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Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry

Nature Protocols volume 9, pages 1131–1145 (2014)Cite this article

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Abstract

Reversible thiol oxidation of cysteine residues occurs in many intracellular catalytic and signaling processes. Here we describe an optimized protocol, which can be completed in ∼5 d, to unambiguously identify specific cysteine residues that are transiently and reversibly oxidized by comparing two complex biological samples obtained from yeast cell cultures at the proteome level. After 'freezing' the in vivo thiol stage of cysteine residues by medium acidification, we first block reduced thiols in extracts with iodoacetamide (IAM), and then we sequentially reduce and label reversible oxidized thiols with the biotin-based heavy or light IAM derivatives, which are known as isotope-coded affinity tag (ICAT) reagents, so that the two samples can be compared at once after combination of the labeled extracts, trypsin digestion, streptavidin-affinity purification of peptides containing oxidized cysteines, and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. For the same protein extracts, before cysteine-containing peptide enrichment, individual relative protein concentrations are obtained by stable-isotope dimethyl labeling.

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Figure 1: Schematic representation of the gel-free strategy to identify and quantify oxidized cysteines by using the ICAT technology on a proteomic scale.
Figure 2: Performance of the ICAT-based biotin switch assay combined with dimethyl labeling to characterize the reversibly oxidized thiol proteome of S. pombe.

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References

  1. Gilbert, H.F. Molecular and cellular aspects of thiol-disulfide exchange. Adv. Enzymol. Relat. Areas Mol. Biol. 63, 69–172 (1990).

    CAS  PubMed  Google Scholar 

  2. Ghezzi, P. Protein glutathionylation in health and disease. Biochim. Biophys. Acta. 1830, 3165–3172 (2013).

    Article  CAS  Google Scholar 

  3. Auclair, J.R. et al. Structural consequences of cysteinylation of Cu/Zn-superoxide dismutase. Biochemistry 52, 6145–6150 (2013).

    Article  CAS  Google Scholar 

  4. Hochgrafe, F. et al. S-cysteinylation is a general mechanism for thiol protection of Bacillus subtilis proteins after oxidative stress. J. Biol. Chem. 282, 25981–2595 (2007).

    Article  Google Scholar 

  5. Salmeen, A. et al. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate. Nature 423, 769–773 (2003).

    Article  CAS  Google Scholar 

  6. van Montfort, R.L., Congreve, M., Tisi, D., Carr, R. & Jhoti, H. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature 423, 773–777 (2003).

    Article  CAS  Google Scholar 

  7. Winterbourn, C.C. & Hampton, M.B. Thiol chemistry and specificity in redox signaling. Free Radic. Biol. Med. 45, 549–561 (2008).

    Article  CAS  Google Scholar 

  8. D'Autreaux, B. & Toledano, M.B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis. Nat. Rev. Mol. Cell Biol. 8, 813–824 (2007).

    Article  CAS  Google Scholar 

  9. Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E. & Stamler, J.S. Protein S-nitrosylation: purview and parameters. Nat. Rev. Mol. Cell Biol. 6, 150–166 (2005).

    Article  CAS  Google Scholar 

  10. Meyer, Y., Buchanan, B.B., Vignols, F. & Reichheld, J.P. Thioredoxins and glutaredoxins: unifying elements in redox biology. Annu. Rev. Genet. 43, 335–367 (2009).

    Article  CAS  Google Scholar 

  11. Vlamis-Gardikas, A. The multiple functions of the thiol-based electron flow pathways of Escherichia coli: Eternal concepts revisited. Biochim. Biophys. Acta. 1780, 1170–1200 (2008).

    Article  CAS  Google Scholar 

  12. Sengupta, R. & Holmgren, A. Thioredoxin and thioredoxin reductase in relation to reversible S-nitrosylation. Antioxid. Redox Signal. 18, 259–269 (2013).

    Article  CAS  Google Scholar 

  13. Benhar, M., Forrester, M.T. & Stamler, J.S. Protein denitrosylation: enzymatic mechanisms and cellular functions. Nat. Rev. Mol. Cell Biol. 10, 721–732 (2009).

    Article  CAS  Google Scholar 

  14. Poole, L.B. & Nelson, K.J. Discovering mechanisms of signaling-mediated cysteine oxidation. Curr. Opin. Chem. Biol. 12, 18–24 (2008).

    Article  CAS  Google Scholar 

  15. Anand, P. & Stamler, J.S. Enzymatic mechanisms regulating protein S-nitrosylation: implications in health and disease. J. Mol. Med. 90, 233–44 (2012).

    Article  CAS  Google Scholar 

  16. Sha, Y. & Marshall, H.E. S-nitrosylation in the regulation of gene transcription. Biochim. Biophys. Acta. 1820, 701–711 (2012).

    Article  CAS  Google Scholar 

  17. Marozkina, N.V. & Gaston, B. S-Nitrosylation signaling regulates cellular protein interactions. Biochim. Biophys. Acta. 1820, 722–729 (2012).

    Article  CAS  Google Scholar 

  18. Cumming, R.C. et al. Protein disulfide bond formation in the cytoplasm during oxidative stress. J. Biol. Chem. 279, 21749–21758 (2004).

    Article  CAS  Google Scholar 

  19. Jaffrey, S.R., Erdjument-Bromage, H., Ferris, C.D., Tempst, P. & Snyder, S.H. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat. Cell Biol. 3, 193–197 (2001).

    Article  CAS  Google Scholar 

  20. Wang, X., Kettenhofen, N.J., Shiva, S., Hogg, N. & Gladwin, M.T. Copper dependence of the biotin switch assay: modified assay for measuring cellular and blood nitrosated proteins. Free Radic. Biol. Med. 44, 1362–1372 (2008).

    Article  CAS  Google Scholar 

  21. Saurin, A.T., Neubert, H., Brennan, J.P. & Eaton, P. Widespread sulfenic acid formation in tissues in response to hydrogen peroxide. Proc. Natl. Acad. Sci. USA 101, 17982–17987 (2004).

    Article  CAS  Google Scholar 

  22. Lind, C. et al. Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis. Arch. Biochem. Biophys. 406, 229–240 (2002).

    Article  CAS  Google Scholar 

  23. Leichert, L.I. et al. Quantifying changes in the thiol redox proteome upon oxidative stress in vivo. Proc. Natl. Acad. Sci. USA 105, 8197–8202 (2008).

    Article  CAS  Google Scholar 

  24. Le Moan, N., Clement, G., Le Maout, S., Tacnet, F. & Toledano, M.B. The Saccharomyces cerevisiae proteome of oxidized protein thiols: contrasted functions for the thioredoxin and glutathione pathways. J. Biol. Chem. 281, 10420–10430 (2006).

    Article  CAS  Google Scholar 

  25. Baty, J.W., Hampton, M.B. & Winterbourn, C.C. Proteomic detection of hydrogen peroxide-sensitive thiol proteins in Jurkat cells. Biochem. J. 389, 785–795 (2005).

    Article  CAS  Google Scholar 

  26. Delahunty, C. & Yates, J.R. III Protein identification using 2D-LC-MS/MS. Methods 35, 248–255 (2005).

    Article  CAS  Google Scholar 

  27. Hao, G., Derakhshan, B., Shi, L., Campagne, F. & Gross, S.S. SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures. Proc. Natl. Acad. Sci. USA 103, 1012–1017 (2006).

    Article  CAS  Google Scholar 

  28. Sinha, V. et al. Proteomic and mass spectroscopic quantitation of protein S-nitrosation differentiates NO-donors. ACS Chem. Biol. 5, 667–680 (2010).

    Article  CAS  Google Scholar 

  29. Foster, M.W. Methodologies for the characterization, identification and quantification of S-nitrosylated proteins. Biochim. Biophys. Acta 1820, 675–683 (2012).

    Article  CAS  Google Scholar 

  30. Bechtold, E. & King, S.B. Chemical methods for the direct detection and labeling of S-nitrosothiols. Antioxid. Redox Signal. 17, 981–991 (2012).

    Article  CAS  Google Scholar 

  31. Martinez-Acedo, P. et al. A novel strategy for global analysis of the dynamic thiol redox proteome. Mol. Cell Proteomics 11, 800–813 (2012).

    Article  CAS  Google Scholar 

  32. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  33. Sethuraman, M. et al. Isotope-coded affinity tag (ICAT) approach to redox proteomics: identification and quantitation of oxidant-sensitive cysteine thiols in complex protein mixtures. J. Proteome Res. 3, 1228–1233 (2004).

    Article  CAS  Google Scholar 

  34. Sethuraman, M. et al. Quantification of oxidative posttranslational modifications of cysteine thiols of p21ras associated with redox modulation of activity using isotope-coded affinity tags and mass spectrometry. Free Radic. Biol. Med. 42, 823–829 (2007).

    Article  CAS  Google Scholar 

  35. Sethuraman, M., McComb, M.E., Heibeck, T., Costello, C.E. & Cohen, R.A. Isotope-coded affinity tag approach to identify and quantify oxidant-sensitive protein thiols. Mol. Cell Proteomics 3, 273–278 (2004).

    Article  CAS  Google Scholar 

  36. Fu, C. et al. Quantitative analysis of redox-sensitive proteome with DIGE and ICAT. J. Proteome Res. 7, 3789–3802 (2008).

    Article  CAS  Google Scholar 

  37. Garcia-Santamarina, S. et al. The oxidized thiol proteome in fission yeast–optimization of an ICAT-based method to identify H2O2-oxidized proteins. J. Proteomics 74, 2476–2486 (2011).

    Article  CAS  Google Scholar 

  38. Garcia-Santamarina, S. et al. Is oxidized thioredoxin a major trigger for cysteine oxidation? Clues from a redox proteomics approach. Antioxid. Redox Signal. 18, 1549–1556 (2013).

    Article  CAS  Google Scholar 

  39. Brandes, N., Reichmann, D., Tienson, H., Leichert, L.I. & Jakob, U. Using quantitative redox proteomics to dissect the yeast redoxome. J. Biol. Chem. 286, 41893–41903 (2011).

    Article  CAS  Google Scholar 

  40. Kumsta, C., Thamsen, M. & Jakob, U. Effects of oxidative stress on behavior, physiology, and the redox thiol proteome of Caenorhabditis elegans. Antioxid. Redox Signal. 14, 1023–1037 (2011).

    Article  CAS  Google Scholar 

  41. Knoefler, D. et al. Quantitative in vivo redox sensors uncover oxidative stress as an early event in life. Mol. Cell 47, 767–776 (2012).

    Article  CAS  Google Scholar 

  42. Christoforou, A.L. & Lilley, K.S. Isobaric tagging approaches in quantitative proteomics: the ups and downs. Anal. Bioanal. Chem. 404, 1029–1037 (2012).

    Article  CAS  Google Scholar 

  43. Boersema, P.J., Raijmakers, R., Lemeer, S., Mohammed, S. & Heck, A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494 (2009).

    Article  CAS  Google Scholar 

  44. Wisniewski, J.R., Zougman, A. & Mann, M. Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J. Proteome Res. 8, 5674–5678 (2009).

    Article  CAS  Google Scholar 

  45. Guo, J. et al. Resin-assisted enrichment of thiols as a general strategy for proteomic profiling of cysteine-based reversible modifications. Nat. Protoc. 9, 64–75 (2014).

    Article  CAS  Google Scholar 

  46. Hansen, R.E. & Winther, J.R. An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations. Anal. Biochem. 394, 147–158 (2009).

    Article  CAS  Google Scholar 

  47. Gilbert, H.F. Thiol/disulfide exchange equilibria and disulfide bond stability. Methods Enzymol. 251, 8–28 (1995).

    Article  CAS  Google Scholar 

  48. Rogers, L.K., Leinweber, B.L. & Smith, C.V. Detection of reversible protein thiol modifications in tissues. Anal. Biochem. 358, 171–184 (2006).

    Article  CAS  Google Scholar 

  49. Held, J.M. & Gibson, B.W. Regulatory control or oxidative damage? Proteomic approaches to interrogate the role of cysteine oxidation status in biological processes. Mol. Cell Proteomics 11, R111 013037 (2012).

    Article  Google Scholar 

  50. Sivaraman, T., Kumar, T.K., Jayaraman, G. & Yu, C. The mechanism of 2,2,2-trichloroacetic acid-induced protein precipitation. J. Protein Chem. 16, 291–297 (1997).

    Article  CAS  Google Scholar 

  51. Bensadoun, A. & Weinstein, D. Assay of proteins in the presence of interfering materials. Anal. Biochem. 70, 241–250 (1976).

    Article  CAS  Google Scholar 

  52. Rajalingam, D., Loftis, C., Xu, J.J. & Kumar, T.K. Trichloroacetic acid-induced protein precipitation involves the reversible association of a stable partially structured intermediate. Protein Sci. 18, 980–993 (2009).

    Article  CAS  Google Scholar 

  53. Boja, E.S. & Fales, H.M. Overalkylation of a protein digest with iodoacetamide. Anal. Chem. 73, 3576–3582 (2001).

    Article  CAS  Google Scholar 

  54. Gundlach, H.G., Moore, S. & Stein, W.H. The reaction of iodoacetate with methionine. J. Biol. Chem. 234, 1761–1764 (1959).

    CAS  PubMed  Google Scholar 

  55. Lapko, V.N., Smith, D.L. & Smith, J.B. Identification of an artifact in the mass spectrometry of proteins derivatized with iodoacetamide. J. Mass Spectrom. 35, 572–575 (2000).

    Article  CAS  Google Scholar 

  56. Crestfield, A.M., Stein, W.H. & Moore, S. Alkylation and identification of the histidine residues at the active site of ribonuclease. J. Biol. Chem. 238, 2413–2419 (1963).

    CAS  PubMed  Google Scholar 

  57. Park, S.S. et al. Effective correction of experimental errors in quantitative proteomics using stable isotope labeling by amino acids in cell culture (SILAC). J. Proteomics 75, 3720–3732 (2012).

    Article  CAS  Google Scholar 

  58. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    Article  CAS  Google Scholar 

  59. Kruger, R., Hung, C.W., Edelson-Averbukh, M. & Lehmann, W.D. Iodoacetamide-alkylated methionine can mimic neutral loss of phosphoric acid from phosphopeptides as exemplified by nano-electrospray ionization quadrupole time-of-flight parent ion scanning. Rapid Commun. Mass Spectrom. 19, 1709–1716 (2005).

    Article  Google Scholar 

  60. Hung, C.W., Schlosser, A., Wei, J. & Lehmann, W.D. Collision-induced reporter fragmentations for identification of covalently modified peptides. Anal. Bioanal. Chem. 389, 1003–1016 (2007).

    Article  CAS  Google Scholar 

  61. Gouw, J.W. & Krijgsveld, J. MSQuant: a platform for stable isotope-based quantitative proteomics. Methods Mol. Biol. 893, 511–522 (2012).

    Article  CAS  Google Scholar 

  62. Mortensen, P. et al. MSQuant, an open source platform for mass spectrometry-based quantitative proteomics. J. Proteome Res. 9, 393–403 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge the Centre for Genomic Regulation-Universitat Pompeu Fabra (CRG-UPF) proteomic facility where the LC-MS/MS experiments were performed. This work was supported by the Spanish Ministry of Science and Innovation (nos. BFU2009-06933 and BFU2012-32045), by PLAN E and Fondo Europeo de Desarrollo Regional (FEDER), by the Spanish program Consolider-Ingenio 2010 (grant no. CSD 2007-0020) and by grant no. SGR2009-195 from Generalitat de Catalunya (Spain) to E.H. E.H. and J.A. are recipients of Institució Catalana de Recerca i Estudis Avançats (ICREA) Academia Awards (Generalitat de Catalunya).

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Authors and Affiliations

  1. Oxidative Stress and Cell Cycle Group, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain

    Sarela García-Santamarina, Susanna Boronat, Alba Domènech, José Ayté & Elena Hidalgo

  2. Proteomics Resource Center, The Rockefeller University, New York, New York, USA

    Henrik Molina

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  1. Sarela García-Santamarina
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Contributions

S.G.-S. and S.B. developed the protocol and conducted the experiments. S.G.-S., H.M. and S.B. interpreted the data. H.M. performed the LC-MS/MS experiments and edited the manuscript. A.D. performed some control experiments. J.A. provided intellectual expertise. S.G.-S. drafted the manuscript. E.H. was responsible for project supervision, data interpretation, manuscript editing and providing grant support.

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Correspondence to Elena Hidalgo.

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Integrated supplementary information

Supplementary Figure 1 TCA followed by IAM, but not only IAM, is required to display the transient H2O2-dependent in vivo oxidation of Pap1.

Wild-type strain 972 was treated or not with 0.2 mM H2O2 for 5 min. Cells were then lysed in the presence of TCA, proteins in the lysates were then alkylated with IAM, and Pap1 redox state was analyzed by non-reducing electrophoresis and Western blot analysis as described before (Vivancos et al. 2005, PNAS 102:8875) (A). Alternatively, 75 mM IAM was added to both cell cultures and cell pellets prior to lysis (B), or only to cell pellets prior to lysis (C), and samples were processed for Western blot analysis as described above.

Supplementary Figure 2 Effect of SDS and Tris-HCl in the labeling of oxidized cysteines and proteins in their N-terminal sites.

Left panel, oxidized cysteines from wild-type (WT) and Δtrr1 cells were labelled with a fluorescent derivative of iodoacetamide (FIAM) as described in García-Santamarina et al. 2011, J. Proteomics 74:2476, using either ICAT buffer (buffer 1; 200 mM Tris-HCl, 0.05% SDS, 6 M urea, 5 mM EDTA) or a buffer without Tris-HCl and SDS (buffer 2; 200 mM Triethyl-ammonium bicarbonate 1 M, pH 8.5 , 6 M urea). Right panel, 100 µg of protein extracts in 50 µl from wild-type (WT) and Δtrr1 cells were labelled at their N-terminal sites with 2.5 µl of 1 mg/ml fluorescein isothiocyanate (FITC), either in ICAT buffer (buffer 1) or in a buffer without Tris-HCl and SDS (buffer 2). In both cases, protein extracts were electrophoretically separated by reducing SDS-PAGE. After electrophoresis fluorescence was scanned in a Typhoon 8600 using λex. 532 nm and λem. 526 nm. Silver staining was used as a protein loading control.

Supplementary information

Supplementary Figure 1

TCA followed by IAM, but not only IAM, is required to display the transient H2O2-dependent in vivo oxidation of Pap1. (PDF 93 kb)

Supplementary Figure 2

Effect of SDS and Tris-HCl in the labeling of oxidized cysteines and proteins in their N-terminal sites. (PDF 319 kb)

Supplementary Table 1

Peptides with oxidized cysteines (ICAT ratio vs protein concentration >1.5 in two biological replicates) identified from wild-type H2O2-treated vs wild-type untreated fission yeast cells. (XLS 48 kb)

Supplementary Table 2

Peptides with oxidized cysteines (ICAT ratio vs protein concentration >1.5 in two biological replicates) identified from Δtrr1 vs wild-type untreated fission yeast cells. (XLS 48 kb)

Supplementary Table 3

Peptides with oxidized cysteines (ICAT ratio vs protein concentration >1.5 in two biological replicates) identified from Δtrx1 vs wild-type untreated fission yeast cells. (XLS 37 kb)

Supplementary Data 1

Description LC gradient with 84 min (marked '120 minutes') and 147 min (marked '180 minutes') resolving gradients. (PDF 40 kb)

Supplementary Data 2

MS and MS/MS parameters for a Q-Exactive mass spectrometer operated in data dependent (dd) mode. (PDF 100 kb)

Supplementary Data 3

Retention time comparison of identical cysteine-containing peptides modified by carbamidomethyl (ca) and/or ICAT measured in the same experiment. (PDF 109 kb)

Supplementary Data 4

MS/MS spectra of the triply charged peptide. (PDF 130 kb)

Supplementary Data 5

MS spectrum of the triply charged peptide. (PDF 143 kb)

Supplementary Data 6

Three dimensional plot of the ICAT-labelled peptide pair shown in Supplementary Data 5. (PDF 283 kb)

Supplementary Data 7

Modifications used to search tandem MS data (MS/MS). (PDF 70 kb)

Supplementary Data 8

Five important steps needed to launch a MaxQuant 1.3.0.5 analysis. (PDF 150 kb)

Supplementary Data 9

The 'peptide.txt' file contains the dimethyl-based quantification results as well as the ICAT-based quantification results. (PDF 52 kb)

Supplementary Data 10

Scatter plot of measured ICAT ratios. (PDF 54 kb)

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García-Santamarina, S., Boronat, S., Domènech, A. et al. Monitoring in vivo reversible cysteine oxidation in proteins using ICAT and mass spectrometry. Nat Protoc 9, 1131–1145 (2014). https://doi.org/10.1038/nprot.2014.065

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