Discovery of a Redox Thiol Switch: Implications for Cellular Energy Metabolism
2020; Elsevier BV; Volume: 19; Issue: 5 Linguagem: Inglês
10.1074/mcp.ra119.001910
ISSN1535-9484
AutoresXing‐Huang Gao, Ling Li, Marc Parisien, Jing Wu, Ilya Bederman, Zhaofeng Gao, Dawid Krokowski, Steven M. Chirieleison, Derek W. Abbott, Benlian Wang, Peter Arvan, Mark J. Cameron, Mark R. Chance, Belinda Willard, Maria Hatzoglou,
Tópico(s)Biochemical effects in animals
ResumoThe redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress. The redox-based modifications of cysteine residues in proteins regulate their function in many biological processes. The gas molecule H2S has been shown to persulfidate redox sensitive cysteine residues resulting in an H2S-modified proteome known as the sulfhydrome. Tandem Mass Tags (TMT) multiplexing strategies for large-scale proteomic analyses have become increasingly prevalent in detecting cysteine modifications. Here we developed a TMT-based proteomics approach for selectively trapping and tagging cysteine persulfides in the cellular proteomes. We revealed the natural protein sulfhydrome of two human cell lines, and identified insulin as a novel substrate in pancreatic beta cells. Moreover, we showed that under oxidative stress conditions, increased H2S can target enzymes involved in energy metabolism by switching specific cysteine modifications to persulfides. Specifically, we discovered a Redox Thiol Switch, from protein S-glutathioinylation to S-persulfidation (RTSGS). We propose that the RTSGS from S-glutathioinylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress. Hydrogen sulfide (H2S) is a gas molecule that can be produced endogenously in many organisms from bacteria to mammals (1Paul B.D. Snyder S.H. H(2)S signalling through protein sulfhydration and beyond.Nat. Rev. Mol. Cell Biol. 2012; 13: 499-507Crossref PubMed Scopus (508) Google Scholar, 2Paul B.D. Snyder S.H. H2S: A Novel Gasotransmitter that Signals by Sulfhydration.Trends Biochem. Sci. 2015; 40: 687-700Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). H2S production at physiological concentrations is cytoprotective, as it reduces blood pressure (3Yang G. Wu L. Jiang B. Yang W. Qi J. Cao K. Meng Q. Mustafa A.K. Mu W. Zhang S. Snyder S.H. Wang R. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase.Science. 2008; 322: 587-590Crossref PubMed Scopus (1716) Google Scholar), prevents neurodegeneration and extends lifespan (2Paul B.D. Snyder S.H. H2S: A Novel Gasotransmitter that Signals by Sulfhydration.Trends Biochem. Sci. 2015; 40: 687-700Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 4Hine C. Harputlugil E. Zhang Y. Ruckenstuhl C. Lee B.C. Brace L. Longchamp A. Trevino-Villarreal J.H. Mejia P. Ozaki C.K. Wang R. Gladyshev V.N. Madeo F. Mair W.B. Mitchell J.R. Endogenous hydrogen sulfide production is essential for dietary restriction benefits.Cell. 2015; 160: 132-144Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 5Kabil O. Banerjee R. Redox biochemistry of hydrogen sulfide.J. Biol. Chem. 2010; 285: 21903-21907Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 6Miller D.L. Roth M.B. Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 20618-20622Crossref PubMed Scopus (159) Google Scholar). H2S reacts with and modifies protein cysteine thiol groups to form persulfide bonds, known as protein S-persulfidation (also referred as S-sulfhydration). Because this gas is unstable, regulation of its synthesis can transiently alter protein cysteine modifications with an impact on cellular metabolism (7Mustafa A.K. Gadalla M.M. Sen N. Kim S. Mu W. Gazi S.K. Barrow R.K. Yang G. Wang R. Snyder S.H. H2S signals through protein S-sulfhydration.Sci. Signaling. 2009; 2: ra72Crossref PubMed Scopus (802) Google Scholar, 8Gao X.H. Krokowski D. Guan B.J. Bederman I. Majumder M. Parisien M. Diatchenko L. Kabil O. Willard B. Banerjee R. Wang B. Bebek G. Evans C.R. Fox P.L. Gerson S.L. Hoppel C.L. Liu M. Arvan P. Hatzoglou M. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.eLife. 2015; 4: e10067Crossref PubMed Scopus (89) Google Scholar). It has been recognized as a great challenge to detect and quantify protein S-persulfidation in vivo, because of the high reactivity and instability of the cysteine persulfide bond in proteins (2Paul B.D. Snyder S.H. H2S: A Novel Gasotransmitter that Signals by Sulfhydration.Trends Biochem. Sci. 2015; 40: 687-700Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Mishanina T.V. Libiad M. Banerjee R. Biogenesis of reactive sulfur species for signaling by hydrogen sulfide oxidation pathways.Nat. Chem. Biol. 2015; 11: 457-464Crossref PubMed Scopus (306) Google Scholar). Earlier, we developed a proteomics approach (8Gao X.H. Krokowski D. Guan B.J. Bederman I. Majumder M. Parisien M. Diatchenko L. Kabil O. Willard B. Banerjee R. Wang B. Bebek G. Evans C.R. Fox P.L. Gerson S.L. Hoppel C.L. Liu M. Arvan P. Hatzoglou M. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.eLife. 2015; 4: e10067Crossref PubMed Scopus (89) Google Scholar) exploiting the biotin thiol assay (BTA) to distinguish other types of cysteine-based PTMs from persulfidated proteins. We used this approach to show that the increase of H2S synthesis during endoplasmic reticulum (ER) stress in pancreatic beta cells promotes selective S-persulfidation of redox sensitive cysteine residues of proteins engaged in specific metabolic pathways (8Gao X.H. Krokowski D. Guan B.J. Bederman I. Majumder M. Parisien M. Diatchenko L. Kabil O. Willard B. Banerjee R. Wang B. Bebek G. Evans C.R. Fox P.L. Gerson S.L. Hoppel C.L. Liu M. Arvan P. Hatzoglou M. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.eLife. 2015; 4: e10067Crossref PubMed Scopus (89) Google Scholar). However, the BTA approach can only be used to compare in parallel two different biological samples. Because of differences in the ionization efficiency and/or detectability of the many peptides in each sample, the changes of the resulting data between two samples cannot accurately reflect relative differences in their persulfidation levels. To cope with the challenges of large-scale proteomics analyses, we combined the BTA assay with the iodoacetyl isobaric tandem mass tag system (iodoTMT) 1The abbreviations used are:iodoTMTiodoacetyl isobaric tandem mass tags3-MST3-mercaptopyruvate sulfurtransferaseAlaAlaineALDOAFructose-bisphosphate aldolaseAspAspartic acidATF4Activating transcription factor 4BioGEEBiotinylated glutathione ethyl esterBTA-TMTBiotin thiol assay conjugated with tandem mass tag systemCBScystathionine β synthaseCitCitric acidCRISPRclustered regularly interspaced short palindromic repeatsCTHcystathionine γ lyaseDAVIDBioinformatics clustering with a pathway annotation programDHAPDihydroxyacetone phosphateENO1Enolase 1FBPFructose-1,6-bisphosphateFDRFalse discovery rateFumFumaric acidG3PGlyceraldehyde 3-phosphateGAPDHGlyceraldehyde 3-phosphate dehydrogenaseGFPGreen fluorescent proteinGluGlutamic acidGSHReduced glutathioneGSISGlucose-stimulated insulin secretionGSSGOxidized glutathioneHPDP-BiotinN-[6-(Biotinamido)hexyl]-3 ′-(2 ′-pyridyldithio) propionamideIAMIodoacetamideIDHIsocitrate dehydrogenaseIHHHuman immortalized human hepatocytesINS1rat pancreatic beta cellsKEGGKyoto Encyclopedia of Genes and GenomesKRBKrebs-Ringer Modified BufferLacLactic acidLCLiquid chromatographyLDHA and LDHBlactate dehydrogenaseLFQLabel-free MS quantificationMalMaleic acidMANFMesencephalic astrocyte-derived neurotrophic factorMAT1AMethionine adenosyltransferase 1AMIN6mouse pancreatic beta cellsMSMass spectrometryNaHSSodium hydrosulfideNEMN-EthylmaleimideNM-BiotinMaleimide-PEG2-BiotinOAAOxaloacetic acidOGDHOxoglutarate dehydrogenasePCK2Mitochondrial phosphoenolpyruvate carboxykinasePKM2Pyruvate kinase 2PLPPyridoxal phosphatePPPPentose phosphate pathwayPyrPyruvateS-SGS-glutathionylated cysteineS-SHS-persulfidated cysteineSerSerinesgRNASingle guide RNASucSuccinic acidTCACitric acid cycleTCEPTris-carboxyethyl phosphine hydrochlorideTEABTriethylammonium bicarbonateTFATrifluoroacetic acidα-KGα-Ketoglutaric acid. 1The abbreviations used are:iodoTMTiodoacetyl isobaric tandem mass tags3-MST3-mercaptopyruvate sulfurtransferaseAlaAlaineALDOAFructose-bisphosphate aldolaseAspAspartic acidATF4Activating transcription factor 4BioGEEBiotinylated glutathione ethyl esterBTA-TMTBiotin thiol assay conjugated with tandem mass tag systemCBScystathionine β synthaseCitCitric acidCRISPRclustered regularly interspaced short palindromic repeatsCTHcystathionine γ lyaseDAVIDBioinformatics clustering with a pathway annotation programDHAPDihydroxyacetone phosphateENO1Enolase 1FBPFructose-1,6-bisphosphateFDRFalse discovery rateFumFumaric acidG3PGlyceraldehyde 3-phosphateGAPDHGlyceraldehyde 3-phosphate dehydrogenaseGFPGreen fluorescent proteinGluGlutamic acidGSHReduced glutathioneGSISGlucose-stimulated insulin secretionGSSGOxidized glutathioneHPDP-BiotinN-[6-(Biotinamido)hexyl]-3 ′-(2 ′-pyridyldithio) propionamideIAMIodoacetamideIDHIsocitrate dehydrogenaseIHHHuman immortalized human hepatocytesINS1rat pancreatic beta cellsKEGGKyoto Encyclopedia of Genes and GenomesKRBKrebs-Ringer Modified BufferLacLactic acidLCLiquid chromatographyLDHA and LDHBlactate dehydrogenaseLFQLabel-free MS quantificationMalMaleic acidMANFMesencephalic astrocyte-derived neurotrophic factorMAT1AMethionine adenosyltransferase 1AMIN6mouse pancreatic beta cellsMSMass spectrometryNaHSSodium hydrosulfideNEMN-EthylmaleimideNM-BiotinMaleimide-PEG2-BiotinOAAOxaloacetic acidOGDHOxoglutarate dehydrogenasePCK2Mitochondrial phosphoenolpyruvate carboxykinasePKM2Pyruvate kinase 2PLPPyridoxal phosphatePPPPentose phosphate pathwayPyrPyruvateS-SGS-glutathionylated cysteineS-SHS-persulfidated cysteineSerSerinesgRNASingle guide RNASucSuccinic acidTCACitric acid cycleTCEPTris-carboxyethyl phosphine hydrochlorideTEABTriethylammonium bicarbonateTFATrifluoroacetic acidα-KGα-Ketoglutaric acid., thus enabling quantitative identification of persulfidated proteins from multiple samples. TMT is one of the most popular multiplexing methods, which uses isobaric tags for simultaneous labeling of peptides for identification and relative quantification by mass spectroscopy (10McAlister G.C. Huttlin E.L. Haas W. Ting L. Jedrychowski M.P. Rogers J.C. Kuhn K. Pike I. Grothe R.A. Blethrow J.D. Gygi S.P. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses.Anal. Chem. 2012; 84: 7469-7478Crossref PubMed Scopus (344) Google Scholar). iodoacetyl isobaric tandem mass tags 3-mercaptopyruvate sulfurtransferase Alaine Fructose-bisphosphate aldolase Aspartic acid Activating transcription factor 4 Biotinylated glutathione ethyl ester Biotin thiol assay conjugated with tandem mass tag system cystathionine β synthase Citric acid clustered regularly interspaced short palindromic repeats cystathionine γ lyase Bioinformatics clustering with a pathway annotation program Dihydroxyacetone phosphate Enolase 1 Fructose-1,6-bisphosphate False discovery rate Fumaric acid Glyceraldehyde 3-phosphate Glyceraldehyde 3-phosphate dehydrogenase Green fluorescent protein Glutamic acid Reduced glutathione Glucose-stimulated insulin secretion Oxidized glutathione N-[6-(Biotinamido)hexyl]-3 ′-(2 ′-pyridyldithio) propionamide Iodoacetamide Isocitrate dehydrogenase Human immortalized human hepatocytes rat pancreatic beta cells Kyoto Encyclopedia of Genes and Genomes Krebs-Ringer Modified Buffer Lactic acid Liquid chromatography lactate dehydrogenase Label-free MS quantification Maleic acid Mesencephalic astrocyte-derived neurotrophic factor Methionine adenosyltransferase 1A mouse pancreatic beta cells Mass spectrometry Sodium hydrosulfide N-Ethylmaleimide Maleimide-PEG2-Biotin Oxaloacetic acid Oxoglutarate dehydrogenase Mitochondrial phosphoenolpyruvate carboxykinase Pyruvate kinase 2 Pyridoxal phosphate Pentose phosphate pathway Pyruvate S-glutathionylated cysteine S-persulfidated cysteine Serine Single guide RNA Succinic acid Citric acid cycle Tris-carboxyethyl phosphine hydrochloride Triethylammonium bicarbonate Trifluoroacetic acid α-Ketoglutaric acid. iodoacetyl isobaric tandem mass tags 3-mercaptopyruvate sulfurtransferase Alaine Fructose-bisphosphate aldolase Aspartic acid Activating transcription factor 4 Biotinylated glutathione ethyl ester Biotin thiol assay conjugated with tandem mass tag system cystathionine β synthase Citric acid clustered regularly interspaced short palindromic repeats cystathionine γ lyase Bioinformatics clustering with a pathway annotation program Dihydroxyacetone phosphate Enolase 1 Fructose-1,6-bisphosphate False discovery rate Fumaric acid Glyceraldehyde 3-phosphate Glyceraldehyde 3-phosphate dehydrogenase Green fluorescent protein Glutamic acid Reduced glutathione Glucose-stimulated insulin secretion Oxidized glutathione N-[6-(Biotinamido)hexyl]-3 ′-(2 ′-pyridyldithio) propionamide Iodoacetamide Isocitrate dehydrogenase Human immortalized human hepatocytes rat pancreatic beta cells Kyoto Encyclopedia of Genes and Genomes Krebs-Ringer Modified Buffer Lactic acid Liquid chromatography lactate dehydrogenase Label-free MS quantification Maleic acid Mesencephalic astrocyte-derived neurotrophic factor Methionine adenosyltransferase 1A mouse pancreatic beta cells Mass spectrometry Sodium hydrosulfide N-Ethylmaleimide Maleimide-PEG2-Biotin Oxaloacetic acid Oxoglutarate dehydrogenase Mitochondrial phosphoenolpyruvate carboxykinase Pyruvate kinase 2 Pyridoxal phosphate Pentose phosphate pathway Pyruvate S-glutathionylated cysteine S-persulfidated cysteine Serine Single guide RNA Succinic acid Citric acid cycle Tris-carboxyethyl phosphine hydrochloride Triethylammonium bicarbonate Trifluoroacetic acid α-Ketoglutaric acid. In the current study, we developed the TMT-BTA approach. With this approach, we show distinct patterns of the natural protein sulfhydromes in human pancreatic beta cells and hepatocytes, with hepatocytes exhibiting higher levels of persulfidated proteins compared with pancreatic beta cells. However, human pancreatic beta cells exhibited a unique landscape of persulfidated proteins, with the most enriched pathways being in intermediary metabolism. Furthermore, we identified insulin, the beta cell specific protein, as a S-persulfidation target. The presence and extent of the conversion of S-persulfidation to other cysteine-based PTMs such as S-glutathionylation and vice versa in the proteome, is largely unknown. However, examples of proteins being glutathionylated and persulfidated on the same cysteine residue have been reported (7Mustafa A.K. Gadalla M.M. Sen N. Kim S. Mu W. Gazi S.K. Barrow R.K. Yang G. Wang R. Snyder S.H. H2S signals through protein S-sulfhydration.Sci. Signaling. 2009; 2: ra72Crossref PubMed Scopus (802) Google Scholar, 11Yun J. Mullarky E. Lu C. Bosch K.N. Kavalier A. Rivera K. Roper J. Chio I.I. Giannopoulou E.G. Rago C. Muley A. Asara J.M. Paik J. Elemento O. Chen Z. Pappin D.J. Dow L.E. Papadopoulos N. Gross S.S. Cantley L.C. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.Science. 2015; 350: 1391-1396Crossref PubMed Scopus (444) Google Scholar, 12Shenton D. Grant C.M. Protein S-thiolation targets glycolysis and protein synthesis in response to oxidative stress in the yeast Saccharomyces cerevisiae.Biochem. J. 2003; 374: 513-519Crossref PubMed Scopus (0) Google Scholar). One such example is the protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which has been shown to have mixed disulfides at the redox sensitive cysteine residue150 (13Hwang N.R. Yim S.H. Kim Y.M. Jeong J. Song E.J. Lee Y. Lee J.H. Choi S. Lee K.J. Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions.Biochem. J. 2009; 423: 253-264Crossref PubMed Scopus (111) Google Scholar). Glutathionylation of GAPDH at cysteine150 destabilizes the tertiary structure of the protein and decreases its enzymatic activity (13Hwang N.R. Yim S.H. Kim Y.M. Jeong J. Song E.J. Lee Y. Lee J.H. Choi S. Lee K.J. Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions.Biochem. J. 2009; 423: 253-264Crossref PubMed Scopus (111) Google Scholar). In contrast, persulfidation of cysteine150, increases the catalytic activity of the protein (7Mustafa A.K. Gadalla M.M. Sen N. Kim S. Mu W. Gazi S.K. Barrow R.K. Yang G. Wang R. Snyder S.H. H2S signals through protein S-sulfhydration.Sci. Signaling. 2009; 2: ra72Crossref PubMed Scopus (802) Google Scholar). It is not known if there is a switch from one modification to another on the same cysteine residue, neither is known the physiological consequence for such a Redox Thiol Switch (RTS). Using the TMT-BTA approach and an in vitro assay, we show the existence of an RTS, which involves H2S-mediated reversal of S-glutathionylation to S-persulfidation (RTSGS) of specific cysteine residues in a subset of glutathionylated proteins. Finally, in order to understand the physiological significance of this RTSGS in cellular energy metabolism, we induced protein S-glutathionylation in pancreatic beta cells treated with diamide and measured metabolic flux of glucose. It is well known that protein S-glutathionylation can inhibit the activities of metabolic enzymes (14Mieyal J.J. Gallogly M.M. Qanungo S. Sabens E.A. Shelton M.D. Molecular mechanisms and clinical implications of reversible protein S-glutathionylation.Antioxidants Redox Signaling. 2008; 10: 1941-1988Crossref PubMed Scopus (411) Google Scholar), and cause a decrease in the metabolic flux of glucose (15Hiranruengchok R. Harris C. Diamide-induced alterations of intracellular thiol status and the regulation of glucose metabolism in the developing rat conceptus in vitro.Teratology. 1995; 52: 205-214Crossref PubMed Scopus (17) Google Scholar). In agreement with the RTSGS mechanism contributing to regulation of energy metabolism, exposure of cells to H2S, rescued the inhibited glucose flux in cells treated with diamide. We conclude that RTSGS, the redox thiol switch from S-glutathionylation to S-persulfidation is a potential mechanism to fine tune cellular energy metabolism in response to oxidative stress. CBS: Abnova, H00000875–001p CTH: Sigma, HPA023300 ATF4: Santa Cruz Biotechnology, SC-200 GAPDH: Santa Cruz Biotechnology, SC-32233 (6C5) GSH: Virogen, 101-A (D8 clone) MANF: Icosagen AS, 310–100 3MST: Santa Cruz Biotechnologies, SC-376168 PCK2: Cell Signaling, #6294 Human pancreatic beta cells (EndoC-BH3) were purchased from the Univercell Biosolutions (Paris, France). The EndoC-BH3 cells were cultured in DMEM containing 5.6 mm glucose, 2% BSA fraction V, 50 μm 2-mercaptoethanol, 10 mm nicotinamide, 5.5 μg/ml transferrin, 6.7 ng/ml sodium selenite, penicillin (100 units/ml) and streptomycin (100 μg/ml). Ten μg/ml of puromycin (selective antibiotic) were added in the complete medium. The cells were seeded onto matrigel- and fibronectin-coated culture plates at 4 × 106 cells/plate. MIN6 and INS1 cells were cultured as described previously (8Gao X.H. Krokowski D. Guan B.J. Bederman I. Majumder M. Parisien M. Diatchenko L. Kabil O. Willard B. Banerjee R. Wang B. Bebek G. Evans C.R. Fox P.L. Gerson S.L. Hoppel C.L. Liu M. Arvan P. Hatzoglou M. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.eLife. 2015; 4: e10067Crossref PubMed Scopus (89) Google Scholar) Glucose-stimulated insulin release was assayed INS1 cells as described previously (16Haataja L. Snapp E. Wright J. Liu M. Hardy A.B. Wheeler M.B. Markwardt M.L. Rizzo M. Arvan P. Proinsulin intermolecular interactions during secretory trafficking in pancreatic beta cells.J. Biol. Chem. 2013; 288: 1896-1906Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) Sample preparation and analysis were based on (8Gao X.H. Krokowski D. Guan B.J. Bederman I. Majumder M. Parisien M. Diatchenko L. Kabil O. Willard B. Banerjee R. Wang B. Bebek G. Evans C.R. Fox P.L. Gerson S.L. Hoppel C.L. Liu M. Arvan P. Hatzoglou M. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.eLife. 2015; 4: e10067Crossref PubMed Scopus (89) Google Scholar). In brief, proteins were extracted from cells with RIPA buffer (150 mm NaCl, 1 mm EDTA, 0.5% Triton X-100, 0.5% deoxycholic acid, and 100 mm Tris pH 7.5) containing protease and phosphatase inhibitors. 2 mg of protein was incubated with 100 μm NM-biotin (Pierce) for 30 min, then mixed with Streptavidin-agarose resin (Thermo Scientific) and kept rotating overnight at 4 °C. The beads were washed and eluted with DTT or TCEP (10 mm). Eluted proteins were concentrated to a final volume of 25–40 μl with using of Amicon Ultracel 10K (Millipore), and used for gel electrophoresis followed by Western blot analysis. Proteins (2 mg) were extracted and biotinylated as described above. Biotinylated 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) diluted with 10 volumes of buffer containing 1 mm CaCl2, 100 mm NaCl and 30 mm HEPES-NaOH pH 8.0, then incubated with MS-grade trypsin (Thermo, 90058) with occasional mixing for 18 h at 37 °C. The ratio of the enzyme to substrate was 1:40 (w/w). After digestion, trypsin was inactivated by incubation at 95 °C for 10 min, then reactions were mixed with streptavidin-agarose beads (0.5–2 ml) and incubated at 4 °C for 18 h following extensive washes in the presence of 0.1% SDS. Peptides were eluted with the buffer containing 10 mm TEAB with 10 mm TCEP. TCEP was removed with using a C18 column (Thermo). Peptides were eluted from the desalting column with 80% methanol, dried under vacuum, and suspended in buffer (50 mm TEAB buffer pH 8.0, Sigma). Free SH groups were alkylated by iodoTMT reagents at final volume of 150 μl. After 1h of TMT labeling under dark at 37 °C, addition of DTT (20 mm) terminated the alkylation reaction. The alkylated peptides were mixed and combined. After desalting through C18 columns, the mixed peptides were eluted with 70% methanol for LC-MS analysis. This iodoTMT 6plex labeled peptide sample was reconstituted into 30 μl 1% acetic acid and was ready for MS analysis. The LC-MS system was a Thermo Ultimate 3000 UHPLC interfaced with a ThermoFisher Scientific Fusion Lumos tribrid mass spectrometer system. The HPLC column was a Dionex 15 cm × 75 μm id Acclaim Pepmap C18, 2 μm, 100 Å reversed-phase capillary chromatography column. Five μl volumes of the extract were injected, and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.3 μl/min were introduced into the source of the mass spectrometer on-line. The nanospray ion source was operated at 1.9 kV. The digest was analyzed using both TMT-MS2 method and TMT-MS3 method. The TMT-MS2 is a data dependent acquisition method using HCD fragmentation for MS/MS scans of the precursor ions selected from MS1 full scan. The MS2 spectra were used for peptide identifications and the low mass region (100–140) of the same spectra was used simultaneously for quantifications of these peptides. The TMT-MS3 method uses a technique called synchronized precursor selection (SPS) that can only be achieved on Thermo Fusion series MS. The method is also a data dependent acquisition using CID fragmentation for the MS2 scan of precursors selected from MS1. Then consecutive MS3 HCD scans at 100–500 m/z were performed on a combination of several of the most abundant ions (set at 10 in this study) selected from the MS2 scans. The CID MS2 spectra were used for peptide identifications and the HCD MS3 spectra were used for peptide quantifications. Data from iodoTMT 6plex samples were searched against UniprotKB protein sequence database of proper species using Sequest program in the Thermo Proteome Discoverer V2.1 software package. Trypsin was used as the protease, and the maximum number of missed cleavage was set to 2. Peptide precursor mass tolerance was set to 10 ppm, and fragment mass tolerance was set to either 0.02 Th if Orbitrap was used as the detector or 0.6 Th if ion trap was used as the detector. Oxidation of Methionine and acetylation of protein N-terminus were set as Dynamic Modifications, and iodoTMT6plex of Cysteine was set as static modification. The false discovery rate (FDR) of peptide identification was set to 1%. Post translational modifications were identified using Sequest program in the Thermo Proteome Discoverer V2.1 software. For the detection of sulfide modification, protease and mass tolerance settings were the same as described in Database Search Parameters section, and in modification settings, Oxidation of Methionine, iodoTMT6plex of Cysteine, Sulfidation of Cysteine, and acetylation of protein N-terminus were set as Dynamic Modifications. The false discovery rate (FDR) of peptide identification was set to 1%. Reporter abundance was quantified using the Thermo Proteome Discoverer V2.1 based on intensity and average reporter S/N threshold was set to 10. Normalization mode was set to total peptide amount. The settings on reporter ions quantifier node were: integration tolerance was set to 20 ppm, integration method was set to most confident centroid. Sample preparation and analysis were based on (17Gao X.H. Bedhomme M. Veyel D. Zaffagnini M. Lemaire S.D. Methods for analysis of protein glutathionylation and their application to photosynthetic organisms.Mol. Plant. 2009; 2: 218-235Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Briefly, pancreatic beta cells were seeded in 102-cm dishes and cultured overnight. Cells were washed once with PBS and then incubated for 2.5 h at 37 °C in culture medium containing 0.03 mm BioGEE for 2.5 h. The cells were then washed twice, and the cells were lysed on ice with lysis buffer containing 10 mm NEM and protease inhibitor. 2 mg of protein was mixed with Streptavidin-agarose resin (Thermo Scientific) and kept rotating overnight at 4 °C. The beads were washed and eluted with TCEP (10 mm). Eluted proteins were concentrated to a final volume of 25–40 μl with using of Amicon Ultracel 10K (Millipore), and used for gel electrophoresis followed by Western blot analysis. For pathway annotation: same as described previously (8Gao X.H. Krokowski D. Guan B.J. Bederman I. Majumder M. Parisien M. Diatchenko L. Kabil O. Willard B. Banerjee R. Wang B. Bebek G. Evans C.R. Fox P.L. Gerson S.L. Hoppel C.L. Liu M. Arvan P. Hatzoglou M. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.eLife. 2015; 4: e10067Crossref PubMed Scopus (89) Google Scholar). The pathways were scored based on persulfidated peptides using DAVID (www.david.ncifcrf.gov) program. Statistical significant of pathways are calculated, and pathways are ranked by the p values based on those tests. For redox cysteine annotation: persulfidated peptide identified by MS, all exact matches in any of the RedoxDB database (https://biocomputer.bio.cuhk.edu.hk/RedoxDB/) on any oxidative modification cysteine sequences were collected. The gel contains two samples derived from IHH and EndoC-BH3 cells. Each gel lane was cut into eight areas, these gel bands were washed/destained in 50% ethanol, 5% acetic acid and then dehydrated in acetonitrile. The bands were then reduced with DTT and alkylated with iodoacetamide prior to the in-gel digestion. All bands were digested in-gel using trypsin, by adding 5 μl 10 ng/μl chymotrypsin in 50 mm ammonium bicarbonate and incubating overnight digestion at room temperature to achieve complete digestion. The peptides that were formed were extracted from the polyacrylamide in two aliquots of 30 μl 50% acetonitrile with 5% formic acid. These extracts were combined and evaporated to <10 μl in Speedvac and then resuspended in 1% acetic acid to make up a finalvolume of ∼30 μl for LC-MS analysis. The LC-MS system was a ThermoScientific Fusion Lumos mass spectrometry system. The HPLC column was a Dionex 15 cm × 75 μm id Acclaim Pepmap C18, 2 μm, 100 Å reversed-phase capillary chromatography column. Five μl volumes of the extract were injected, and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 0.3 μl/min were introduced into the source of the mass spectrometer on-line. The microelectrospray ion source is operated at 2.5 kV. The digest was analyzed using the data dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans. These digests were analyzed utilizing LC gradient from 2 to 70% acetonitrile i
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