cysTMTRAQ—An Integrative Method for Unbiased Thiol-based Redox Proteomics

Abstract

Protein redox regulation plays important roles in many biological processes. Protein cysteine thiols are sensitive to redox changes and may function as redox switches, which turn signaling and metabolic pathways on or off to ensure speedy responses to environmental stimuli or stresses. Here we report a novel integrative proteomics method called cysTMTRAQ that combines two types of isobaric tags, cysteine tandem mass tags and isobaric tag for relative and absolute quantification, in one experiment. The method not only enables simultaneous analysis of cysteine redox changes and total protein level changes, but also allows the determination of bona fide redox modified cysteines in proteins through the correction of protein turnover. Overlooking the factor of protein-level changes in the course of protein posttranslational modification experiments could lead to misleading results. The capability to analyze protein posttranslational modification dynamics and protein-level changes in one experiment will advance proteomic studies in many areas of biology and medicine. Protein redox regulation plays important roles in many biological processes. Protein cysteine thiols are sensitive to redox changes and may function as redox switches, which turn signaling and metabolic pathways on or off to ensure speedy responses to environmental stimuli or stresses. Here we report a novel integrative proteomics method called cysTMTRAQ that combines two types of isobaric tags, cysteine tandem mass tags and isobaric tag for relative and absolute quantification, in one experiment. The method not only enables simultaneous analysis of cysteine redox changes and total protein level changes, but also allows the determination of bona fide redox modified cysteines in proteins through the correction of protein turnover. Overlooking the factor of protein-level changes in the course of protein posttranslational modification experiments could lead to misleading results. The capability to analyze protein posttranslational modification dynamics and protein-level changes in one experiment will advance proteomic studies in many areas of biology and medicine. Changes in the redox states of protein cysteine thiols serve as regulatory switches in diverse biological processes (1.Go Y.M. Jones D.P. Mapping the cysteine proteome: analysis of redox-sensing thiols.J. Biol. Chem. 2013; 288: 26512-26520Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The redox cycle is regulated by well-known factors such as the ferredoxin-thioredoxin and glutathione-glutaredoxin systems, which reduce oxidized cysteines. Other oxidoreductases and oxidants such as reactive oxygen species act primarily to oxidize cysteine thiol groups (2.Ghezzi P. Bonetto V. Redox proteomics: identification of oxidatively modified proteins.Proteomics. 2003; 3: 1145-1153Crossref PubMed Scopus (218) Google Scholar, 3.Barty J.W. Hampton M.B. Winterbourn C.C. Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells.Biochem. J. 2005; 389: 785-795Crossref PubMed Scopus (127) Google Scholar). In order to map and quantify cysteine redox modifications on the proteome scale, several approaches and methods have been developed, mostly using thiol-specific reagents and isotope tags. Two-dimensional gel electrophoresis technology combined with fluorescent dye labeling (e.g. monobromobimane (4.Alkhalfioui F. Renard M. Vensel W.H. Wong J. Tanaka C.K. Hurkman W.J. Buchanan B.B. Montrichard F. Thioredoxin-linked proteins are reduced during germination of Medicago truncatula seeds.Plant Physiol. 2007; 144: 1559-1579Crossref PubMed Scopus (131) Google Scholar, 5.Alvarez S. Wilson G.H. Chen S. Proteomic analysis of in vivo redox regulated proteins in Arabidopsis.J. Chromatogr. B. 2009; 877: 101-104Crossref PubMed Scopus (14) Google Scholar) and cyanine dyes (6.Fu C. Hu J. Liu T. Ago T. Sadoshima J. Li H. Quantitative analysis of redox-sensitive proteome with DIGE and ICAT.J. Proteome Res. 2008; 7: 3789-3802Crossref PubMed Scopus (90) Google Scholar, 7.Zhu M. Zhu N. Song W. Harmon A. Assmann S.M. Chen S. Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.Plant J. 2014; 78: 491-515Crossref PubMed Scopus (56) Google Scholar)) and gel-free technology with isotope tagging (e.g. isotope-coded affinity tagging (6.Fu C. Hu J. Liu T. Ago T. Sadoshima J. Li H. Quantitative analysis of redox-sensitive proteome with DIGE and ICAT.J. Proteome Res. 2008; 7: 3789-3802Crossref PubMed Scopus (90) Google Scholar, 7.Zhu M. Zhu N. Song W. Harmon A. Assmann S.M. Chen S. Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.Plant J. 2014; 78: 491-515Crossref PubMed Scopus (56) Google Scholar, 8.Go Y.M. Roede J.R. Orr M. Liang Y. Jones D.P. Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of Cd toxicity.Toxicol. Sci. 2014; 139: 59-73Crossref PubMed Scopus (77) Google Scholar), cysTMT 1The abbreviations used are:cysTMTcysteine reactive tandem mass tagsiTRAQisobaric tags for relative and absolute quantitationcysTMTRAQcysteine reactive tandem mass tags with isobaric tags for relative and absolute quantitationHCDhigh-energy collision dissociation. 1The abbreviations used are:cysTMTcysteine reactive tandem mass tagsiTRAQisobaric tags for relative and absolute quantitationcysTMTRAQcysteine reactive tandem mass tags with isobaric tags for relative and absolute quantitationHCDhigh-energy collision dissociation. (9.Parker J. Zhu N. Zhu M. Chen S. Profiling thiol redox proteome using isotope tagging mass spectrometry.J. Vis. Exp. 2012; 61: 3766-3791Google Scholar), and iTRAQ labeling of enriched cysteine-containing peptides (10.McDonagh B. Martínez-Acedo P. Várquez J. Padilla A. Sheehan D. Bárcena J.A. Application of iTRAQ reagents to relatively quantify the reversible redox state of cysteine residues.Int. J. Proteomics. 2012; 2012: 1-9Crossref Google Scholar, 11.Tambor V. Hunter C.L. Seymour S.L. Kacerovsky M. Stulik J. Lenco J. cysTRAQ—a combination of iTRAQ and enrichment of cysteinyl peptides for uncovering and quantifying hidden proteomes.J. Proteomics. 2012; 75: 857-867Crossref PubMed Scopus (37) Google Scholar, 12.Mermelekas G. Makridakis M. Koeck T. Vlahou A. Redox proteomics: from residue modifications to putative biomarker identification by gel- and LC-MS-based approaches.Expert Rev. Proteomics. 2013; 10: 537-549Crossref PubMed Scopus (13) Google Scholar, 13.Liu P. Zhang H. Wang H. Xia Y. Identification of redox-sensitive cysteines in the Arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method.Proteomics. 2014; 14: 750-762Crossref PubMed Scopus (70) Google Scholar, 14.Su D. Gaffrey M.J. Guo J. Hatchell K.E. Chu R.K. Clauss T.R.W. Aldrich J.T. Wu S. Purvine S. Camp D.G. Smith R.D. Thrall B.D. Qian W. Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling.Radic. Biol. Med. 2014; 67: 460-470Crossref PubMed Scopus (77) Google Scholar)) are often used to identify potential redox-sensitive cysteine residues and quantify redox changes. cysteine reactive tandem mass tags isobaric tags for relative and absolute quantitation cysteine reactive tandem mass tags with isobaric tags for relative and absolute quantitation high-energy collision dissociation. cysteine reactive tandem mass tags isobaric tags for relative and absolute quantitation cysteine reactive tandem mass tags with isobaric tags for relative and absolute quantitation high-energy collision dissociation. In addition to the well-known capabilities and limitations associated with two-dimensional gel electrophoresis–based and gel-free approaches (15.Chen S. Harmon A. Advance in plant proteomics.Proteomics. 2006; 6: 5504-5516Crossref PubMed Scopus (198) Google Scholar), each method has its strengths and weaknesses in redox proteomics. For example, the two-dimensional gel electrophoresis methods allow the inspection of spot patterns related to redox and protein-level changes. However, spot-volume-based quantification becomes problematic, as each spot often contains more than one protein species from complex samples. In addition, the limited number of fluorescent reagents compromises multiplexing capability, and the use of cyanine dyes does not allow mapping of the modified cysteines (6.Fu C. Hu J. Liu T. Ago T. Sadoshima J. Li H. Quantitative analysis of redox-sensitive proteome with DIGE and ICAT.J. Proteome Res. 2008; 7: 3789-3802Crossref PubMed Scopus (90) Google Scholar, 7.Zhu M. Zhu N. Song W. Harmon A. Assmann S.M. Chen S. Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.Plant J. 2014; 78: 491-515Crossref PubMed Scopus (56) Google Scholar). Other thiol labeling approaches such as the use of N-ethylmaleimide, biotin-N-[6-(Biotinamido)hexyl]-3-(2-pyridyldithio) propionamide (16.Izquierdo-Alvarez A. Ramos E. Villanueva J. Hernansanz-Augustín P. Fernández-Rodriguez R. Tello D. Carrascal M. Martínez-Ruiz A. Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia.J. Proteomics. 2012; 75: 5449-5462Crossref PubMed Scopus (35) Google Scholar), and isotope-coded affinity tags allow specific enrichment of cysteine-containing peptides, mapping of cysteine modification sites, and duplex experiments in the case of isotope-coded affinity tags (6.Fu C. Hu J. Liu T. Ago T. Sadoshima J. Li H. Quantitative analysis of redox-sensitive proteome with DIGE and ICAT.J. Proteome Res. 2008; 7: 3789-3802Crossref PubMed Scopus (90) Google Scholar, 7.Zhu M. Zhu N. Song W. Harmon A. Assmann S.M. Chen S. Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.Plant J. 2014; 78: 491-515Crossref PubMed Scopus (56) Google Scholar). To enable multiplexing, 4- or 8-plex iTRAQ tags were recently used to label cysteine-containing peptides isolated from thiol-affinity chromatography (10.McDonagh B. Martínez-Acedo P. Várquez J. Padilla A. Sheehan D. Bárcena J.A. Application of iTRAQ reagents to relatively quantify the reversible redox state of cysteine residues.Int. J. Proteomics. 2012; 2012: 1-9Crossref Google Scholar, 11.Tambor V. Hunter C.L. Seymour S.L. Kacerovsky M. Stulik J. Lenco J. cysTRAQ—a combination of iTRAQ and enrichment of cysteinyl peptides for uncovering and quantifying hidden proteomes.J. Proteomics. 2012; 75: 857-867Crossref PubMed Scopus (37) Google Scholar, 14.Su D. Gaffrey M.J. Guo J. Hatchell K.E. Chu R.K. Clauss T.R.W. Aldrich J.T. Wu S. Purvine S. Camp D.G. Smith R.D. Thrall B.D. Qian W. Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling.Radic. Biol. Med. 2014; 67: 460-470Crossref PubMed Scopus (77) Google Scholar, 16.Izquierdo-Alvarez A. Ramos E. Villanueva J. Hernansanz-Augustín P. Fernández-Rodriguez R. Tello D. Carrascal M. Martínez-Ruiz A. Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia.J. Proteomics. 2012; 75: 5449-5462Crossref PubMed Scopus (35) Google Scholar). Another multiplexing technology, cysTMT, was developed to specifically label cysteines with free thiol groups of proteins from six different samples (9.Parker J. Zhu N. Zhu M. Chen S. Profiling thiol redox proteome using isotope tagging mass spectrometry.J. Vis. Exp. 2012; 61: 3766-3791Google Scholar). Although these multiplexing technologies have found utility, they do not address the issue of protein turnover in the course of experiments, and many researchers have overlooked this important factor that could lead to misleading results (8.Go Y.M. Roede J.R. Orr M. Liang Y. Jones D.P. Integrated redox proteomics and metabolomics of mitochondria to identify mechanisms of Cd toxicity.Toxicol. Sci. 2014; 139: 59-73Crossref PubMed Scopus (77) Google Scholar, 13.Liu P. Zhang H. Wang H. Xia Y. Identification of redox-sensitive cysteines in the Arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method.Proteomics. 2014; 14: 750-762Crossref PubMed Scopus (70) Google Scholar, 14.Su D. Gaffrey M.J. Guo J. Hatchell K.E. Chu R.K. Clauss T.R.W. Aldrich J.T. Wu S. Purvine S. Camp D.G. Smith R.D. Thrall B.D. Qian W. Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling.Radic. Biol. Med. 2014; 67: 460-470Crossref PubMed Scopus (77) Google Scholar, 17.Muthuramalingam M. Matros A. Scheibe R. Mock H.P. Dietz K.J. The hydrogen-sensitive proteome of the chloroplast in vitro and in vivo.Front. Plant Sci. 2013; 19: 4-54Google Scholar). Only a small number of researchers have attempted to compare potential redox changes determined in proteomics experiments to total protein-level changes obtained from parallel or different studies (6.Fu C. Hu J. Liu T. Ago T. Sadoshima J. Li H. Quantitative analysis of redox-sensitive proteome with DIGE and ICAT.J. Proteome Res. 2008; 7: 3789-3802Crossref PubMed Scopus (90) Google Scholar, 7.Zhu M. Zhu N. Song W. Harmon A. Assmann S.M. Chen S. Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.Plant J. 2014; 78: 491-515Crossref PubMed Scopus (56) Google Scholar, 16.Izquierdo-Alvarez A. Ramos E. Villanueva J. Hernansanz-Augustín P. Fernández-Rodriguez R. Tello D. Carrascal M. Martínez-Ruiz A. Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia.J. Proteomics. 2012; 75: 5449-5462Crossref PubMed Scopus (35) Google Scholar). However, the success of this strategy is often low because many proteins quantified in redox experiments are either absent or not quantified with confidence in total proteomics experiments as a result of experimental variation and mass spectrometry stochastical sampling issues (18.Chong P.K. Gan C.S. Pham T.K. Wright P.C. Isobaric tags for relative and absolute quantification (iTRAQ) reproducibility: implication of multiple injections.J. Proteome Res. 2006; 5: 1232-1240Crossref PubMed Scopus (169) Google Scholar, 19.Lee J. Koh H.J. A label-free quantitative shotgun proteomics analysis of rice grain development.Proteome Sci. 2011; 9: 51-61Crossref PubMed Scopus (44) Google Scholar). To overcome this challenge, we have developed a double-labeling strategy that uses iTRAQ and cysTMT in one experiment for the simultaneous determination of quantifiable cysteine redox changes and protein-level changes. This new strategy, named cysTMTRAQ, utilizes each of the tags for their specific chemical properties. cysTMT tags (m/z 126, 127, 128, 129, 130, and 131 for six samples) were used to label protein thiols responsive to a treatment, and iTRAQ tags (m/z 114, 115, 116, 117, 119, and 121 for six samples) were used to label the N termini of peptides for analysis of protein-level changes during the experiments. By taking advantage of the different mass tags and their labeling specificities, one can quantify changes in protein redox and total levels in the same experiment. As protein redox regulation is a ubiquitous process (1.Go Y.M. Jones D.P. Mapping the cysteine proteome: analysis of redox-sensing thiols.J. Biol. Chem. 2013; 288: 26512-26520Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 2.Ghezzi P. Bonetto V. Redox proteomics: identification of oxidatively modified proteins.Proteomics. 2003; 3: 1145-1153Crossref PubMed Scopus (218) Google Scholar, 12.Mermelekas G. Makridakis M. Koeck T. Vlahou A. Redox proteomics: from residue modifications to putative biomarker identification by gel- and LC-MS-based approaches.Expert Rev. Proteomics. 2013; 10: 537-549Crossref PubMed Scopus (13) Google Scholar), the utility of this new integrative cysTMTRAQ method is expected to greatly advance redox proteomic studies in many fields of biology and medicine, and thus benefit a broad range of scientists. A six-protein mixture containing bovine serum albumin, α-lactabumin, β-galactosidase, lysozyme, apotransferrin, and β-lactoglobulin from the AB Sciex iTRAQ labeling kit (AB Sciex Inc., Foster City, CA) was used for optimization of the double-labeling procedure and oxidation experiments. Proteins were solubilized in a lysis buffer containing 6 m urea, 50 mm Tris, and 1 mm ethylenediaminetetraacetic acid, pH 7.5, reduced with 5 mm tris(2-carboxyethyl)phosphine for 1 h at room temperature. Oxidation treatment was performed with 1 mm H2O2 for 1 h at room temperature. Zeba columns (Thermo Scientific Inc.) were utilized after treatment to remove the reducing and oxidizing agents. Ellman's test for free thiols was performed to determine the free thiol concentration as described previously (20.Bulaj G. Kortemme T. Goldenber D.P. Ionization-reactivity relationships for cysteine thiols in polypeptides.Biochem. 1998; 37: 8965-8972Crossref PubMed Scopus (317) Google Scholar). Samples for optimization of the labeling procedure were prepared in and in order to of each concentration were Samples for the identification of redox proteins and cysteines were prepared at a of and of combined oxidized and reduced and and oxidized samples. of was in to of at and with for 1 Protein was as previously described F. T. M. J.R. P.C. changes in the thiol redox proteome in Sci. 2008; PubMed Scopus Google Scholar). and labeling of thiols were performed as by the (Thermo Scientific Inc., and J. Zhu N. Zhu M. Chen S. Profiling thiol redox proteome using isotope tagging mass spectrometry.J. Vis. Exp. 2012; 61: 3766-3791Google Scholar), Protein samples used for optimization of the labeling procedure were after treatment with tris(2-carboxyethyl)phosphine and Zeba samples with a with and with a with and and with a with and 131 labeling was performed for the redox experiments. The reduced and oxidized samples with mm for 1 h at thiols for labeling were through treatment with 5 mm tris(2-carboxyethyl)phosphine for 1 h at room temperature. A with a Zeba was performed for samples cysTMT Samples were combined in of oxidized to reduced the of reduced with each a total of and reduced and oxidized samples were with and 131 For each labeling a of cysTMT tag to free thiols was and labeling was performed at room for A was performed to the iTRAQ (AB Sciex Inc.) at for h (9.Parker J. Zhu N. Zhu M. Chen S. Profiling thiol redox proteome using isotope tagging mass spectrometry.J. Vis. Exp. 2012; 61: 3766-3791Google Scholar). were with columns Inc., and to For iTRAQ samples were in m Samples were to the (AB Sciex and a concentration of was used for the iTRAQ tags. the samples were with and Samples for optimization of the double-labeling procedure labeling with and to a and for a and and 121 for a Samples for the redox the reduced and oxidized were with and 121 For the E. biological were with cysTMT tags and and with iTRAQ tags and The biological were with cysTMT tags and 131 and with iTRAQ tags and 121 were in A mm and acid, pH and using with a A 5 were with a of to and mm pH 50 by to in 5 were by the at The were and in of and a Inc., The at A acid, acid, and at each the was with and with A. analysis was on mass in a and (Thermo (m/z were in the with at analysis of the with detection in for was at at with a at collision and for with a of mass a of and a of a of a of and of The mass was performed to the using a mixture of the and Proteome with the (Thermo Scientific Inc., was used to a of of the six-protein mixture (AB Sciex Inc.) in the of on 2012; for the six-protein samples and a from on 2013; for the E. samples. Proteome for and were to with the to a and mass of and 5 The was used for protein were to two of absolute of and at were to a mass and a mass modifications oxidation and The modification was for was used for protein identification with of a of and a of The was with a of and the used mass at with a of was performed for cysTMT and iTRAQ results were to only peptides with confidence and cysteine and protein cysTMT and iTRAQ were and changes were the The of peptides were and were were as described previously H. Y. H.J. S. J.W. for detection using on mass spectrometry 2008; PubMed Scopus Google Scholar). during the optimization experiment were relative to and were to their mass for proteome 2013; 10: PubMed Scopus Google Scholar). during the of redox treatment were relative to and for the reduced were from were also to their mass for proteome 2013; 10: PubMed Scopus Google Scholar). For protein quantification, at peptides were Protein quantification was performed by the of peptides B. C. S. R. for to relative protein PubMed Scopus Google Scholar), and were as described To analyze the from biological we the from the as test on the was and the were the method A to R. B. Scopus Google Scholar). A with a than and a than or than was The peptides with cysTMT were with the proteins quantified test was the of peptides and the of the proteins quantified A correction factor was to the of peptides, taking the of the protein quantified iTRAQ The of peptides and proteins can found in the and To test the of double-labeling of peptides from a six-protein mixture with cysTMT and iTRAQ tags, a labeling method was and the of different samples were in order to the experimental changes samples Proteins were six two at two at and two at the cysTMT tags were used to label the six samples. The samples and iTRAQ labeling by to changes of were from Proteome and tagging was determined by the of the of the from the expected for peptides and and changes to the double-labeling we found that cysteine-containing peptides with cysTMT the expected changes with iTRAQ tags also the expected changes The in the iTRAQ quantification relative to the cysTMT quantification to the in the number of peptides only the cysteine-containing peptides were the in cysTMT and iTRAQ quantification were the quantification can to the protein level the protein-level changes also the expected analysis of cysteine peptides that the changes of iTRAQ tags in the were to the changes of cysTMT tags iTRAQ and cysTMT tags the peptides at different groups with the same analysis of the iTRAQ quantitative and the cysTMT quantitative that the two were results that double-labeling of peptides is using the different isobaric tags and The expected changes of the samples can determined at and protein To the double-labeling method in a redox two of proteins were reduced and oxidized and combined in different a total protein in each labeling procedure was used in which the six protein thiol of oxidized and labeling with the thiol-specific cysTMT tags and iTRAQ tags of the samples the relative of oxidized proteins in the and the experimental to the expected for the samples of iTRAQ tags that the not the samples and the results at the protein level were with at the level analysis a in cysTMT and iTRAQ labeling of cysteine-containing peptides and in iTRAQ labeling in peptides results that the isobaric tag labeling procedure allows one to cysteine redox changes protein level changes, the of cysTMTRAQ in redox proteomics of complex samples. To the cells to we E. cells with for 1 h to redox and protein-level changes was This is important because the addition of not only the cells but also which Protein thiol modifications in Biol. PubMed Scopus Google Scholar). In we cysteine-containing peptides, and of iTRAQ and cysTMT of the cysteine-containing peptides, were a of and either a or a the (e.g. protein and were previously to have redox-sensitive cysteines the protein-level was 5 of the peptides containing redox-sensitive cysteines also changes and were as redox regulated only cysTMT quantification was However, the correction was on iTRAQ these proteins not redox regulation The results have that double-labeling of proteins with two different of isobaric tags, cysTMT and is and can used to protein redox and total changes in one experiment. enrichment of cysteine peptides is the method has the potential to the of redox However, enrichment can to redox proteins after the double-labeling In this the enriched and the will to with redox proteomics approaches (e.g. C. Hu J. Liu T. Ago T. Sadoshima J. Li H. Quantitative analysis of redox-sensitive proteome with DIGE and ICAT.J. Proteome Res. 2008; 7: 3789-3802Crossref PubMed Scopus (90) Google Scholar, 7.Zhu M. Zhu N. Song W. Harmon A. Assmann S.M. Chen S. Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.Plant J. 2014; 78: 491-515Crossref PubMed Scopus (56) Google Scholar, 16.Izquierdo-Alvarez A. Ramos E. Villanueva J. Hernansanz-Augustín P. Fernández-Rodriguez R. Tello D. Carrascal M. Martínez-Ruiz A. Differential redox proteomics allows identification of proteins reversibly oxidized at cysteine residues in endothelial cells in response to acute hypoxia.J. Proteomics. 2012; 75: 5449-5462Crossref PubMed Scopus (35) Google Scholar), this developed cysTMTRAQ method is because allows researchers to identify and quantify redox peptides, and proteins and in the redox changes and total protein changes. cysTMT and iTRAQ are in the same experiment with the same the variation and of protein-level changes in redox determination are confidence and in the determination of the redox of cysteine peptides can with this method redox and total protein-level samples in small such as and can In to the use of two different experiments that lead to low and variation of peptides and this cysTMTRAQ method is and However, that the of associated with tandem mass tag and iTRAQ technologies has R. W. in isobaric quantitative 2011; PubMed Scopus Google Scholar, enables proteome quantification with isobaric 2011; PubMed Scopus Google Scholar). The function on the new of mass (e.g. the Scientific can overcome this this cysTMTRAQ will in redox as as posttranslational modification in many areas of biology and medicine. The mass spectrometry have been to the the with the number with