) GO enrichment analyses based on BPs (D), MFs (E), and CCs (F) observed…

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  Figure 3. Exclusive cysteine redoxome analysis in RT and Cold groups with KEGG and motif analysis (A and B) We identified ten relevant enriched KEGG pathways associated with exclusive cysteine residues in BAT under RT (A) and cold (B). The complete list of enriched KEGG metabolic pathways is presented in Tables S16 and S17. (C–F) Sequence motif analyses were conducted for exclusively oxidized cysteine residues in RT (C), exclusive oxidized cysteine residues in Cold (D), exclusive reduced cysteine residues in RT (E), and exclusive reduced cysteine residues in Cold (F), spanning proximal positions (±seven). Horizontal red lines denote a significance level of p-value 0.05. (G and H) Heatmaps illustrating amino acid sequences surrounding reduced cysteine sites in RT (G) and Cold (H). These heatmaps depict the position weight matrix (PWM) values reflecting amino acid frequencies proximal to cysteine, categorized by amino acid properties.