Fig 2: Orthogonal protein and peptide-based biotin thiol assay (BTA) approaches show CGL dependence for DR-induced persulfidation shifts.a Graphical presentation of experimental setup. Kidney and brain extracts from 12-month-old CGL WT and KO mice under 1 week 50% DR were subjected to protein level or peptide level BTA, LC-MS/MS, and analyzed via the given software platform (n = 3 mice/group). Furthermore, an additional peptide level BTA was subjected to iodoTMT labeling prior to LC-MS/MS for quantification and site-level analysis of cysteine residues (n = 3 mice/group). b–g Volcano plots showing CGL-dependent intensity shifts in DTT-eluted & unlabeled (b–e) or TCEP-eluted & iodoTMT labeled (f, g) persulfidated proteins. b, c Derived from unlabeled protein level BTA for kidney (b) and brain (c). d, e Derived from unlabeled peptide level BTA for kidney (d) and brain (e). f, g Derived from iodoTMT labeled peptide level BTA for kidney (f) and brain (g). The log2(Fold Change WT:KO) X-axis displays the average fold change in intensity for each protein, and the -log10 Y-axis displays the calculated P value from a two-sided Student’s t test when comparing the individual intensity values for each protein from WT versus KO mice. The non-axial red dotted vertical lines highlight the biological significance threshold of + /- twofold change in intensity, while the non-axial red dotted horizontal line with asterisk highlights the statistical significance threshold of P < 0.05. Blue (KO enriched) and green (WT enriched) dots indicate proteins reaching both biological- and statistical-thresholds. The percentage and direction of proteins skewed toward CGL WT is provided above the tissue label. See also Supplementary Fig. 9.

Fig 3: CGL dependent and independent persulfidation and pathway enrichment in muscle, brain, and heart.a–c Volcano plots showing differentially abundant persulfidated proteins in muscle (a), brain (b), and heart (c) from AL (n = 3 CGL KO mice/group) versus DR (n = 3 CGK KO mice/group). The log2(Fold Change DR:AL) X-axis displays the average fold change in spectral counts for each identified persulfidated protein while the -log10 Y-axis displays the calculated P value from a two-sided Student’s t test when comparing the individual spectral count values for each identified persulfidated protein from AL versus DR fed mice. Blue (AL enriched) and green (DR enriched) dots and text indicate persulfidated proteins reaching both biological (twofold enrichment)- and statistical (P < 0.05)- thresholds. Gray dots indicate persulfidated proteins not reaching the criteria for both biological and statistical significance under either diet. The percentage and direction of proteins skewed toward one diet type is provided above the tissue label. d–f Shared and unshared persulfidated proteins between CGL WT and KO muscle (d), brain (e), and heart (f). g–i Pathway analysis of persulfidated proteins unique to CGL WT muscle (g), brain (h), and heart (i). Numbers indicate the proteins involved in that specific pathway. Significance for pathway enrichment plotted as the adjusted -log10 (P value) and was auto-calculated via the g:Profiler g:SCS algorithm for KEGG database that utilizes multiple testing corrections. j, k Volcano plots and l dot plot re-imaging data from Figs. 2, 4, 7, and 8 showing differentially abundant persulfidated proteins in liver, kidney, muscle, brain, and heart from CGL WT AL versus CGL WT DR mice (j) and from CGL KO AL versus CGL KO DR mice (k) with the calculated P values from a two-sided Student’s t test when comparing individual spectral count values for each identified persulfidated protein from AL versus DR fed mice. In (l), the mean + /- SEM is plotted for % persulfidated protein enrichment under DR compared to AL feeding from each solid tissue in CGL WT and KO mice as determined through spectral counting or MS1 intensity. Source data are provided as a Source Data file. See also Supplementary Fig. 8.

Fig 4: Model depicting the impact of dietary restriction (DR) on endogenous H2S production and protein persulfidation in tissue-specific and CGL-dependent manners. Arrows pointing up indicate an increase, arrows pointing down indicate a decrease, horizontal arrows indicate no change, and + or - relate to CGL dependence for the indicated DR-induced changes relative to ad libitum fed controls.

Fig 5: Dietary restriction enriches the persulfidation profiles in liver, kidney, muscle, and brain.a–d Volcano plots showing differentially abundant persulfidated proteins in liver (a), kidney (b), quadriceps muscle (c), and whole brain (d) from ad libitum (AL; n = 4 mice/group) versus 50% dietary restriction (DR; n = 5 mice/group) fed cystathionine ?-lyase (CGL) wildtype (WT) mice. The log2(Fold Change DR:AL) X-axis displays the average fold change in spectral counts for each identified persulfidated protein while the -log10 Y-axis displays the calculated P value when comparing the individual spectral count values for each identified persulfidated protein in a specific tissue from AL versus DR fed mice via a two-sided Student’s t test. The non-axial red dotted vertical lines highlight the biological significance threshold of + /- twofold change in spectral counts between DR versus AL, while the non-axial red dotted horizontal line with asterisk highlights our statistical significance threshold of P < 0.05. The number of total persulfidated proteins identified in each tissue are given next to the tissue name, while blue (AL enriched) and green (DR enriched) colored dots and text indicate persulfidated proteins reaching both biological- and statistical-thresholds. Gray color dots indicate persulfidated proteins not reaching the criteria for both biological and statistical significance for enrichment under either diet. The percentage of proteins skewed toward DR is provided above the tissue label. See also Supplementary Fig. 2.