Dietary Restriction Transforms the Mammalian Protein Persulfidome in a Tissue-specific and Cystathionine γ-lyase-dependent Manner

Title: Dietary restriction transforms the mammalian protein persulfidome in a tissue-specific and cystathionine γ-lyase-dependent manner

Journal: Nature Communications

Published: 2021

Background

This study presents the first systematic mapping of the S-persulfidome across multiple murine tissues using high-resolution chemical proteomics. By employing biotin-switch labeling and LC-MS/MS analysis, the research team quantitatively compared alterations in protein S-persulfidation levels in liver, brain, and muscle tissues between normal diet and caloric restriction conditions. The results demonstrated that caloric restriction significantly enhances S-persulfidation of mitochondrial enzymes and antioxidant proteins, suggesting this modification plays a pivotal role in regulating energy metabolism, antioxidant responses, and lifespan extension.

The study highlights the robust potential of persulfidome analysis in elucidating metabolic regulatory mechanisms, and validates the applicability of mass spectrometry for site-specific quantification. This work establishes a reproducible experimental paradigm for persulfidome profiling in disease models or pharmacological interventions.

Materials & Methods

S-persulfidation Figure 1: 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) H2S 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.

Protein level biotin thiol assay

Mouse tissues were lysed in RIPA buffer containing protease inhibitors, and protein concentrations were normalized using a BCA assay. Approximately 7 mg of total protein were incubated with 343 µM Maleimide–PEG2–biotin in RIPA buffer (pH 7.4) for 30 minutes at room temperature. Maleimide specifically reacted with thiol groups (–SH and –SSnH). Proteins were precipitated with cold acetone, washed, and resuspended in RIPA/SDS suspension buffer, then neutralized in Tris/EDTA/NaCl/Triton X-100 buffer. Biotin-labeled proteins were incubated overnight with streptavidin–agarose resin at 4 ℃. Bound proteins were washed sequentially with buffers of increasing salt concentration to remove non-specific binders. Elution was first performed without DTT (–DTT control), followed by elution with 20 mM DTT to selectively release persulfidated proteins by reducing S–S bonds. Eluted proteins were concentrated with Amicon filters (10 kDa cutoff), then analyzed by SDS-PAGE and LC–MS/MS for identification and quantification of persulfidated proteins.

Results

S-persulfidation Figure 2: 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.

S-persulfidation Figure 3: 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).

S-persulfidation Figure 4: 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. 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.

Conclusions

This study provides a comprehensive characterization of protein S-persulfidation using a refined Biotin Thiol Assay (BTA) combined with high-resolution proteomics. By optimizing selective thiol labeling and DTT-dependent elution, the authors successfully isolated and quantified persulfidated proteins across multiple tissues and physiological conditions. The findings highlight S-persulfidation as a widespread and dynamic post-translational modification that modulates redox signaling, energy metabolism, and stress response. This work establishes a robust analytical framework for studying redox-dependent cysteine modifications and underscores the biological importance of persulfidation in maintaining cellular homeostasis and metabolic adaptation.

Reference

Bithi, N., Link, C., Henderson, Y. O., Kim, S., Yang, J., Li, L., Wang, R., Willard, B., & Hine, C. (2021). Dietary restriction transforms the mammalian protein persulfidome in a tissue-specific and cystathionine γ-lyase-dependent manner. Nature communications, 12(1), 1745. https://doi.org/10.1038/s41467-021-22001-w

* For Research Use Only. Not for use in diagnostic procedures.

Our customer service representatives are available 24 hours a day, 7 days a week. Inquiry

From Our Clients

"I recently used their proteomics service for a project analyzing protein interactions in yeast models. The team was very responsive and helped clarify the methodology they employed, which made me feel confident in the results. The data quality was solid, with clear identification of several key proteins involved in our study. Their thorough analysis enabled me to pinpoint specific interactions that I hadn't considered before, which significantly improved the direction of my research. I appreciate their professionalism and support throughout the process."

Sarah Thompson, University of California, Berkeley

"Our lab collaborated with them on a project studying cancer biomarkers. The proteomics analysis provided was detailed and focused, specifically highlighting the differential expression of proteins between healthy and tumor samples. Their clear explanations of the data helped my team understand the biological implications. I also appreciated their willingness to revise the reports based on our feedback, ensuring that we had everything we needed for our publication. This collaborative spirit was invaluable."

Emily Rodriguez, Stanford University

"Our lab worked with them on a project studying the effects of diet on gut microbiota using proteomics. They used a label-free quantification method to analyze proteins in fecal samples before and after dietary intervention. The results showed significant changes in protein expression linked to microbial activity. This was pivotal for our hypothesis about diet-microbiota interactions. The clarity of their data presentation made it easy for our team to integrate these findings into our ongoing research."

Dr. Lisa Wong, University of Toronto

"My experience with Creative Proteomics during the mass spectrometry analysis was excellent. We sent in human saliva and mouse brain tissue samples, which they expertly analyzed using both LC-MS and GC-MS techniques. The results were invaluable, revealing key metabolites in the saliva and identifying biomarkers linked to brain function in the brain tissue."

Dr. Emily Carter, Senior Research Scientist

"The overall service from Creative Proteomics was outstanding. They made the entire process seamless and efficient, allowing us to focus on our research. We worked with leaf and root samples from various Arabidopsis genotypes for targeted metabolomics analysis. Their thorough profiling of primary and secondary metabolites gave us important insights into how the plants respond metabolically to environmental stress."

Dr. Laura Henderson, Plant Physiologist

"We had a pleasant collaboration with Creative Proteomics on mass spectrometry analysis of lipids. They conducted a detailed analysis of lipid species, providing us with important insights into lipid metabolism and its relationship with metabolic syndrome disease states."

Dr. Sarah Mitchell, Research Scientist

Online Inquiry

Please submit a detailed description of your project. We will provide you with a customized project plan to meet your research requests. You can also send emails directly to for inquiries.

Great Minds Choose Creative Proteomics