Abstract

   Hydrogen sulfide (H[2]S) is a cytoprotective redox-active metabolite
   that signals through protein persulfidation (R-SS[n]H). Despite the
   known importance of persulfidation, tissue-specific persulfidome
   profiles and their associated functions are not well characterized,
   specifically under conditions and interventions known to modulate H[2]S
   production. We hypothesize that dietary restriction (DR), which
   increases lifespan and can boost H[2]S production, expands
   tissue-specific persulfidomes. Here, we find protein persulfidation
   enriched in liver, kidney, muscle, and brain but decreased in heart of
   young and aged male mice under two forms of DR, with DR promoting
   persulfidation in numerous metabolic and aging-related pathways. Mice
   lacking cystathionine γ-lyase (CGL) have overall decreased tissue
   protein persulfidation numbers and fail to functionally augment
   persulfidomes in response to DR, predominantly in kidney, muscle, and
   brain. Here, we define tissue- and CGL-dependent persulfidomes and how
   diet transforms their makeup, underscoring the breadth for DR and H[2]S
   to impact biological processes and organismal health.

   Subject terms: Proteomics, Post-translational modifications, Ageing,
   Metabolism
     __________________________________________________________________

   Dietary restriction (DR) can increase protein persulfidation but the
   tissue specificity of this process is not well understood. Here, the
   authors compare organ-specific protein persulfidomes in young and aged
   mice under DR, and show that DR-dependent persulfidome changes depend
   on cystathionine γ-lyase.

Introduction

   Established interventions and models to extend both lifespan and
   healthspan across evolutionary boundaries include dietary restriction
   (DR)^[40]1 and disruption of the growth hormone (GH)/insulin-like
   growth factor 1 (IGF-1) axis^[41]2. Largely defined as reduced nutrient
   intake without malnutrition, DR encompasses decreased total daily
   caloric intake, the removal of specific macronutrients such as amino
   acids, and/or intermittent fasting. In addition to defending against
   aging-related diseases^[42]3–[43]5, DR also provides stress
   resistance^[44]6 and metabolic fitness^[45]7,[46]8. Similar to DR, mice
   lacking adequate GH production^[47]9 or GH receptor (GHRKO)
   signaling^[48]10 have increased lifespans and metabolic fitness^[49]11.
   It is hypothesized that common molecular pathways enriched or affected
   by DR and GH/IGF-1 axis disruption are central to improved
   aging^[50]12. Identifying these shared pathways and elucidating their
   mechanism of action will shed light onto the underpinnings of aging and
   usher targeted diagnostics and therapies into the clinic.

   A common molecular phenomenon amongst several dietary and endocrine
   models of longevity is the altered metabolism of sulfur-containing
   amino acids methionine and cysteine, which entails flux through
   transsulfuration^[51]13–[52]15 and increased production capacity and/or
   bioavailability of hydrogen sulfide (H[2]S) gas^[53]16. H[2]S and its
   HS^– anion and S^2− ion, herein referred simply as H[2]S, is
   historically classified as an environmental and occupational
   hazard^[54]17–[55]19. However, in the later part of the 20th and early
   part of the 21st centuries, functional and beneficial production of
   H[2]S via enzymatic catalysis of sulfur-containing amino acids was
   discovered in brain, aorta, and vascular smooth muscle
   cells^[56]20–[57]22. Exogenously provided H[2]S increases lifespan in
   model organisms^[58]23, prevents ischemia-reperfusion
   injury^[59]24,[60]25, improves cardiovascular health^[61]26,[62]27, and
   is recognized as a potential therapeutic against aging-associated
   diseases^[63]28. Conversely, deficiencies in endogenous H[2]S levels or
   production capacity correlate with and/or are causative of
   hypertension^[64]27 and neurodegeneration^[65]29. Thus, enhancing H[2]S
   production serves as a therapeutic target for numerous clinically
   relevant endpoints.

   Due to the multiphasic dose response of H[2]S, life evolved with
   mechanisms for its controlled production and utilization. In mammals,
   H[2]S is enzymatically produced during the catabolism of
   sulfur-containing amino acids, primarily cysteine and homocysteine, via
   the activities of cystathionine β-synthase (CBS), cystathionine γ-lyase
   (CGL), and 3-mercaptopyruvate sulfurtransferase (3-MST)^[66]30. CGL,
   CBS, and 3-MST are differentially expressed and active in
   tissue-specific manners, with CGL contributing the majority of
   enzymatic H[2]S production in the kidney, liver, and
   endothelium^[67]31–[68]33. Lack of CGL expression and/or activity is
   attributed to hypertension^[69]27, neurodegeneration^[70]34, and the
   inability to properly respond to dietary and endocrine
   cues^[71]25,[72]33,[73]35–[74]37. Remarkably, increased CGL expression
   in the liver is a hallmark of numerous dietary, genetic, hormonal, and
   pharmaceutical models of an extended lifespan and may serve as a
   molecular biomarker associated with longevity^[75]38.

   Downstream mechanisms to how CGL and subsequent H[2]S production impart
   cellular and systemic benefits rest on the redox versatility of
   sulfur^[76]39. H[2]S performs several non-mutually exclusive
   biochemical reactions, including mitochondrial electron transfer,
   alterations of iron–sulfur and heme centers, antioxidant activity, and
   protein posttranslational modification via persulfidation—aka
   sulfhydration—of reactive cysteine residues to form persulfide tails
   (R-SS[n]H)^[77]40. While the benefits of a diffusible antioxidant to
   counter oxidative damage along with improvement in bioenergetics to
   delay the onset of aging-related disorders are easily identifiable, the
   act of persulfidation and its extent to improve fitness and lifespan is
   less understood.

   Like other posttranslational modifications, persulfidation potentially
   alters a protein’s structure, function, stability, and/or
   macromolecular interactions^[78]39,[79]41,[80]42. The persulfidation
   process generally involves: (1) oxidation such as sulfenylation of the
   cysteine residue(s) on the targeted protein followed by nucleophilic
   attack of the oxidized thiol and/or disulfide by H[2]S, or (2) reaction
   between H[2]S derived polysulfides (H[2]S[n]) with a reduced or
   glutathione-attached protein thiol^[81]41,[82]43–[83]45. It is
   estimated that 10 to 25% of proteins in the rodent liver are
   persulfidated^[84]42 and this modification typically increases the
   reactivity of the modified cysteine^[85]46. Studies examining
   biochemical functions of persulfidation are primarily constructed on
   cell or purified protein models in vitro. Few studies have examined the
   total number and identity of persulfidated proteins, or
   persulfidome—aka sulfhydrome—of an organism in vivo. One such study was
   completed in the plant Arabidopsis thaliana, with the discovery of 2015
   proteins, or ∼5% of the entire proteome, as persulfidated^[86]47.
   Recently, it was elegantly shown using a dimedone switch method that
   aging attenuates and long-term DR enhances overall liver persulfidation
   abundance in mice^[87]44. However, a detailed analysis of mammalian
   multi-organ persulfidomes, the individual proteins and their cysteine
   sites of modification contained within, and the enrichment of specific
   biological pathways with these persulfidated proteins have not been
   described, particularly under anti-aging and CGL-perturbed conditions
   known to impact endogenous H[2]S production.

   Taking advantage of the relative specificity for maleimide to alkylate
   protein thiols and persulfides^[88]48–[89]50, particularly in whole
   tissue preparations^[90]51, we utilized biotin-conjugated maleimide in
   a modified Biotin-Thiol-Assay (BTA) developed by Gao, et
   al.^[91]43,[92]45, to isolate and identify persulfidated proteins and
   peptides from mammalian tissues. We set out to test the hypothesis that
   interventions known to increase lifespan, improve metabolic and stress
   resiliency, and boost endogenous H[2]S production would essentially
   expand and/or alter tissue-specific persulfidome profiles in vivo. To
   address this hypothesis, we employed two dietary interventions: (A)
   Short-term 1-week 50% caloric restriction in young adult (6 and 12
   month old) male CGL wildtype (WT) and total body CGL knockout (KO)
   mice, and (B) Long-term 2.5-month every other day (EOD) fasting in aged
   adult (20 month old) C57BL/6 male mice. In the 50% caloric restriction
   scenario, we examined for diet-, tissue-, and genotype-specific changes
   in H[2]S production (i), protein persulfidation (ii), and biological
   pathways impacted by persulfidation (iii) (Fig. [93]1a). This type and
   duration of dietary intervention was chosen as it was previously shown
   to induce CGL expression and functional CGL-dependent hepatic H[2]S
   production in WT mice^[94]25.

Fig. 1. Diet and cystathionine γ-lyase (CGL) status impact H[2]S production
in a tissue specific manner.

   [95]Fig. 1
   [96]Open in a new tab

   a Graphical presentation of the overarching experimental setup.
   Six-month-old male CGL wildtype (WT) and total body knock out (KO) mice
   were placed on ad libitum (AL) or 50% dietary restriction (DR) diets
   for 1 week prior to tissue harvest. Tissues were analyzed for (i) H[2]S
   production capacity via the lead acetate/lead sulfide method, (ii)
   protein persulfidation profiles via the biotin thiol (BTA) assay, and
   (iii) biological pathway enrichment/function of the identified
   persulfidated proteins. b, c H[2]S production capacity in tissues in AL
   fed CGL WT (n = 3 mice/group) and CGL KO (n = 3 mice/group) mice at
   short 6 h (b) and long 16 h (c) exposures of the same assay plate.
   Means are shown and error bars are ± SEM. The asterisk indicates the
   significance of the difference in the average tissue-specific H[2]S
   production capacity between CGL WT and KO mice after subtracting the
   (–) Tissue negative controls as background; with P values obtained from
   two-sided Student’s t test. d–i H[2]S production capacity in tissues
   harvested from CGL WT mice after 1 week of AL (n = 4 mice/group) or 50%
   DR (n = 5 mice/group) feeding. j, k H[2]S production capacity in liver
   (j) and kidney (k) in CGL KO mice after 1 week of AL (n = 3 mice/group)
   or 50% DR (n = 3 mice/group) feeding. Means are shown and error bars
   are ± SEM. The asterisks indicate the significance of the difference
   between the dietary treatment groups; with P values obtained from
   two-sided Student’s t test. ns not significant. Negative values after
   subtracting background have been converted to zero. The dash mark in
   the lead acetate/lead sulfide images in (d–i) indicates no sample
   loaded in that location. Source data are provided as a Source Data
   file. See also Supplementary Fig. [97]1.

   In this work, we identify persulfidated proteins and their associated
   biological pathways in the liver, kidney, heart, muscle (quadriceps),
   brain, and plasma. DR enriches the number of persulfidated proteins in
   the liver, kidney, muscle, and brain while it decreases these in the
   heart and has minimal impact on the plasma in WT mice. CGL KO mice have
   an overall decrease in persulfidated proteins identified in all tissues
   and fail to appropriately augment their persulfidome in response to DR
   relative to WT mice, chiefly in kidney, muscle, and brain. Likewise,
   long-term EOD fasting in aged mice enriches tissue-specific
   persulfidomes to a similar extent as 1-week 50% caloric restriction in
   young adult mice. These findings suggest a transformation of the
   protein persulfidome may be one mechanism as to how DR imparts its
   multi-tissue benefits to improve organismal health.

Results

Diet and CGL status impact tissue-specific H[2]S production

   Ad libitum (AL) fed mice were provided 24-h access to food and the 50%
   DR (DR) mice were fed their calculated allotment near the start of
   their dark phase at 7 pm to avoid disturbances in circadian rhythms and
   feeding patterns between the two groups^[98]52,[99]53. Average daily
   food intake monitored over the 1-week intervention showed the AL group
   consumed 0.2 g food/g mouse, or ∼12 kcal/mouse, per day while the DR
   group consumed 0.1 g food/g mouse, or ∼6 kcal/mouse, per day
   (Supplementary Fig. [100]1a, b). DR for 7 days reduced body mass
   similarly in both CGL WT and KO mice by 5–10% of initial weight
   (Supplementary Fig. [101]1c, d).

   After the 1-week dietary intervention, liver, kidney, heart, muscle
   (quadriceps), brain, and plasma were collected for downstream H[2]S
   production capacity analysis via the lead acetate/lead sulfide endpoint
   method^[102]54 and for persulfidation profiling (Fig. [103]1a). Similar
   to previous results obtained in our laboratory regarding the tissue
   specificity of CGL, CBS, and 3MST protein expression, H[2]S production,
   and the dependence on CGL for H[2]S production^[104]32, and in line
   with unbiased ENCODE transcriptome data of H[2]S producing and
   consuming genes obtained from NCBI Gene Database^[105]55 (Supplementary
   Fig. [106]1e–h), we show here H[2]S production capacity in AL fed mice
   is highest in liver and kidney, and this is dependent on CGL
   (Fig. [107]1b, c). Heart, muscle, brain, and plasma showed low H[2]S
   production capacities with little to no dependence on CGL
   (Fig. [108]1b, c). DR increased H[2]S production capacity in the liver
   (Fig. [109]1d) and kidney (Fig. [110]1e), but had little to no impact
   in heart, muscle, brain, or plasma (Fig. [111]1f–i). As CGL status is
   important for liver and kidney H[2]S production, we next focused on the
   impact of DR on H[2]S production in these two organs in CGL KO mice. DR
   failed to increase detectable H[2]S production capacity in the liver
   and kidney of CGL KO mice (Fig. [112]1j, k). Thus, H[2]S production
   capacity is highest and malleable via DR in a CGL-dependent manner in
   the liver and kidney, while the other whole-tissue homogenates tested
   have relatively low H[2]S production capacity independent of CGL status
   or diet.

DR enriches the persulfidome in the liver, kidney, muscle, and brain

   To verify the ability of the modified BTA^[113]43 (Supplementary
   Fig. [114]2a) to detect persulfidated proteins from tissues, we ran
   Western blot analysis on the liver BTA elution −/+ DTT for α-Tubulin
   and GAPDH, two hepatic proteins previously shown to be
   persulfidated^[115]42 (Supplementary Fig. [116]2b). α-Tubulin and GAPDH
   were detected in the persulfidation-specific +DTT elution^[117]43 from
   WT mice but not CGL KO mice nor in the –DTT elution lanes
   (Supplementary Fig. [118]2b). Pre-treatment of 1 mM NaHS on WT livers
   ex vivo prior to performing BTA resulted in decreased detection. This
   is indicative that ex vivo addition of 1 mM NaHS may act as a reducing
   equivalent, similar to DTT, and remove the in vivo formed
   persulfidation modifications prior to the BTA (Supplementary
   Fig. [119]2b). Thus, utilizing this modified BTA protein level
   isolation technique followed by mass spectrometry-based proteomics
   analysis for identification and label-free quantification
   (Supplementary Fig. [120]2a), we set out to examine tissue-, dietary-,
   and CGL-dependent persulfidome profiles. Two types of label-free mass
   spectrometry quantification analysis approaches were used for this
   study: peptide spectral counting and MS1 intensity. While the
   scientific narrative outlined in these Figures is based on the spectral
   counting method due to it affording relatively simple and fast data
   analysis in addition to its ability to quantify relative protein
   abundance over a dynamic range of 2 to 60 orders of
   magnitude^[121]56–[122]58, we have also supplied all MS1
   intensity-based analyses in parallel. The presentation of
   intensity-based analyses is included in the [123]Supplementary Data
   files as the second tab in the Excel sheets, with the first tab
   containing the respective spectral counting data. In these sheets are
   the quantitative and qualitative data with corresponding visual graphs
   and plots for both spectral counting and intensity analyses. Including
   data in this format has two benefits: (1) Ease of data access and
   visualization from both approaches, and (2) Ability to compare and
   utilize intermediate and end results from the two approaches, which
   ultimately show similar outcomes and emphasize the reported results in
   the narrative as robust and rigorously obtained. Additionally,
   side-by-side comparisons of these two approaches are described in the
   Peer Review File.

   Of the six tissues tested in WT mice, four had enriched persulfidome
   profiles in response to DR when compared to AL feeding (Fig. [124]2a–d,
   Supplementary Fig. [125]2c–f, Supplementary Data [126]1–[127]4). These
   tissues included liver (Fig. [128]2a, Supplementary Fig. [129]2c,
   Supplementary Data [130]1), kidney (Fig. [131]2b, Supplementary
   Fig. [132]2d, Supplementary Data [133]2), muscle (Fig. [134]2c,
   Supplementary Fig. [135]2e, Supplementary Data [136]3), and brain
   (Fig. [137]2d, Supplementary Fig. [138]2f, Supplementary Data [139]4).
   Total persulfidated proteins identified in each tissue included 977 in
   liver, 1086 in kidney, 431 in muscle, and 884 in brain. Identified
   proteins enriched under DR, displayed as green dots, included 30 in
   liver, 16 in kidney, 34 in muscle, and 369 in brain that met a
   biological- and statistical-significance threshold of at least a
   twofold increase in DR:AL spectral count ratio with a P value < 0.05,
   respectively (Fig. [140]2a–d). Likewise, proteins enriched under AL
   feeding in these four tissues, displayed as blue dots, included 16 in
   liver, 5 in kidney, 0 in muscle, and 42 in brain that met a biological-
   and statistical-significance threshold of at least a twofold increase
   in AL:DR spectral count ratio with a P value < 0.05, respectively
   (Fig. [141]2a–d). In total, 72%, 76%, 86%, and 87% of persulfidated
   proteins were skewed for enrichment under DR feeding in liver, kidney,
   muscle, and brain, respectively (Fig. [142]2a–d).

Fig. 2. Dietary restriction enriches the persulfidation profiles in liver,
kidney, muscle, and brain.

   [143]Fig. 2
   [144]Open in a new tab

   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 γ-lyase (CGL)
   wildtype (WT) mice. The log[2](Fold Change DR:AL) X-axis displays the
   average fold change in spectral counts for each identified
   persulfidated protein while the −log[10] 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. [145]2.

Numerous pathways enriched with persulfidated proteins dependent on diet and
organ

   In the four organs positively enriched for persulfidated proteins after
   DR, 1854 individual proteins were identified, with 209, or 11.3%,
   shared amongst all four tissues (Fig. [146]3a, Supplementary
   Data [147]5). Biological function and pathway enrichment of these 209
   via g:Profiler analysis^[148]59 utilizing the KEGG database^[149]60
   revealed 15 pathways, with carbon metabolism, proteasome, and valine,
   leucine, and isoleucine degradation as the top three hits
   (Fig. [150]3b). Of the 1854 proteins, 63 unique proteins were enriched
   under AL feeding (Fig. [151]3c) and 429 unique proteins under DR
   feeding (Fig. [152]3d), with none of these, shared amongst all four
   tissues.

Fig. 3. Multiple pathways enriched with persulfidated proteins dependent on
diet and organ.

   [153]Fig. 3
   [154]Open in a new tab

   a Four-way Venn diagram showing the number of shared and non-shared
   persulfidated proteins identified in liver, kidney, muscle, and brain
   in cystathionine γ-lyase (CGL) wildtype (WT) mice. A total of 209
   persulfidated proteins were shared amongst all four organs. n = 9 mice
   total; 4/AL group and 5/DR group. b KEGG biological function and
   pathway enrichment of the shared 209 proteins found in liver, kidney,
   muscle, and brain via g:Profiler analysis. c, d Four-way Venn diagrams
   showing shared and non-shared persulfidated proteins enriched under AL
   (c) and DR (d) feeding. e–g KEGG biological function and pathway
   enrichment of AL enriched (blue bars) or DR enriched (green bars)
   persulfidated proteins in liver (e), kidney (f), and brain (g). The
   numbers within the bars indicate individual persulfidated proteins
   identified for that specific pathway. Statistical significance for
   pathway enrichment plotted as the adjusted −log[10] (P value) and was
   auto-calculated via the g:Profiler g:SCS algorithm for KEGG database
   that utilizes multiple testing corrections. See also Supplementary
   Fig. [155]3.

   As no common persulfidated proteins were enriched as a function of diet
   in all four tissues, we next examined tissue-specific pathway
   enrichment. The liver contained two pathways enriched under AL;
   selenocompound metabolism and terpenoid backbone biosynthesis, and one
   pathway enriched under DR; metabolic pathways (Fig. [156]3e,
   Supplementary Data [157]6). Kidney contained one pathway enriched under
   AL; glycine, serine, and threonine metabolism, and one pathway under
   DR; nitrogen metabolism (Fig. [158]3f, Supplementary Data [159]7). No
   pathways were enriched from the diet-specific persulfidated proteins in
   the muscle (Supplementary Data [160]8). Brain contained 12 pathways
   enriched under AL and 13 enriched under DR feeding (Fig. [161]3g,
   Supplementary Data [162]9). The top three pathways enriched under AL
   feeding in the brain included valine, leucine, and isoleucine
   degradation, fatty acid degradation, and metabolic pathways, while the
   top three pathways enriched under DR feeding in the brain included
   carbon metabolism, biosynthesis of amino acids, and
   glycolysis/gluconeogenesis.

   Persulfidated proteins not significantly impacted by diet, which
   primarily compose the majority of the persulfidomes in liver, kidney,
   muscle, and brain, still trended for enrichment under DR
   (Fig. [163]2a–d; gray dots). Function and pathway enrichment of these
   proteins revealed 34, 41, 15, and 13 pathways in liver, kidney, muscle,
   and brain, respectively (Supplementary Fig. [164]3a–d). Ten of the
   pathways identified were shared amongst all four tissues, and include
   metabolic processes, carbon metabolism, valine, leucine, and isoleucine
   degradation, biosynthesis of amino acids, glyoxylate and dicarboxylate
   metabolism, cysteine and methionine metabolism, pyruvate metabolism,
   proteasome, citrate cycle (TCA cycle), and glycolysis/gluconeogenesis.
   While not shared amongst all tissues, pathways related to aging-related
   neurodegenerative Parkinson and Huntington diseases were enriched with
   persulfidated proteins in both kidney and muscle (Supplementary
   Fig. [165]3b, c). Thus, these four major metabolic organs respond to DR
   by augmenting protein persulfidation, with many of the proteins falling
   into specific biological pathways and functions.

DR decreases heart and negligibly impacts plasma persulfidome profiles

   While DR enriched the persulfidomes of liver, kidney, muscle, and
   brain, it failed to do so in heart and plasma. In heart, 459
   persulfidated proteins were identified with 45 enriched under AL
   feeding and 17 enriched under DR feeding (Fig. [166]4a, Supplementary
   Fig. [167]4a, and Supplementary Data [168]10). In plasma, 160
   persulfidated proteins were identified with three enriched under AL
   feeding and zero enriched under DR feeding (Fig. [169]4b, Supplementary
   Fig. [170]4b, and Supplementary Data [171]11). Between heart and
   plasma, 78 common persulfidated proteins were identified (Fig. [172]4c,
   Supplementary Data [173]12), of which four biological
   functions/pathways were enriched, and include complement and
   coagulation cascades, staphylococcus aureus infection, HIF-1 signaling,
   and glycolysis/gluconeogenesis (Fig. [174]4d). Of the persulfidated
   proteins enriched under AL feeding in heart and plasma, zero were
   common between the two tissues (Fig. [175]4e).

Fig. 4. Persulfidomes of heart and plasma respond differently to diet
compared to those of liver, kidney, muscle, and brain.

   [176]Fig. 4
   [177]Open in a new tab

   a, b Volcano plots showing differentially abundant persulfidated
   proteins in heart (a) and plasma (b) from AL (n = 4 mice/group) versus
   DR (n = 5 mice/group) cystathionine γ-lyase (CGL) wildtype (WT) mice.
   The log[2](Fold Change DR:AL) X-axis displays the average fold change
   in spectral counts for each identified persulfidated protein while the
   −log[10] 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 with P values
   obtained from two-sided Student’s t test. 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 and
   direction of proteins skewed toward AL is provided above the tissue
   label. c Venn diagram of shared and non-shared persulfidated proteins
   in heart and plasma. d KEGG biological pathway enrichment of the shared
   78 proteins found in heart and plasma via g:Profiler analysis. e
   Overlap of AL enriched persulfidated proteins in heart and plasma. f
   KEGG biological function and pathway enrichment of AL enriched (blue
   bars) or DR enriched (green bars) persulfidated proteins in heart. g
   Venn diagram of shared and unshared persulfidated proteins amongst all
   6 tissues. h KEGG biological function and pathway enrichment of the
   shared 28 proteins found in all six tissues via g:Profiler analysis.
   The numbers in the bars indicate individual persulfidated proteins
   identified for that specific pathway. Statistical significance for
   pathway enrichment plotted as the adjusted −log[10] (P value) and was
   auto-calculated via the g:Profiler g:SCS algorithm for KEGG database
   that utilizes multiple testing corrections. See also Supplementary
   Fig. [178]4.

   Examination of tissue-specific pathway enrichment revealed heart
   contained 12 pathways under AL with the top three being carbon
   metabolism, valine, leucine, and isoleucine degradation, and
   citrate/TCA cycle, and three pathways under DR; complement and
   coagulation cascades, ferroptosis, and staphylococcus aureus infection
   (Fig. [179]4f, Supplementary Data [180]13). Plasma had no diet-specific
   pathway enrichment (Supplementary Data [181]14). Pathway analysis of
   the persulfidated proteins not significantly impacted by diet revealed
   27 and 6 pathways enriched in the heart and plasma, respectively. In
   heart, the top three pathways enriched were carbon metabolism,
   metabolic pathways, and pyruvate metabolism (Supplementary
   Fig. [182]4c). In plasma, the top three pathways enriched were
   complement and coagulation cascades, staphylococcus aureus infection,
   and prion diseases (Supplementary Fig. [183]4d).

   To examine shared persulfidated proteins amongst all six tissues, we
   compared the 209 proteins identified as shared in liver, kidney,
   muscle, and brain with the 78 proteins identified as shared in heart
   and plasma. A total of 28 proteins were identified as common in all six
   tissues (Fig. [184]4g, Supplementary Data [185]15). These 28 were
   involved in two blood-centric biological functions/pathways, which
   included complement and coagulation cascades, and platelet activation
   (Fig. [186]4h), indicative of potential blood/plasma carryover in the
   non-perfused tissues and thus suggestive that little to no common
   persulfidated proteins are inherent to all tissues.

Late-life initiated long-term EOD fasting transforms tissue-specific
persulfidomes

   While 1 week of 50% DR tested in the young adult mice is sufficient to
   temporarily enhance acute tissue-specific H[2]S production capacity and
   stress resistance^[187]25, it is most likely not long enough to extend
   longevity nor represent a tool for probing resiliency and adaptation at
   older ages. The impact of DR on longevity parameters sometimes arises
   more latently to the effects of reduced calories and/or timed feeding.
   Previously, Zivanovic, et al. showed that persulfidation in rat brain
   and heart tissues decreased with advanced age at 12–24 months, and that
   long-term daily 30% DR in mice between 2 to 20 months of age rescued
   aging-related persulfidation loss in the liver^[188]44. However,
   studies examining the effects of long-term daily DR on older humans are
   somewhat difficult to execute due to patient adherence^[189]61,[190]62
   and need for medical oversight to ensure caloric intake guidelines are
   safe followed^[191]63–[192]67. Given these limitations, the relatively
   implementable yet effective DR regimen of intermittent fasting has
   gained substantial attention as an alternative method to continuous,
   chronic DR^[193]68,[194]69. There are several types of intermittent
   fasting regimens, with every-other-day (EOD) fasting being one of the
   most popular^[195]70,[196]71. In EOD fasting, animals and/or
   participants abstain from food for a day (i.e., 24 h) followed by food
   intake ad libitum (AL) for the next 24 h. This method is feasible in
   older adult humans^[197]72, and in aged rodents augments
   lifespan^[198]73–[199]77 and improves metabolic flexibility^[200]74.

   Therefore, we next utilized late-life initiated EOD fasting for 2.5
   months in 20-month-old male C57BL/6 mice to test if this more
   translatable form of DR is capable of altering tissue-specific
   persulfidomes in aged subjects (Fig. [201]5a). Similar to our findings
   in young adult mice, EOD fasting augmented protein persulfidation in
   liver (Fig. [202]5b, Supplementary Fig. [203]5a, Supplementary
   Data [204]16), kidney (Fig. [205]5c, Supplementary Fig. [206]5b,
   Supplementary Data [207]17), muscle (Fig. [208]5d, Supplementary
   Fig. [209]5c, Supplementary Data [210]18), and brain (Fig. [211]5e,
   Supplementary Fig. [212]5d, Supplementary Data [213]19), while it
   decreased persulfidation in the heart (Fig. [214]5f, Supplementary
   Fig. [215]5e, Supplementary Data [216]20). Overall numbers of
   persulfidated proteins identified in each tissue from these aged mice
   were on the same order to what was found in the young adult mice. While
   the shift in protein persulfidation enrichment under 2.5 months EOD
   fasting in aged mice for kidney (Fig. [217]5c) and muscle
   (Fig. [218]5d) was not as robust as that found under 1-week 50% DR in
   young adult mice, a greater number of proteins meeting the biological-
   and statistical-thresholds were found in the EOD fasting group. Thus,
   despite being completely different forms and durations of DR, in
   addition to being tested on mice of different ages and strain
   background/source, long-term EOD fasting and short-term 50% DR alter
   tissue-specific persulfidomes in an analogous manner.

Fig. 5. Late-life initiated every other day (EOD) fasting for 2.5 months
transforms tissue-specific persulfidomes similar to short-term DR in young
adult mice.

   [219]Fig. 5
   [220]Open in a new tab

   a Graphical presentation of the overarching experimental setup.
   Twenty-month-old male C57BL/6 mice were placed on ad libitum (AL) or
   Every Other Day (EOD) fasting for 2.5 months prior to tissue
   collection. Tissues were analyzed for protein persulfidation via the
   biotin thiol (BTA) assay. b–f Volcano plots showing differentially
   abundant persulfidated proteins in liver (b), kidney (c), quadriceps
   muscle (d), whole brain (e), and heart (f) from AL fed (n = 5
   mice/group) versus 2.5 months of EOD fasting (n = 5 mice/group)
   20-month-old C57BL/6 male mice. The log[2](Fold Change DR:AL) X-axis
   displays the average fold change in spectral counts for each identified
   persulfidated protein while the −log[10] 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 in a
   specific tissue from AL versus EOD fed mice. The non-axial red dotted
   vertical lines highlight the biological significance threshold
   of + /− twofold change in spectral counts between EOD 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 (EOD 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 and
   direction of proteins skewed toward one diet type are provided above
   the tissue label. See also Supplementary Fig. [221]5.

CGL and regulation of its enzymatic activity as targets for persulfidation

   H[2]S generating enzymes CGL and CBS were identified as targets for
   persulfidation in liver (Supplementary Data [222]1 and [223]16) and
   kidney (Supplementary Data [224]2 and [225]17) from young adult and
   aged mice in this current study, and in liver in a previous study by
   Mustafa, et al.^[226]42. However, functional consequences for CGL
   persulfidation have not been established. Given that reduction of
   redox-active cysteine residues in CBS increases H[2]S production
   capacity^[227]78, we set out to determine if treatment of CGL with
   sodium hydrosulfide (NaHS) would impact its enzymatic H[2]S production
   capacity. To do so, we first adapted an in vitro lead acetate/lead
   sulfide headspace assay^[228]54 to measure H[2]S production capacity
   from purified recombinant CGL protein. There was a linear increase in
   H[2]S production as a function of CGL concentration between 1–10 µg
   after a 2 h incubation at 37 °C (Fig. [229]6a, b). Next, we added NaHS
   between 0–1 mM to reaction mixtures with and without 1.125 µg purified
   CGL prior to incubation at 37 °C under normal room oxygen to examine
   for dose-dependent increases in enzymatic H[2]S production
   (Fig. [230]6c, d and Supplementary Fig. 6a–c). As NaHS itself gives an
   immediate positive lead sulfide result as it quickly releases HS^− upon
   placement in aqueous solutions at physiological pH, two additional
   controls were utilized to diminish this noise and allow for accurate
   detection of CGL-derived H[2]S. First, multiple successive incubation
   periods each with a fresh lead acetate embedded detection paper were
   used to allow the NaHS to off-gas HS^− during the early 1 h incubation
   period (Supplementary Fig. [231]6a) so as to not interfere with
   detection of enzymatic production during the subsequent 2.5, 5, and
   6.5 h incubation periods (Fig. [232]6c, d and Supplementary
   Fig. [233]6b, c). Second, the (–) CGL lead sulfide integrated density
   values were subtracted from their dose-corresponding (+)CGL lead
   sulfide integrated density values. Taking these controls into account,
   we determined NaHS pretreatment of CGL prior to incubation at 37 °C
   resulted in dose-dependent increases up to 10–20% in enzymatic H[2]S
   production capacity (Fig. [234]6c, d and Supplementary Fig. [235]6b,
   c). Thus, the persulfidation of CGL detected in vivo combined with
   these in vitro results suggest a potential positive feedback loop
   regulating CGL activity and downstream H[2]S production.

Fig. 6. NaHS pretreatment augments H[2]S production capacity of CGL.

   [236]Fig. 6
   [237]Open in a new tab

   a, b Detection via the lead acetate/lead sulfide method (a) and
   quantification via integrated densitometry (b) of CGL-derived H[2]S
   production in reaction mixtures containing L-cysteine and pyridoxal
   phosphate after 2 h incubation at 37 °C. c, d Pretreatment with NaHS
   prior to incubation at 37 °C dose dependently increases CGL-derived
   H[2]S production capacity after correcting for detection of
   non-enzymatic H[2]S off-gassed in the corresponding (–)CGL wells (c).
   Plot of the percent change in H[2]S production relative to the (+)CGL
   w/0 µM NaHS control. Error bars are ± SEM. The given individual P
   values are calculated from two-sided t test without multiple comparison
   test, while asterisks indicate P < 0.05 calculated from one-way ANOVA
   with Dunnet’s Multiple Comparison Test. n = 4/reaction condition.
   Source data are provided as a Source Data file. See also Supplementary
   Fig. [238]6.

CGL deficiency limits the persulfidome under AL and/or DR feeding in a
tissue-dependent manner

   We next tested the requirement for CGL in diet-induced persulfidome
   changes. As basal and enhanced diet-induced H[2]S production in liver
   and kidney were primarily CGL driven, we first examined persulfidation
   in these two tissues from 6-month-old CGL KO mice under AL and 1 week
   50% DR feeding. In the liver, 698 persulfidated proteins were detected;
   ~30% reduction compared to CGL WT mice (Fig. [239]7a, Supplementary
   Fig. [240]7a, and Supplementary Data [241]21). Despite this overall
   reduction, DR still enriched the liver persulfidome in CGL KO mice (61%
   skewed toward DR; Fig. [242]7a) similarly to WT mice (72% skewed toward
   DR; Fig. [243]2a). However, in comparing total persulfidated proteins
   of WT and CGL KO mice, we discovered ~33% unique to WT mice, ~6% unique
   to KO mice, and ~62% shared (Fig. [244]7b). Pathway enrichment for the
   CGL-dependent proteins (Supplementary Data [245]22) included metabolic
   pathways, oxidative phosphorylation, Parkinson’s disease, Huntington’s
   disease, non-alcoholic fatty liver disease, lysosome, Alzheimer’s
   disease, and thermogenesis (Fig. [246]7c). In comparing DR-enriched
   persulfidated proteins in WT and CGL KO mice, we found 55% unique to WT
   mice, 46% unique to KO mice, and zero shared (Fig. [247]7d). The
   DR-enriched proteins unique to the KO livers failed to account for any
   pathway enrichment. Thus, despite CGL being dispensable for DR-induced
   overall enrichment of the liver persulfidome (suggesting a potential
   involvement of CBS and 3MST), it may be required for targeted
   protein-specific persulfidation.

Fig. 7. Diet induced persulfidation changes in the liver and kidney of
cystathionine γ-lyase (CGL) knockout (KO) mice.

   [248]Fig. 7
   [249]Open in a new tab

   a Volcano plot showing differentially abundant persulfidated proteins
   in liver from AL (n = 3 mice/group) versus DR (n = 3 mice/group) fed
   CGL KO mice. The log[2](Fold Change DR:AL) X-axis displays the average
   fold change in spectral counts for each identified persulfidated
   protein while the −log[10] 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 in liver 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 biological and
   statistical significance under either diet. The percentage and
   direction of proteins skewed toward one diet type is provided above the
   tissue label. b Venn diagram of shared and unshared persulfidated
   proteins between CGL WT and KO livers. c Enriched function/pathway
   analysis of the 339 persulfidated proteins unique to CGL WT liver. d
   Venn diagram to examine shared and unshared DR enriched persulfidated
   proteins in WT and CGL KO livers. e–h are similar to a–d, but analysis
   was performed in kidneys. e Volcano plot showing differentially
   abundant persulfidated proteins in kidney from AL (n = 3 mice/group)
   versus DR (n = 3 mice/group) fed CGL KO mice. f Shared and unshared
   persulfidated proteins between CGL WT and KO kidneys. g Enriched
   function/pathway analysis of the 333 persulfidated proteins unique to
   CGL WT kidney. h Shared and unshared DR enriched persulfidated proteins
   in WT and CGL KO kidneys. Significance for pathway enrichment plotted
   as the adjusted −log[10] (P value) and was auto-calculated via the
   g:Profiler g:SCS algorithm for KEGG database that utilizes multiple
   testing corrections. See also Supplementary Fig. [250]7.

   In the kidney, 869 persulfidated proteins were detected in CGL KO mice;
   ~20% reduction compared to WT mice (Fig. [251]7e, Supplementary
   Fig. [252]7b, and Supplementary Data [253]23). Loss of CGL resulted in
   failure to enrich the persulfidome under DR, with 74% of persulfidated
   proteins skewed toward AL feeding (Fig. [254]7e), which was an equal
   and opposite response detected in WT mice, which had 76% skewed toward
   DR (Fig. [255]2b). In comparing total kidney persulfidated proteins of
   WT and CGL KO mice, ~28% were unique to WT mice, ~10% unique to KO
   mice, and ~63% shared (Fig. [256]7f). Pathway enrichment for the
   CGL-dependent proteins (Supplementary Data [257]24) included metabolic
   pathways and valine, leucine, and isoleucine degradation
   (Fig. [258]7g). In comparing DR-enriched persulfidated proteins in WT
   and CGL KO kidneys, ~76% were unique to WT mice, ~24% unique to KO
   mice, and zero shared (Fig. [259]7h), with the DR-enriched proteins
   unique to KO kidneys failing to account for any pathway enrichment.
   Thus, CGL is required for DR-induced persulfidation enrichment in the
   kidney.

   We next examined tissues with DR-induced shifts in persulfidome
   profiles yet no discernable dependence for CGL or diet in their H[2]S
   production capacities. Muscle, brain, and heart from CGL KO mice had
   decreased numbers of total persulfidated proteins compared to WT mice
   by 56, 39, and 59%, respectively (Fig. [260]8a–c, Supplementary
   Fig. 8a–c, and Supplementary Data [261]25, [262]26, and [263]27). DR
   failed to enrich the muscle persulfidome in CGL KO mice (Fig. [264]8a),
   however, DR still enriched brain (Fig. [265]8b) and attenuated heart
   (Fig. [266]8c) persulfidomes in CGL KO mice. In comparing total
   persulfidated proteins from these three tissues, we discovered: (1) 472
   in muscle with 284 (~60%) unique to WT mice, 41(~9%) unique to KO mice,
   and 147 (~31%) shared (Fig. [267]8d), (2) 998 in the brain with 459
   (~46%) unique to WT mice, 114 (~11%) unique to KO mice, and 425 (~43%)
   shared (Fig. [268]8e), (3) 479 in the heart with 292 (~61%) unique to
   WT mice, 20 (~4%) unique to KO mice, and 167 (~35%) shared
   (Fig. [269]8f). The top ten biological pathways enriched by
   CGL-dependent persulfidated proteins in the muscle (Supplementary
   Data [270]28), brain (Supplementary Data [271]29), and heart
   (Supplementary Data [272]30) are shown in Fig. [273]8g–i, respectively,
   and include carbon metabolism, oxidative phosphorylation, and
   neurodegenerative-related pathways.

Fig. 8. CGL dependent and independent persulfidation and pathway enrichment
in muscle, brain, and heart.

   [274]Fig. 8
   [275]Open in a new tab

   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 log[2](Fold Change
   DR:AL) X-axis displays the average fold change in spectral counts for
   each identified persulfidated protein while the −log[10] 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 −log[10] (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. [276]2, [277]4, [278]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. [279]8.

   Further examining the extent for CGL in shaping the persulfidome under
   DR, we compiled individual and shared persulfidated proteins from the
   five solid tissues in WT (Fig. [280]8j) and CGL KO mice (Fig. [281]8k).
   A total of 3837 proteins were plotted in WT and 2481 proteins in KO,
   resulting in an ~35% decrease due to loss of CGL. While the fold
   difference between the numbers of DR-enriched and AL-enriched
   persulfidated proteins meeting the statistical and biological
   thresholds were similar in WT (4.3-fold) and KO (4.2-fold), the
   proteins in CGL KO mice failed to fall under specific functional
   pathway enrichments. Proteins not meeting statistical and biological
   thresholds are prominently shifted towards enrichment under DR in WT
   mice, with 71% +/−20% with a DR:AL spectral count ratio above 1
   (Fig. [282]8j, Supplementary Data [283]31), while this same subset of
   persulfidated proteins is shifted towards enrichment under AL in CGL KO
   mice, with only 42% +/−30% having a DR:AL spectral count ratio above 1
   (Fig. [284]8k, Supplementary Data [285]31). Importantly, similar
   results were obtained via MS1 intensity analysis of this subset of
   persulfidated proteins from the five solid tissues (Fig. [286]8l), with
   72% +/−25% having a DR:AL intensity ratio above 1 in CGL WT mice
   (Supplementary Data [287]31), while in CGL KO mice only 38% +/−34% had
   a DR:AL intensity ratio above 1 (Supplementary Data [288]31). Thus,
   both label-free quantification mass spectrometry analysis techniques
   utilized ultimately produced similar results and highlighted the tissue
   specificity and dependence on CGL for transforming persulfidomes under
   DR.

Orthogonal protein and labeled peptide approaches show CGL dependence for
DR-induced persulfidation shifts

   Persulfidome profiles generated thus far were performed on protein
   level pulldown and subsequent label-free quantification (LFQ). While
   suitable for protein persulfidation identification and showing relative
   shifts between groups, these methods are not without their drawbacks.
   Mainly, protein-level pulldown lends to potential false positive
   identification due to intermolecular disulfide bonds, thus allowing a
   non-persulfidated protein to be co-captured with a persulfidated
   protein and eluted during the final reducing step^[289]39,[290]79.
   Conversely, false negatives are possible due to NM-Biotin bound
   non-persulfidated intramolecular thiols (RS-NM-Biotin) on the same
   protein as a labeled persulfidated cysteine residue (RS-S[n]-NM-Biotin)
   preventing final elution with a reducing agent^[291]79. While these
   caveats are valid, it has been hypothesized that concentrations of
   disulfide bonds in intracellular proteins are low and not expected to
   be a major source of false positives in the approach utilized^[292]39.
   Nonetheless, we pursued an orthogonal approach (Fig. [293]9a and
   Supplementary Fig. [294]9a) to avoid these limitations and further
   validate and expand our findings utilizing kidney and brain obtained
   from an independent cohort of 12-month-old CGL WT and KO mice fed 1
   week 50% DR.

Fig. 9. Orthogonal protein and peptide-based biotin thiol assay (BTA)
approaches show CGL dependence for DR-induced persulfidation shifts.

   [295]Fig. 9
   [296]Open in a new tab

   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 log[2](Fold Change WT:KO) X-axis displays the average fold
   change in intensity for each protein, and the −log[10] 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. [297]9.

   Direct comparison between WT and KO kidney utilizing protein level BTA
   revealed the persulfidome enriched in WT with 87% of all proteins
   having a higher LFQ intensity in WT over KO (Fig. [298]9b,
   Supplementary Fig. [299]9b, and Supplementary Data [300]32). Similarly,
   123 proteins in WT met the biological- and statistical-significance LFQ
   thresholds, versus only seven meeting similar thresholds in KO
   (Fig. [301]9b). Direct comparison between WT and KO brains utilizing
   protein level BTA resulted in a near-even split in total persulfidated
   proteins, with 55% skewed toward WT (Fig. [302]9c, Supplementary
   Fig. [303]9c, and Supplementary Data [304]33). These results are not
   surprising given: (1) The equal but opposite direction the kidney
   persulfidome is shifted under DR in 6-month-old CGL WT (76% toward DR;
   Fig. [305]2b) and KO mice (74% toward AL; Fig. [306]7e), and (2) The
   comparable brain persulfidome enrichments in 6-month-old CGL WT (87%
   toward DR; Fig. [307]2d) and KO mice (86% toward DR; Fig. [308]8b).

   We next examined CGL dependence for DR-induced shifts utilizing peptide
   level BTA (Fig. [309]9a). Tissue lysates underwent trypsinization to
   digest proteins and produce smaller peptides prior to affinity
   purification, thus overcoming potential intermolecular and
   intramolecular cysteine-related issues experienced in protein level
   BTA^[310]79. In kidney, 70% of peptides and resultant protein ID’s had
   higher LFQ intensities in WT over KO (Fig. [311]9d, Supplementary
   Fig. [312]9b, and Supplementary Data [313]34). Seven proteins in WT met
   the biological- and statistical-significance LFQ thresholds, versus
   only two meeting similar thresholds in KO (Fig. [314]9d). In brain, 74%
   of the peptides and resultant protein ID’s were skewed toward WT
   (Fig. [315]9e, Supplementary Fig. [316]9c, and Supplementary
   Data [317]35). Six proteins in WT met the biological- and
   statistical-significance LFQ thresholds, versus only one meeting
   similar thresholds in KO (Fig. [318]9e). Thus, peptide level pulldown
   showed CGL-dependent kidney persulfidation enrichment under DR similar
   to protein level pulldown, while it revealed a CGL dependence for brain
   persulfidation enrichment under DR that was not detected by protein
   level BTA analyses.

   Advancing the peptide level BTA, we applied multiplex iodoacetyl
   isobaric tandem mass tag (iodoTMT) labeling of sulfhydryl groups after
   final tris(2-carboxyethyl)phosphine (TCEP) elution of persulfidated
   (Fig. [319]9a and Supplementary Fig. [320]9a). These steps and
   approaches advance upon non-labeled peptide BTA as TCEP reduction and
   subsequent iodoTMT labeling provide quantitative identification of
   persulfidated proteins and peptide site-level analysis for the modified
   cysteine residues^[321]45,[322]80,[323]81. In both kidneys
   (Fig. [324]9f and Supplementary Data [325]36) and brain (Fig. [326]9g
   Supplementary Data [327]37) total, persulfidated peptides and resultant
   protein ID’s had higher iodoTMT intensities in WT over KO, with 100%
   enrichment in WT kidney and 80% enrichment in WT brain. Twelve proteins
   in WT kidney and two proteins in WT brain met the biological- and
   statistical-significance for WT:KO iodoTMT intensity ratio, while no
   proteins reached similar thresholds in KO tissues (Fig. [328]9f, g).
   Interestingly, when examining kidneys from AL fed WT and KO mice, the
   shift in iodoTMT intensities favored the KO group (68%; Supplementary
   Fig. [329]9d and Supplementary Data [330]38), despite decreased H[2]S
   production capacity in KO kidneys (Fig. [331]1b, c). This suggests: (1)
   Basal persulfidation activity under AL feeding or unstressed conditions
   is independent of CGL and/or CGL derived H[2]S, and (2) Enhanced
   persulfidation in select tissues relies not only on CGL derived H[2]S
   but also its coordination with other cysteine- and thiol-redox
   modifications induced by DR^[332]82,[333]83 or other
   stressors^[334]45,[335]84. Site-level analysis for the modified
   cysteine residues from peptide iodoTMT labeling is provided in the
   second tabs of Supplementary Data [336]36 for DR kidney, Supplementary
   Data [337]37 for DR brain, and Supplementary Data [338]38 for AL
   kidney.

Discussion

   Enhanced H[2]S production and signaling, particularly via CGL activity,
   has only recently been recognized as a common phenomenon and proposed
   mechanisms of action in models of longevity^[339]16,[340]36,[341]38.
   However, the mechanisms underlying the pleiotropic cellular,
   physiological, and systemic benefits of H[2]S have remained unclear. As
   aging is a multifaceted and complex decline in numerous physiological
   and metabolic functions, the role H[2]S plays in combating these
   declines theoretically should not be limited to one pathway or
   mechanism. Most likely, it is through H[2]S performing several
   non-mutually exclusive functions related to antioxidant and redox
   homeostasis^[342]85, mitochondrial electron transfer^[343]86, and
   protein modification and signaling via
   persulfidation^[344]39,[345]42,[346]43. In the current study, we
   focused on the latter mechanism, as little has been revealed regarding
   the entirety and scope of persulfidation in mammals across several
   organ systems while under an anti-aging intervention.

   Here, we found CGL dependent alterations in H[2]S production capacity
   and persulfidome enrichment induced by DR to vary based on tissue,
   summarized in Fig. [347]10. Utilizing three independent experimental
   mouse cohorts differing in age, diet type, diet duration, and/or strain
   background along with several variations of the BTA, we found DR
   enhanced the persulfidomes of liver, kidney, muscle, and brain while
   contracted those of heart. Dependence for CGL in diet-induced
   persulfidome expansion was most prominent in kidney and muscle, and to
   a certain extent in brain. While this is not the first study to
   recognize relative changes in mammalian protein persulfidation as a
   function of CGL or diet^[348]44,[349]87, it is the first to reveal
   individual identities and enrichment shifts of these proteins, their
   biological pathway involvement, sites of modification, and the
   interdependence of tissue-type, diet, and CGL to impact persulfidation
   profiles.

Fig. 10. Model depicting the impact of dietary restriction (DR) on endogenous
H[2]S production and protein persulfidation in tissue-specific and
CGL-dependent manners.

   Fig. 10
   [350]Open in a new tab

   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.

   While the results obtained in liver and kidney were predictable based
   upon their diet-malleable H[2]S production capacities, we did not
   expect increased muscle and brain persulfidation under DR. As both of
   these tissues fail to augment detectable H[2]S production capacity
   under DR, this suggests DR-mediated enhanced persulfidation may not be
   tissue autonomous and/or more complex than previously thought. The
   sources of H[2]S that enhance persulfidation in these weak-producing
   tissues may arise distally from liver or kidney, or from
   diet-modifiable H[2]S producing endothelial cells residing within the
   tissue^[351]33. While the idea of H[2]S being a circulating
   gasotransmitter in the blood is somewhat controversial^[352]88, our
   data show these weak producing tissues still somehow augment their
   persulfidomes under DR. The failure to detect increased H[2]S
   production capacity or enriched persulfidation in plasma may suggest DR
   does not induce systemic increases in sulfide availability, and local
   enrichment of persulfidation may be tissue autonomous via direct
   enzymatic persulfidation of proteins via CGL, CBS, and/or 3-MST or
   through local endothelial production. However, only plasma was assayed
   and not white and red blood cells, of which the latter act as a sink
   for H[2]S via exchange in the microcirculation^[353]89. Thus, we cannot
   entirely rule out DR-enhanced H[2]S in circulation as the mechanism for
   augmented persulfidation in marginally H[2]S producing tissues. Future
   studies are needed to test the requirement of direct protein–protein
   interactions for CGL, CBS, and 3-MST in DR-induced persulfidation
   changes.

   Additionally, the absolute number of persulfidated proteins in the
   brain and their expansion under DR was surprising due to the purported
   sensitivity of the central nervous system (CNS) to H[2]S-induced
   toxicity related to low or non-existent sulfide quinone oxidoreductase
   (SQR) expression^[354]90,[355]91. SQR is a key mitochondrial enzyme
   catalyzing initial steps of H[2]S oxidation, detoxification, and
   removal^[356]92,[357]93. However, H[2]S serves beneficial roles in the
   brain, as decreased H[2]S or disruption of H[2]S producing enzymes in
   the brain or neuronal cells are associated with aging-related
   Parkinson’s, Huntington’s, and Alzheimer’s disease-like
   phenotypes^[358]94, while controlled H[2]S exposure protects against
   cerebral ischemia-reperfusion injury^[359]95. This suggests the lack of
   SQR enables H[2]S that reaches or is produced in the CNS to be utilized
   primarily for protein persulfidation.

   Higher SQR activity in liver, kidney, or heart potentially limits
   protein persulfidation via the oxidation and removal of H[2]S. This may
   explain the decrease in persulfidation in heart upon DR, as H[2]S may
   impart its beneficial cardiac functions^[360]96,[361]97, particularly
   under ischemia-reperfusion injury, via SQR-mediated mitochondrial
   oxidation of H[2]S rather than persulfidation-centric mechanisms.
   Conversely, the decrease in cardiac persulfidation upon DR may be
   through potential depersulfidation of proteins via the thioredoxin
   (Trx) system^[362]79,[363]87. The function and potential benefit of
   removing sulfhydryl groups on proteins in cardiac tissue upon DR is not
   well understood or described in our study. However, Trx mediated
   cleavage of cysteine persulfides results in H[2]S release^[364]87.
   Thus, a potential hypothesis is that depersulfidation in the heart
   releases free H[2]S and/or polysulfides for potential beneficial use
   elsewhere, perhaps via SQR for mitochondrial protection and appropriate
   changes in metabolism and fuel usage during ischemia reperfusion.
   Further examination into Trx depersulfidase activity during DR, and if
   it is stunted in tissues with enhanced persulfidome profiles and
   boosted in tissues with decreased persulfidome profiles, is warranted.
   This also offers an alternative explanation for the changes in
   tissue-specific persulfidomes as a function of DR that is mutually
   exclusive of H[2]S production capacities.

   Reactive oxygen and nitrogen species (RONS), much like H[2]S, display
   hormetic dose responses and act as secondary messengers. Similarly,
   RONS production during DR through increased mitochondrial fatty acid
   oxidation is thought to drive many of the benefits of DR and
   DR-mimetics^[365]82,[366]98. In light of the requirement for cysteine
   residue activation via oxidation or a redox thiol switch to occur prior
   to persulfidation by H[2]S^[367]43–[368]45, it is appropriate to
   consider the interdependence of DR-mediated augmentation in RONS and
   H[2]S for promoting changes in the persulfidome during DR. Likewise,
   non-dietary means to extend lifespan in the worm Caenorhabditis elegans
   triggered generation of RONS and H[2]S, with both factors responsible
   for stress resistance and slowed aging^[369]84. Thus, metabolic and
   stress cues leading to increased endogenous H[2]S production also boost
   RONS production, and these RONS potentially prime cysteine residues for
   persulfidation. Additionally, enhanced H[2]S production may prevent
   further irreversible oxidation of cysteine residues while under
   stressful conditions such as DR. This hypothesis is further supported
   by our observations showing basal persulfidation activity in the kidney
   under AL feeding is independent of CGL despite there being a large
   differential in H[2]S production capacity in kidneys from AL fed CGL WT
   and KO mice, and it is not until DR is applied that concomitant
   enhanced persulfidation activity and H[2]S production capacity via CGL
   is detected. These findings also bring to light the importance of
   oxygen tension during mammalian cell culture experiments being factored
   in as a variable for persulfidation assays, as the typical 20% O[2]
   cell cultures are exposed to, rather than 1–11% found in vivo^[370]99,
   is hyperoxic and in itself may pose as a redox stressor impacting
   persulfidation readouts.

   There are several biological-related limitations to our study. First,
   we examined only whole organ lysates, thus region-specific
   persulfidation enrichments, particularly in highly structured and
   region-specific organs such as the brain, are not reported. Second, the
   pathway enrichment analyses simply show involvement of the
   persulfidated proteins in specific pathways, but the direction of
   metabolic flux or pathway activation/silencing is not inferred. Third,
   our experimental animal setup may limit or influence the persulfidomes
   identified, as we only used male mice and under conditions of
   fitness-improving diets. Changing sex, variables related to circadian
   patterns and time of tissue collection, and diet lipid composition
   could expand or contract persulfidation^[371]37,[372]100,[373]101.
   Likewise, it was recently reported that enhanced H[2]S production
   capacity under DR was only detected in mouse strains that gain
   longevity benefits under DR, so it would be interesting to study how
   persulfidation changes under DR in non-responding or
   negative-responding mouse strains^[374]102.

   There are also several technical-related limitations to our study. The
   first to address is the ratio of maleimide-biotin to protein added
   during the alkylation step in protein level BTA. Lower concentrations
   of maleimide-biotin result in selective labeling of the more highly
   reactive persulfidated cysteine groups versus the less reactive
   non-persulfidated cysteine thiol^[375]43. Increasing the ratio of
   maleimide-biotin to protein may underestimate persulfidated proteins
   identified by labeling both persulfidated cysteines and
   non-persulfidated cysteines on the same protein, thus preventing
   elution with DTT from the avidin columns. At the same time, this
   strategy offers more selective isolation of proteins with multiple
   persulfidation modifications. Here, we used a slightly higher ratio of
   maleimide-biotin to protein than utilized previously^[376]43. Thus, the
   latter scenario may hold true and altering maleimide-biotin
   concentrations during alkylation would give different persulfidome
   results. Second, similar to intramolecular cysteines providing
   challenges in protein level BTA, intermolecular disulfide bonds can
   result in potential false-positive identification due to
   non-persulfidated proteins co-captured with a persulfidated protein and
   elute during the reducing step^[377]39,[378]79. However, it has been
   hypothesized that this issue is not expected to be a major source of
   false positives^[379]39. Nonetheless, it still has the potential to
   artificially expand or mask persulfidome shifts. For these reasons, we
   pursued predigested peptide level BTA in Fig. [380]9, in which we
   detected diet- and CGL-dependent enrichments in tissue-specific
   persulfidomes similar to our protein level BTA, thus validating and
   expanding upon the data generated from the latter method.

   The third variable to address is the choice of alkylating agent. The
   use of maleimide derivatives to alkylate thiols and persulfides is well
   established^[381]45,[382]48,[383]50,[384]103,[385]104. Yet, a few
   studies indicate they also bind to sulfenic and sulfinic acids under
   specific experimental conditions^[386]105,[387]106, making way for
   potential false-positive persulfidation hits. However, these false
   positives may be minor as the interaction between maleimide derivatives
   and oxidized cysteine residues are not as stable under pH 7–8^[388]106,
   which is optimal pH for thiol and persulfide alkylation with
   maleimide^[389]107 and what we used in the current study. In addition,
   the reported low presence of oxidized cysteine species relative to
   thiols and persulfides limits the potential for false positives in the
   analysis of persulfides in the proteome^[390]105. Importantly, the
   concentration of maleimide derivatives used in the studies reporting
   interaction with oxidized cysteines ranged from 5 mM to 20 mM, which is
   14- to 58-fold higher than what was used in the present study,
   suggesting our lower concentration may avoid off-target labeling.
   Furthermore, in larger scale mammalian whole tissue cysteine
   modification studies, much like ours, maleimide derivatives
   preferentially alkylate and block R–SH groups and not R–SOH groups on
   proteins^[391]51. Given the enzymatic activity of CGL^[392]108 is
   likely to only prompt alterations of cysteines with a persulfide and
   not sulfenic, sulfinic, or sulfonic modifications, the relatively
   minimal impact of oxidized cysteines giving false-positive results
   within our methodology is leveraged by our use of CGL KO mice, in
   which: (1) There is a reduction in total protein persulfidation numbers
   in all tissues from KO mice, and (2) There is an attenuation of DR
   induced persulfidation in kidney and brain of KO mice as detected in
   direct WT versus KO studies utilizing protein and peptide level BTA.
   Thus, while false positives are conceivable, the diet-induced and
   tissue-specific proteomic shifts detected in a CGL-dependent manner in
   this study most likely infer changes in persulfidation.

   Thus, due to the dynamic rather than static nature of the persulfidome
   and the biological or technical experimental factors listed above, the
   generation of alternative persulfidome profiles is likely. Despite
   these limitations, our work provides a snapshot of these proteins and
   their persulfidation changes specific to tissue, diet, peptide site,
   and CGL status. Future work is needed to identify how these proteins
   are persulfidated; i.e., passive or targeted, commonalities or motifs
   in the peptide sequence that prompt for persulfidation, and the
   functional changes in protein stability, location, and activity induced
   by these modifications.

   In summary, we reveal the global persulfidation profile alterations in
   major metabolic organs under ad libitum and dietary restriction. We
   show the importance of CGL enzyme for protein persulfidation in
   multiple major organs. In addition to testing the necessity for CGL to
   shape baseline and diet-induced changes in persulfidomes, we also
   identified CGL as a target for persulfidation and its potential impact
   on increasing H[2]S generating capacity. Ultimately, this study
   establishes a data resource for dietary restriction and H[2]S fields
   while prompting the need to decipher downstream effects of these
   persulfidated proteins in metabolism, stress resistance, and longevity.

Methods

Animal husbandry and diet intervention

   All experiments were performed with approval of the Cleveland Clinic
   Institutional Animal Care and Use Committee, protocol # 2016–1778, and
   followed the National Institutes of Health Guide for the Care and Use
   of Laboratory Animals. In experiments utilizing male cystathionine
   gamma-lyase (CGL) wildtype (WT) and knockout (KO) mice, the mice were
   bred and weaned at 21–23 days of age and maintained under standard
   barrier housing in the Cleveland Clinic Lerner Research Institute on a
   14-h light/10-h dark cycle, temperature between 20–23 °C, 30–70%
   relative humidity, and with initial ad libitum access to standard
   rodent food (Envigo #2918) and drinking water until the dietary
   intervention. Experimental cystathionine γ-lyase (CGL) mice were
   obtained from initial parental CGL Het x Het breeding to generate CGL
   WT and KO F1s, and then breeding CGL WT x WT or CGL KO x KO F1s to
   generate the littermate and/or age-matched F2 experimental animals.
   Mice were group-housed with 3–5 mice per cage. The CGL WT and KO mice
   were originally generated on a mixed 129/C57BL/6 background as
   described previously^[393]25,[394]27 and then subsequently rederived
   into pathogen-free C57BL/6 mice at Jackson Laboratories prior to colony
   establishment at Cleveland Clinic. When mice were 6 or 12 months old
   they were switched to the experimental AIN-93G-based diet (Research
   Diets D10012G-2V-Formula 1) and exposed to ad libitum access for
   several days to adapt to the new food and for monitoring intake. The
   experimental diet consists of 20% of calories from protein
   (casein-based containing ∼2.9% methionine and 0.4% cysteine, with
   additional L-cystine supplemented to a final 1.5 g/1000 g final diet
   composition by Research Diets, Inc.), 64% of calories from
   carbohydrate, and 16% calories from fat, and importantly has 2x
   concentrations of mineral mix S10022G, vitamin mix V10037, and choline
   bitartrate to avoid potential micronutrient malnutrition during 50%
   dietary restriction. The powdered food mix was added in a 1:1 ratio
   (gram:mL) to a 2% agar (Sigma #A1296) solution in water before
   solidification to a semi-solid consistency that lessens the potential
   for food hoarding in group housing and increases the accuracy of food
   consumption measurement. Ad libitum food intake per cage was measured
   daily for up to 4 days to determine the correct amount to restrict to
   achieve 50% reduction in food intake. After randomly assigning ad
   libitum (AL) (n = 3–4/genotype/experiment) or diet restriction (DR)
   (n = 3–5/genotype/experiment) feeding to the cages, food intake, and
   body mass were measured over the 1-week intervention. AL fed mice were
   provided 24-h access to the diet and the DR mice fed their calculated
   allotment near the start of their dark phase at 7 pm to limit
   disturbances in circadian rhythms and feeding patterns between the two
   groups^[395]52,[396]53.

   For aged mice experiments testing every-other-day (EOD) fasting, male
   C57BL/6 mice were obtained between 60–65 weeks of age (Stock no.
   000664, Jackson Laboratories, Bar Harbor, ME) and group-housed (up to
   five same-sex mice per cage) in the Cleveland Clinic Lerner Research
   Institute Biological Resource Unit. The mice had ad libitum (AL) access
   to standard rodent chow (18.6% protein, 44.2% carbohydrate, and 6.2%
   fat; Teklad Global Rodent Diet #2918, Envigo, Madison, WI). At ∼20
   months of age, cages were randomly assigned to either EOD fasting
   (n = 5) or AL access (n = 5) to the standard rodent chow. The EOD
   fasting regimen consisted of repeated cycles of 24 h consecutive
   removal of food access with water always available (fast day) followed
   by 24-h access to food and water (fed day). The EOD fasting
   intervention proceeded for 2.5 months. To circumvent possible
   disturbances in circadian rhythms and feeding patterns in the Chow EOD
   group, the food was provided to or removed from the Chow EOD group just
   prior to the dark cycle onset at 7 pm.

   After the noted dietary intervention periods, mice were euthanized in
   the late morning (between 9am–12pm) via isoflurane anesthesia overdose
   followed by cervical dislocation prior to collecting liver, kidney,
   heart, muscle (quadriceps), brain, and plasma. Plasma was collected via
   retro-orbital bleed and the blood immediately placed into
   lithium-heparin-coated tubes (Terumo #T-MLH). Tubes were centrifuged to
   separate RBCs from the plasma. Collected tissues were immediately
   placed in 1.5 mL centrifuge tubes, flash frozen in liquid nitrogen, and
   then stored in the dark at −80 °C until further analysis. Subsequent
   analysis of tissues was by researchers not involved in the live animal
   experiments and was initially done blinded in regards to individual
   animal identify then unblinded for downstream assays and data analysis
   for grouping purposes.

Lead acetate/lead sulfide assay to detect enzymatic H[2]S production

   Tissue H[2]S production capacity was measured by the lead acetate/lead
   sulfide method^[397]25,[398]54. Briefly, ~80 mg of tissue is first
   placed in 1.5 mL microcentrifuge tubes containing 250 µL of 1x passive
   lysis buffer (Promega) and homogenized, followed by multiple rounds of
   flash freezing/thawing using liquid nitrogen. After homogenization and
   lysis, protein concentration was measured with bicinchoninic acid assay
   (BCA) kit (Bio-Rad) followed by normalization of proteins via
   additional 1x passive lysis buffer. Next, the lead acetate/lead sulfide
   assay is setup by initially preparing the reaction mixture of 10mM
   L-cysteine (Sigma #168149) and 1 mM pyridoxal phosphate (PLP) (Sigma
   #9255) in PBS, with 150 µL placed into each well of a 96-well plate.
   Hundred micrograms of protein from each tissue or 20 µL of plasma are
   added to each respective well, then the plate overlaid with lead
   acetate embedded filter paper and incubated at 37 °C until lead sulfide
   is detected for quantification using ImageJ (version: 1.51n)
   densitometry analysis via the IntDen function after subtracting
   background levels obtained from reaction mixture-only wells with no
   tissue or protein added. A similar approach was used to examine
   purified recombinant CGL-derived H[2]S production in vitro, with
   reactions run in 50 mM Tris (pH ~7.8) + 10 mM L-Cys, + 1 mM PLP,
   and + /– purified recombinant human CGL (Cayman Chemical Company
   #10329 + /– NaHS treatment in the given amounts and concentrations
   noted in the figure and figure legend.

Mouse gene expression profiles

   RNA expression graphs of H[2]S producing and consuming proteins were
   generated utilizing the Mouse ENCODE project^[399]55 with values
   extracted from the NCBI Mouse Gene Database at the following NCBI
   webpages: CGL: [400]https://www.ncbi.nlm.nih.gov/gene/107869, CBS:
   [401]https://www.ncbi.nlm.nih.gov/gene/12411, 3-MST:
   [402]https://www.ncbi.nlm.nih.gov/gene/246221, and SQR
   [403]https://www.ncbi.nlm.nih.gov/gene/59010.

Protein level biotin thiol assay (BTA)

   The isolation and detection of persulfidated proteins were performed
   using an adaptation to the biotin thiol assay (BTA) as previously
   described by Gao, et al.^[404]43. To isolate persulfidated proteins,
   tissues were first lysed with RIPA lysis buffer (Thermo Fisher
   Scientific, #89900) containing protease inhibitor cocktail (Thermo
   Fisher Scientific, #78415). Protein concentration was determined via
   BCA kit (Bio-Rad) and all concentrations normalized. Next, 7 mg of
   protein were incubated with 343 µM Maleimide-PEG2-biotin (Thermo Fisher
   Scientific, #21901BID) in RIPA buffer in a total volume of 500 µL at pH
   7.4 at room temperature for 30 min with agitation. Maleimide was chosen
   as the alkylating agent because it predominantly interacts with
   sulfhydryl groups (-SH and -SS[n]H) of cysteines versus other cysteine
   modifications in non-reducing buffers at physiological pH of
   7–7.5^[405]50,[406]103,[407]104. Precipitation of the proteins was done
   by adding 1 mL of 100% cold acetone at –20 °C for 30 min followed by
   centrifugation. Additional washes were performed with 1 mL of 75% cold
   acetone followed by centrifugation and removal of acetone. Proteins
   were then resuspended in 0.25 mL of suspension buffer (RIPA + 1% SDS,
   pH 7.5) followed by adding 0.75 mL of neutralization buffer (30 mM
   Tris, 1 mM EDTA, 150 mM NaCl, 0.5 % Triton X-100, pH 7.5). Prior to
   adding the alkylated proteins to the streptavidin-agarose resin
   containing spin columns, the columns (Pierce, Catalog no. 69705) were
   first washed with PBS two times, followed by adding 0.39 mL
   streptavidin-agarose resin (Thermo Scientific, #20347) to the column
   and washing the resin 1x with PBS. The resin was then equilibrated with
   a 1:4 suspension buffer:neutralization buffer washes four times prior
   to plugging the outlet of the column. Alkylated proteins were then
   added to the streptavidin-agarose resin containing spin columns and
   kept rotating overnight at 4 °C. After the overnight incubation, the
   protein resin mix in the columns was washed six times in 0.85 mL of
   wash buffer 1 (30 mM Tris, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100,
   pH 7.5) followed by another six washes with 0.85 mL of wash buffer 2
   (30 mM Tris, 1 mM EDTA, 600 mM NaCl, 0.5% Triton X-100, pH 7.5) and
   finally another three washes with 0.85 mL of wash buffer 3 (30 mM Tris,
   1 mM EDTA, 100 mM NaCl, pH 7.5). All washes were done using the Vac-Man
   Laboratory Vacuum Manifold (Promega A7231). Resin with bound proteins
   was first incubated with freshly prepared 500 µL elution buffer (30 mM
   Tris, 1 mM EDTA, 100 mM NaCL, pH 7.5) without DTT for 30 min at 25 °C
   followed by centrifugation of the spin tube inside of a 2 mL collection
   tube at 2000 g for 2 min to collect the (–) DTT negative control
   eluate. The same spin column was then placed into a new 2 mL collection
   tube and 500 µL of freshly prepared elution buffer containing 20 mM DTT
   was added for 30 min at 25 °C followed by centrifugation at 2000 g for
   2 min to elute persulfidated proteins. The addition of DTT in the final
   elution step selectively elutes persulfidated proteins, as it breaks
   the S–S bond but not the S–NM–Biotin bond at pH between
   7–7.5^[408]43,[409]48,[410]109, as can be seen with the –DTT control
   gel lanes having lower detectable proteins compared to the +DTT gel
   lanes (Supplementary Fig. 2b–f), supporting the effectiveness of the
   BTA method. Eluted proteins were run through Amicon Ultracel 10 K
   (Millipore, #UFC501096) to concentrate in a final volume of 50 µL.
   After obtaining these final eluates, subsequent gel electrophoresis
   analysis and mass spectrometry-based label-free protein identification
   is commenced to visualize, identify, and quantify the persulfidated
   proteins.

Peptide level BTA

   Seven milligrams of proteins were extracted and alkylated with 343 µM
   Maleimide-PEG2- biotin as described above in the protein level BTA
   assay. Alkylated proteins were precipitated with ice-cold acetone,
   resuspended in denaturation buffer (8 M urea, 1 mM MgSO4 and 30 mM
   Tris–HCl, pH 7.5). After denaturation for 10 min at 90 °C proteins were
   diluted with seven volumes of dilution buffer (1 mM CaCl2, 100 mM NaCl,
   and 30 mM HEPES-NaOH pH 7.5), followed by incubating with trypsin
   (Promega, #V5111) (ratio of 1:50 W/W) overnight at 37 °C. After
   overnight digestion, trypsin was heat inactivated at 95 °C for 10 min.
   Prior to adding the digested peptide to the streptavidin-agarose resin
   containing spin columns, the columns (Pierce, Catalog no. 69705) were
   first washed with PBS two times, followed by adding 0.400 mL
   streptavidin-agarose resin (Thermo Scientific, #20347) to the column
   and washing the resin 1x with PBS. The resin was then equilibrated with
   a 1:7 denaturation buffer: dilution buffer wash four times prior to
   plugging the outlet of the column and incubated at 4 °C with continuous
   rotation to mix for 18 h. After the overnight incubation, the protein
   resin mix in the columns was washed six times in 0.85 mL of wash buffer
   1 (30 mM Hepes, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, pH
   7.5) followed by another six washes with 0.85 mL of wash buffer 2
   (30 mM Hepes, 1 mM EDTA, 600 mM NaCl, 0.5% Triton X-100, 0.1% SDS, pH
   7.5) and finally another three washes with 0.85 mL of wash buffer 3
   (30 mM Hepes, 1 mM EDTA, 100 mM NaCl, pH 7.5). All washes were done
   using the Vac-Man Laboratory Vacuum Manifold (Promega A7231). Resin
   with bound peptide was eluted with or without DTT as described above in
   the protein level BTA, with the exception that no trypsin was added
   during the gel processing steps as the proteins were predigested prior
   to streptavidin capture.

Peptide level iodoTMT-BTA

   Seven milligrams of proteins were extracted and alkylated with 343 µM
   Maleimide-PEG2- biotin as described above in the protein level BTA
   assay. Alkylated proteins were precipitated with ice-cold acetone,
   resuspended in denaturation buffer (8 M urea, 1 mM MgSO4 and 30 mM
   Tris–HCl, pH 7.5). After denaturation for 10 min at 90 °C proteins were
   diluted with seven volumes of dilution buffer (1 mM CaCl2, 100 mM NaCl,
   and 30 mM HEPES-NaOH pH 7.5), followed by incubating with trypsin
   (Promega, #V5111) (ratio of 1:50 W/W) overnight at 37 °C. After
   overnight digestion, trypsin was heat inactivated at 95 °C for 10 min.
   Prior to adding the digested peptide to the streptavidin-agarose resin
   containing spin columns, the columns (Pierce, Catalog no. 69705) were
   first washed with PBS two times, followed by adding 0.400 mL
   streptavidin-agarose resin (Thermo Scientific, #20347) to the column
   and washing the resin 1x with PBS. The resin was then equilibrated with
   a 1:7 denaturation buffer: dilution buffer washes four times prior to
   plugging the outlet of the column and incubated at 4 °C with continuous
   rotation for better mixing for 18 h. After the overnight incubation,
   the protein resin mix in the columns was washed six times in 0.85 mL of
   wash buffer 1 (30 mM Hepes, 1 mM EDTA, 150 mM NaCl, 0.5% Triton X-100,
   0.1% SDS, pH 7.5) followed by another six washes with 0.85 mL of wash
   buffer 2 (30 mM Hepes, 1 mM EDTA, 600 mM NaCl, 0.5% Triton X-100, 0.1%
   SDS, pH 7.5) and finally another three washes with 0.85 mL of wash
   buffer 3 (30 mM Hepes, 1 mM EDTA, 100 mM NaCl, pH 7.5). All washes were
   done using the Vac-Man Laboratory Vacuum Manifold (Promega A7231).
   Resin with bound peptide was first incubated with freshly prepared
   500 µL elution buffer (30 mM Hepes, 1 mM EDTA, 100 mM NaCl, 10 mM TCEP,
   10 mM TEAB, pH 7.5) for 30 min at 25 °C followed by centrifugation of
   the spin tube inside of a 2 mL collection tube at 2000 g for 2 min to
   collect the eluate. A C18 column (Thermo #89870) was used to remove
   TCEP followed by eluting the peptides with 70% ACN. Eluted peptides
   were dried and suspended with 100 µl of 50 mM TEAB buffer. Thiol groups
   were alkylated and labeled with 2.95 mM iodoTMT (Thermo Scientific
   #90101) for 1 h in the dark. Reactions were then quenched by vortexing
   and incubating for 15 min at 37 °C in 20 mM DTT in the dark. Each of
   the six samples labeled with a unique iodoTMT tag was then mixed into a
   single tube and dried. The iodoTMT tags were iodoTMT^6-126,
   iodoTMT^6-127, iodoTMT^6-128, iodoTMT^6-129, iodoTMT^6-130, and
   iodoTMT^6-131. Finally, the combined labeled peptides were
   reconstituted with 1% Acetic Acid for further analyses via LC/MS.

Gel electrophoresis and in-gel digestion

   1.5 mm thick sodium dodecyl sulfate (SDS)-12% polyacrylamide gels were
   used to separate and visualize purified persulfidated proteins via
   staining or Western Blot. 0.75 mm thick one-dimensional (SDS)-12%
   polyacrylamide gels were used to separate persulfidated proteins for
   in-gel digestion and downstream HPLC-MS/MS analysis. For both of these
   processes, 11 µL of proteins from input, eluted –DTT samples, and +DTT
   samples, were mixed and denatured by boiling at 100 °C with 2 µL of 5x
   Laemmli loading buffer (Fisher Scientific, Catalog no. 39001) and
   loaded into each lane, with 7 µL of PageRuler Plus Prestained (Thermo
   Fisher #26619) protein ladder in the first lane. Gels were run at 135
   volts for 2 h for imaging only and Western blot endpoints, and for
   13–15 min for gels purposed for obtaining persulfidated proteins for
   in-gel digestion and HPLC-MS/MS analysis. Gels for imaging only were
   washed with ddH[2]O for 10 min, protein bands stained with colloidal
   blue staining kit (Thermo Fisher, #LC6025) for 3 h at room temperature,
   and followed by another 3 h of washing with ddH[2]O. Gels were then
   scanned (Epson Perfection V500 scanner) to obtain images. Proteins in
   gels used for Western Blotting were then transferred to polyvinylidene
   difluoride membranes (Whatman), blotted for GAPDH (Abcam #ab8245,
   1:1000 in 5% milk) followed by horseradish peroxidase-conjugated goat
   anti-mouse secondary antibody (Invitrogen #62-6520, diluted 1:5000 in
   5% milk) or α-tubulin (Abcam #ab4074, 1:1000 in 5% milk) followed by
   horseradish peroxidase-conjugated goat anti-rabbit IgG secondary
   antibody (Abcam #97051, 1:5000 in 5% milk) and visualized using
   SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific
   #34096) on an Amersham Imager 600 (General Electric). Gels used for
   in-gel digestion and downstream HPLC-MS/MS analysis were first cut for
   size and then washed for half an hour with ddH[2]O at room temperature.
   Protein bands were visualized by staining with gel code blue stain
   reagent (Thermo Fisher scientific, #24590) for 1 h followed by washing
   with ddH[2]O for one hour at room temperature. Gels were scanned by
   using the Epson Scanner. A scalpel was used to excise the entire band,
   and then the band was further cut into smaller bands of ∼1 mm^3. All of
   the smaller gel pieces from the original band were transferred to
   0.2 mL of wash buffer (50% ethanol and 5% acetic acid) in a 1.5 mL tube
   and washed overnight at room temperature. The following day gel pieces
   were dehydrated with acetonitrile and dried in a SpeedVac for 6 min.
   Proteins in the dried bands were reduced with 0.1 mL of 65 mM
   dithiothreitol (DTT) (Fisher Scientific # BP172–5) at room temperature
   for 30 min, followed by alkylation for another 30 min at room
   temperature with 0.1 mL of 162 mM iodoacetamide (Fisher Scientific,
   #AC122270050). Gel pieces were dehydrated two times with 0.2 mL
   acetonitrile and rehydration with 0.2 mL of 100 mM ammonium
   bicarbonate. Then, 40 µL of 10 ng/µL trypsin (Promega, #V5111) was
   added into the dried gel pieces with an additional 20 µL of 50 mM
   ammonium bicarbonate added to completely cover the gel pieces and kept
   overnight at room temperature for tryptic digestion. 0.08 mL of
   extraction buffer (50% acetonitrile + 5% formic acid) was added twice
   for 10 min at room temperature each time to extract the digested
   peptides from the gel pieces followed by micro centrifuging for 10 s.
   Supernatants were combined and transferred into a new 0.5 mL centrifuge
   tube and dried at room temperature in a Speedvac for 3 h. Thirty
   microliters of 1% acetic acid was added into the dried peptides and
   transferred into an HPLC vial w/cap (Sun-Sri, #200 050 & 501 313) for
   further mass spectrometric analysis.

HPLC-tandem mass spectrometric analysis

   Digested peptides were analyzed on a ThermoFisher Scientific UltiMate
   3000 HPLC system (ThermoFisher Scientific, Bremen, Germany) interfaced
   with a ThermoFisher Scientific Orbitrap Fusion Lumos Tribrid mass
   spectrometer (Thermo Scientific, Bremen, Germany). Liquid
   chromatography was performed prior to MS/MS analysis for peptide
   separation. The HPLC column used is a Dionex 15 cm × 75 µm Acclaim
   Pepmap C18, 2 μm, 100 Å reversed-phase capillary chromatography column.
   Five microliter volumes of the peptide extract were injected and
   peptides eluted from the column by a 90-min acetonitrile/0.1% formic
   acid gradient at a flow rate of 0.30 μL/min and introduced to the
   source of the mass spectrometer on-line. Nano electrospray ion source
   was operated at 2.3 kV. Tissue extracted non-labeled protein digests
   were analyzed using the data-dependent multitask capability of the
   instrument acquiring full scan mass spectra using a Fourier Transform
   (FT) in the Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo
   Scientific, Bremen, Germany) to determine peptide molecular weights and
   collision-induced dissociation (CID) MS/MS product ion spectra with an
   ion-trap analyzer to further determine the amino acid sequence in
   successive instrument scans. The MS method used in this study was a
   data-dependent acquisition (DDA) with 3 s duty cycle. It includes one
   full scan at a resolution of 120,000 followed by as many MS/MS scans as
   possible on the most abundant ions in that full scan. Dynamic exclusion
   was enabled with a repeat count of 1 and ions within 10 ppm of the
   fragmented mass were excluded for 60 s. Data using the iodoTMT 6-plex
   labeled peptide samples were also collected using a TMT-MS2 method in
   the same Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo
   Scientific, Bremen, Germany). This was a data dependent acquisition
   method using higher-energy collisional dissociation (HCD) as the
   fragmentation method for MS/MS scans. It included one full scan
   followed by as many MS/MS scans as possible at collision energy 38 in a
   3 s duty cycle on the most abundant ions in that full scan. The
   detector of MS full scans was the Orbitrap at a resolution of 120,000,
   and the detector of MS/MS scans was the Orbitrap at a resolution of
   50,000. Dynamic exclusion was enabled with a repeat count of 1, and
   ions within 10 ppm of the fragmented mass were excluded for 60 s.
   Instrumental quality performance, aka quality control 1 (QC1), was
   periodically monitored via Pierce Retention Time Calibration Mixture
   (Pierce #88320) ([411]Supplementary Data File 39, with raw data
   uploaded to Figshare at
   [412]https://figshare.com/s/941a9d9bc9cd39b0dac6 and
   [413]https://figshare.com/s/f613f80460bce4eb722d).

Bioinformatics for peptide identification and quantification

   Mascot, Sequest, and Scaffold software packages were used for
   label-free quantitative and qualitative proteomics analysis. These
   software tools were used for protein identification by converting raw
   spectrometric data into protein IDs and for relative quantification of
   protein abundance via label-free spectral counting. For protein
   identification, three search engines were used including Mascot,
   Sequest which is bundled into Proteome Discoverer 1.4, and X!Tandem
   which is bundled into Scaffold 4.8.7. For the Mascot searches, primary
   raw MS/MS data were converted into its MGF (Mascot Generic File) format
   files by using Discoverer Daemon 1.4 software (Licensed under Cleveland
   Clinic Proteomics Core). The parameters selected for these searches
   include the following: Database: SwissProt
   ([414]https://www.ebi.ac.uk/uniprot/), Taxonomy: Mus Musculus, Enzyme:
   Trypsin, Fixed modification: Carbamidomethyl(C), Variable modification:
   Oxidation of Methionine, Precursor Mass Tolerance: 10 ppm, Fragment
   Mass Tolerance: 0.8 Da, Peptide charge: 2+, 3+, and 4+, with the
   monoisotopic and decoy database present. Scaffold software (version
   Scaffold_4.8.7, Proteome Software Inc, Portland, OR)^[415]110 was used
   to validate peptide and protein identifications and create
   comprehensive lists of target proteins. Protein and peptide
   identifications were validated using a decoy database strategy (reverse
   sequence of each protein for use as a decoy) and the decoy rate was set
   to zero, which may limit the total number of proteins identified but
   enables a higher confidence in protein identity for those that do meet
   all stringent criteria. Other settings for Scaffold included only
   allowing positive IDs if they could be established at greater than
   99.9% probability and contained at least two identified peptides by the
   Peptide Prophet algorithm^[416]111,[417]112 with Scaffold delta-mass
   correction. Proteins that contained similar peptides and could not be
   differentiated based on MS/MS analysis alone were grouped to satisfy
   the principles of parsimony. Additionally, to remove any false positive
   hits, we individually analyzed all of the proteins identified to ensure
   each one contained at least one cysteine residue by using the mouse
   protein amino acid sequences from UniProt Knowledgebase protein
   database (Proteome_ID/Tax_ID: UP000000589/10090
   [[418]https://www.uniprot.org/proteomes/UP000000589]). Those that did
   not contain at least one cysteine residue, and thus could not
   theoretically be persulfidated (~2–4% of our initial findings), were
   removed from our database and from further analysis. Peptide
   identifications were also required to exceed specific database search
   engine thresholds, including Mascot identifications required at least
   40 ion score, Sequest identifications required at least XCorr (+1) is
   1.5, XCorr (+2) is 2, XCorr (+3) is 2.25, and XCorr(+4) is 2.5, and
   X!Tandem identifications required at least 2.

   Label-free spectral counting was used to determine relative differences
   in persulfidated protein abundance between AL versus DR fed groups
   within a specific tissue^[419]57. The spectral counts, defined as the
   total number of spectra identified for a specific protein^[420]113,
   have integer values ranging from 0 for proteins below the level of
   detection up to 753. Theoretically, higher spectral counts for a given
   protein equates to higher abundance of that protein in a given tissue
   sample, where the number of spectra matched to peptides from a specific
   protein is used as a surrogate measure of protein abundance^[421]114.
   The average spectral counts for each protein in each tissue were
   calculated, and the ratio of average spectral counts amongst the
   control group (e.g., AL, WT) compared to the experimental group (e.g.,
   DR, EOD, KO) were used to determine fold-change between the two groups.
   Spectral count values below the level of detection were originally
   automatically assigned a value of 0, however, these were changed to 0.1
   for purposes of statistical analysis and plotting in log scale in
   volcano plots.

   We also used MaxQuant version 1.6.14.0 with Perseus version 1.6.12.0
   for further analysis and validation of raw data files for label-free
   intensity-based quantitation rather than spectral counting with the
   following settings. For variable modifications, we allowed acetyl
   (protein N-term) and methionine oxidation. As a fixed modification we
   selected carbamidomethylation on cysteine residues as all samples were
   treated with iodoacetamide and a maximum number of modifications
   allowed were 5. For the digestion mode, we used the enzymatic rule of
   trypsin/P with a maximum of two missed cleavages. We used Maxquant
   standard settings for instrument type setup. We used label-free
   quantification with the minimum ratio count of 2 and the normalization
   was set to none. We used the 17,053 reviewed proteins present in the
   mouse proteome downloaded from Uniprot on April 13, 2019, as a search
   FASTA file ([422]https://www.ebi.ac.uk/uniprot/). We selected minimum
   peptide length as 6 and maximum peptide mass as 5000 Da with minimum
   peptide length for unspecified search as 8. Protein FDR was set at
   0.01, minimum peptides were set as 2 and minimum unique peptides set as
   1. For protein quantification we used unique and razor peptides and
   allowed oxidation (M) and acetyl (protein N-term) modifications.
   Intensity values below the level of detection or given a value of 0
   were changed to 10,000 for purposes of statistical analysis and
   plotting in log scale in volcano plots.

   MS raw data files of iodoTMT labeled peptides were analyzed by Proteome
   Discoverer version 2.4.1.15 (Thermo Fisher Scientific) with the
   following settings. We used Mus musculus in SwissProt version 2019
   (Swiss Institute of Bioinformatics, Switzerland) FASTA file. For
   quantification method, we used iodoTMT6plex with the TMT reporter ion
   isotope distribution (lot number VH308730). For processing setup, we
   used integration tolerance as 20 ppm and integration method set to the
   most confident centroid. FTMS mass analyzer was used, and we selected
   MS1 precursor for the precursor selection section. For the enzyme name,
   we used trypsin (full) with a maximum missed cleavage site of 2.
   Precursor mass tolerance was set to 10 ppm and fragment mass tolerance
   was set to 0.2 Da. For dynamic modification, we allowed methionine
   oxidation (+15.995 Da). We selected static modification as
   iodoTMT6plex/+329.227 Da of cysteine. For consensus setup, we used
   intensity as reporter abundance. Validation mode was set to automatic
   (control peptide level error rate if possible). Target FDR (strict) for
   PSMs and target FDR (strict) for peptide were set at 0.01, while Target
   FDR (relaxed) for PSMs and target FDR (relaxed) for peptide set at
   0.05. Peptide confidence was set to high with minimum peptide length
   allowed to 6. For confidence threshold, we used target FDR (strict) and
   target FDR (relaxed) at 0.01 and 0.05, respectively. Minimum PSM
   confidence level was set to high.

   Raw proteomics-related data have been uploaded to the ProteomeXchange
   Consortium via the PRIDE partner repository with the following
   accession codes [423]PXD022888 (Proteome Discoverer 2.4 based
   analysis), [424]PXD022956 (MaxQuant 1.6.14.0 based analysis), and
   [425]PXD022954 (Scaffold 4.8.7 based analysis).

Biological function and pathway enrichment

   The online-based web server g:Profiler
   ([426]https://biit.cs.ut.ee/gprofiler/gost)^[427]59 was used for
   functional enrichment analysis of identified persulfidated proteins.
   Gene ID’s or accession numbers of enriched proteins were used in the
   g:GOSt (Gene Group Functional Profiling) identifier tool to detect
   biological pathways from the KEGG database^[428]60
   ([429]https://www.genome.jp/kegg/) significantly enriched with
   persulfidated proteins. These significance values are given a threshold
   of p < 0.05 and were auto-calculated by the software’s proprietary
   built-in g:SCS algorithm
   ([430]https://biit.cs.ut.ee/gprofiler/page/docs) that utilizes multiple
   testing correction for P values gained from pathway enrichment
   analysis. In setting the boundaries for significance determination, it
   corresponds to an experiment-wide threshold of a = 0.05, with the g:SCS
   threshold pre-calculated for list sizes up to 1000 accession number or
   gene ID terms and analytically approximates a threshold t corresponding
   to the 5% upper quantile of randomly generated queries of that size. As
   per the g:Profiler description, all actual P values resulting from the
   query are automatically transformed to corrected p values by
   multiplying these to the ratio of the approximate threshold t and the
   initial experiment-wide threshold a = 0.05 with consideration to the
   underlying gene sets annotated to terms from the KEGG database, and,
   therefore, gives a tighter threshold to significant results and is more
   conservative than the Benjamini–Hochberg False Discovery Rate
   (FDR)^[431]59.

Statistical analysis

   Statistical significance and data display was generated in Microsoft
   Excel (version 2013), GraphPad Prism (version 5), Origin (version
   2018), and Venny 2.1.0 software. The differences between two diet
   groups within the same genotype in regards to H[2]S production or
   spectral counts for individual persulfidated proteins were analyzed by
   two-sided Student’s t test, with a level of significance set to
   P < 0.05. N values for the number of individual mice used are as
   follows (unless noted in the figure legend): (1) 6-month old CGL WT and
   KO mice: 4 mice for WT AL, 5 mice for WT DR, 3 mice for KO AL, and 3
   mice for KO DR, (2) 12-month-old CGL WT and KO mice: 3 mice for WT DR,
   and 3 mice for KO DR, and (3) 20-month C57BL/6 mice: 5 mice for AL, and
   5 mice for EOD fasting. Data were arranged in Microsoft Excel to show
   both relative fold-changes in average spectral counts for an individual
   protein, as well as the two-sided t test P value, and these data are
   found in the [432]Supplementary Data files. Bar-graph data in
   Fig. [433]1 are displayed as means + /–SEM with n values between 3 and
   5 as indicated in the figure legend. Analysis of recombinant CGL H[2]S
   production was by one-way ANOVA with Dunnet’s Multiple Comparison Test
   against the +CGL w/0 NaHS group after subtracting against the –CGL spot
   corresponding for that given dose of NaHS. Data are plotted as
   individual points with the mean and SEM shown. Origin software version
   2018 ([434]https://www.originlab.com/) was used to generate volcano
   plots to display differentially abundant persulfidated proteins in
   tissues from AL versus DR fed mice. Venny 2.1.0 software
   ([435]https://bioinfogp.cnb.csic.es/tools/venny/) was used to generate
   Venn diagrams and determine differences and commonalities in
   persulfidated proteins between tissues, diets, and genotypes.
   Statistical analysis for pathway enrichment via g:SCS algorithm is
   described in the above section regarding the g:Profiler online
   functional enrichment analysis tool. The individual calculated/adjusted
   P values and identities of proteins for each enriched pathway generated
   by g:Profiler is found in the [436]Supplementary Data files. All
   measurements in this study were taken from distinct individual
   biological samples/animals and not the same sample measured repeatedly
   nor multiple samples taken from the same animal.

Reporting summary

   Further information on research design is available in the [437]Nature
   Research Reporting Summary linked to this article.

Supplementary information

   [438]Supplementary Information^ (1.8MB, pdf)
   [439]Peer Review File^ (3.2MB, pdf)
   [440]41467_2021_22001_MOESM3_ESM.docx^ (20.7KB, docx)

   Description of Additional Supplementary Files
   [441]Supplementary Data 1^ (343.9KB, xlsx)
   [442]Supplementary Data 2^ (389.2KB, xlsx)
   [443]Supplementary Data 3^ (173KB, xlsx)
   [444]Supplementary Data 4^ (318.1KB, xlsx)
   [445]Supplementary Data 5^ (301.4KB, xlsx)
   [446]Supplementary Data 6^ (22.6KB, xlsx)
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   [458]Supplementary Data 18^ (219.8KB, xlsx)
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   [464]Supplementary Data 24^ (109.6KB, xlsx)
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   [470]Supplementary Data 30^ (109.8KB, xlsx)
   [471]Supplementary Data 31^ (13.1KB, xlsx)
   [472]Supplementary Data 32^ (159.3KB, xlsx)
   [473]Supplementary Data 33^ (126.7KB, xlsx)
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   [475]Supplementary Data 35^ (47KB, xlsx)
   [476]Supplementary Data 36^ (169.9KB, xlsx)
   [477]Supplementary Data 37^ (128KB, xlsx)
   [478]Supplementary Data 38^ (179.1KB, xlsx)
   [479]Supplementary Data 39^ (28.7KB, xlsx)
   [480]Reporting Summary^ (390KB, pdf)

Acknowledgements