Supplement to Discovery of a redox thiol switch: implications for cellular energy metabolism

Supplemental Data

Table S2: Assessment of the TMT-BTA assay - MIN6 cell extracts treated NaHS (100 μM) were subjected to the TMT-BTA assay. After biotinylation and trypsin digestion, the peptide sample was divided into six equal fractions. Each fraction was incubated with the same amount of streptavidin beads; the biotinylated proteins were incubated with or without TCEP for 45 min. 3 replicates for each condition. The eluates were labeled with iodoTMT 6plex reagents, and then subjected for LC-MS analysis. The samples treated with TCEP were labeled by iodoTMT129-131, the samples treated without TCEP were labeled by iodoTMT 126-128.
Table S3: Quantitative profiling of protein sulfhydromes in MIN6 cells by the TMT-BTA assay. - Mouse pancreatic beta cells were incubated with increasing concentrations of NaHS followed by the TMT-BTA method. Control without NaHS (iodoTMT126), 5uM (iodoTMT127), 20 uM (iodoTMT128), 80 uM (iodoTMT129), 200 uM (iodoTMT131).
Table S4: Quantitative profiling of protein sulfhydromes between human hepatocytes and pancreatic beta cells by the TMT-BTA approach. - 3 biological replicates of each cell type. Human hepatocytes (IHH, iodoTMT126-128),human pancreatic beta cells (endoC-BH3, iodoTMT129-131).
Table S5: KEGG pathway of highly enriched persulfided proteins from IHH and EndoC-BH3 cells. - KEGG pathway of highly enriched persulfided proteins from IHH and EndoC-BH3 cells.
Table S6: Label-free MS quantification (LFQ) to determine the relative protein abundance in human hepatocytes (IHH) and pancreatic beta cells (EndoC). - Label-free MS quantification (LFQ) to determine the relative protein abundance in human hepatocytes (IHH) and pancreatic beta cells (EndoC).
Table S7: KEGG pathway of most abundant proteins in IHH and EndoC-BH3 cells. - The most abundant proteins with log2 ratios greater than 2 were selected for the pathway analysis.
Table S8: Quantitative profiling of protein sulfhydromes in rat pancreatic beta cells (INS1) expressed ATF4 transcription factor. - 3 biological replicates of each condition. The TMT-BTA assay was applied to IINS1 cells expressed ATF4 (iodoTMT129-131) or GFP (iodoTMT126-128).
Table S9: Quantitative profiling of protein sulfhydromes in response to the depletion of CTH in ATF4-expressing INS1 cells. - 3 biological replicates. The TMT-BTA assay was applied to the CTH KO (sgCTH, iodoTMT129-131) or control (EV, iodoTMT126-128) INS1 cells expressed ATF4.
Table S10: Identification of S-glutathionylated proteins by the BST assay. - Identification of S-glutathionylated proteins by the BST assay.
Table S11: Absolute metabolite flux of metabolites from MIN6 cells - Absolute metabolite flux of metabolites from MIN6 cells treated as follows: 0.1 mM diamide, 0.1 mM diamide and 10 μM NaHS. Control as untreated.
Table S12: Evaluation of the changes in protein S-persulfidation and S-glutathionylation in rat pancreatic beta cells (INS1) upon exposure to diamide followed by H2S treatment. - The BioGEE and BTA assays were employed to identify S-glutathionylated and S-persulfidated proteins. Eluates were labeled by iodoTMT 6plex tags followed a LC-MS quantification. The BTA assay was employed for identification of S-persulfidated proteins. Control of untreated (iodoTMT126), Diamide-treated (iodoTMT127), diamide and NaHS-treated (iodoTMT128). BioGEE assay was employed for identification of S-glutathionylated proteins. Control of untreated (iodoTMT129), diamide-treated (iodoTMT130), diamide and NaHS-treated (iodoTMT131).
Table S13: Identification and quantification of RTSGS protein targets from S-glutathionylation to S-persulfidation. - Lysates from MIN6 cells were exposed to GSSG. An equal amount of GSSG-treated MIN6 cell extracts were incubated with NaHS at different concentrations 10 (iodoTMT126-127), 50 (iodoTMT128-129) and 100 μM (iodoTMT130-131) in duplicates, then subjected to the TMT-BTA assay for LC-MS analysis.
Table S14: Most enriched KEGG pathway among all the TMT-labeled proteins from BioGEE and BTA assays - Most enriched KEGG pathway among all the TMT-labeled proteins from BioGEE and BTA assays.
Table S15: KEGG pathway of the RTSGS target proteins with great redox sensitive cysteine residues to H2S - KEGG pathway of the RTSGS target proteins with great redox sensitive cysteine residues to H2S
supplemental figures - All supplemental figures and supplemental legends from the main manuscript PDF