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Characteristic Tandem Mass Spectral Features Under Various Collision Chemistries for Site-Specific Identification of Protein S-Glutathionylation

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Journal of The American Society for Mass Spectrometry

Abstract

Protein S-glutathionylation is a reversible post-translational modification widely implicated in redox regulated biological functions. Conventional biochemical methods, however, often do not allow such a mixed disulfide modification to be reliably identified on specific cysteine residues or be distinguished from other related oxidized forms. To develop more efficient mass spectrometry (MS)-based analytical strategies for this purpose, we first investigated the MS/MS fragmentation pattern of S-glutathionylated peptides under various dissociation modes, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD), using synthetic peptides derived from protein tyrosine phosphatase as models. Our results indicate that a MALDI-based high energy CID MS/MS on a TOF/TOF affords the most distinctive spectral features that would facilitate rapid and unambiguous identification of site-specific S-glutathionylation. For more complex proteomic samples best tackled by LC-MS/MS approach, we demonstrate that HCD performed on an LTQ-Orbitrap hybrid instrument fairs better than trap-based CID and ETD in allowing more protein site-specific S-glutathionylation to be confidently identified by direct database searching of the generated MS/MS dataset using Mascot. Overall, HCD afforded more peptide sequence-informative fragment ions retaining the glutathionyl modification with less neutral losses of side chains to compromise scoring. In conjunction with our recently developed chemo-enzymatic tagging strategy, our nanoLC-HCD-MS/MS approach is sufficiently sensitive to identify endogenous S-glutathionylated peptides prepared from non-stressed cells. It is anticipated that future applications to global scale analysis of protein S-glutathionylation will benefit further from current advances in both speed and mass accuracy afforded by HCD MS/MS mode on the Orbitrap series.

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References

  1. Dalle-Donne, I., Rossi, R., Giustarini, D., Colombo, R., Milzani, A.: S-glutathionylation in protein redox regulation. Free Radic. Biol. Med. 43, 883–898 (2007)

    Article  CAS  Google Scholar 

  2. Forman, H.J., Fukuto, J.M., Torres, M.: Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. Am. J. Physiol. Cell Physiol. 287, C246–C256 (2004)

    Article  CAS  Google Scholar 

  3. Giustarini, D., Rossi, R., Milzani, A., Colombo, R., Dalle-Donne, I.: S-glutathionylation: from redox regulation of protein functions to human diseases. J. Cell. Mol. Med. 8, 201–212 (2004)

    Article  CAS  Google Scholar 

  4. Biswas, S., Chida, A.S., Rahman, I.: Redox modifications of protein-thiols: emerging roles in cell signaling. Biochem. Pharmacol. 71, 551–564 (2006)

    Article  CAS  Google Scholar 

  5. Klatt, P., Lamas, S.: Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur. J. Biochem. 267, 4928–4944 (2000)

    Article  CAS  Google Scholar 

  6. Melissa, D., Shelton, P., Chock, B., Mieyal, J.J.: Glutaredoxin: role in reversible protein S-glutathionylation and regulation of redox signal transduction and protein translocation. Antioxid. Redox Signal. 7, 348–366 (2005)

    Article  Google Scholar 

  7. Fratelli, M., Demol, H., Puype, M., Casagrande, S., Eberini, I., Salmona, M., Bonetto, V., Mengozzi, M., Duffieux, F., Miclet, E., Bachi, A., Vandekerckhove, J., Gianazza, E., Ghezzi, P.: Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc. Natl. Acad. Sci. U. S. A. 99, 3505–3510 (2002)

    Article  CAS  Google Scholar 

  8. Söderdahl, T., Enoksson, M., Lundberg, M., Holmgren, A., Ottersen, O.P., Orrenius, S., Bolcsfoldi, G., Cotgreave, I.A.: Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells. FASEB J. 17, 124–126 (2003)

    Google Scholar 

  9. Dalle-Donne, I., Rossi, R., Giustarini, D., Colombo, R., Milzani, A.: Actin S-glutathionylation: evidence against a thiol-disulphide exchange mechanism. Free Radic. Biol. Med. 35, 1185–1193 (2003)

    Article  CAS  Google Scholar 

  10. Mawatari, S., Murakami, K.: Different types of glutathionylation of hemoglobin can exist in intact erythrocytes. Arch. Biochem. Biophys. 421, 108–114 (2004)

    Article  CAS  Google Scholar 

  11. Landino, L.M., Moynihan, K.L., Todd, J.V., Kennett, K.L.: Modulation of the redox state of tubulin by the glutathione/glutaredoxin reductase system. Biochem. Biophys. Res. Commun. 314, 555–560 (2004)

    Article  CAS  Google Scholar 

  12. Craghill, J., Cronshaw, A.D., Harding, J.J.: The identification of a reaction site of glutathione mixed-disulphide formation on S-crystallin in human lens. Biochem. J. 379, 595–600 (2004)

    Article  CAS  Google Scholar 

  13. Aracena-Parks, P., Goonasekera, S.A., Gilman, C.P., Dirksen, R.T., Hidalgo, C., Hamilton, S.L.: Identification of cysteines involved in S-nitrosylation, S-glutathionylation, and oxidation to disulfides in ryanodine receptor type 1. J. Biol. Chem. 281(52), 40354–40368 (2006)

    Article  CAS  Google Scholar 

  14. Barbirz, S., Happersberger, H.P., Przybylski, M., Glocker, M.O.: Selective cyanylation of cysteinyl residues as an approach for the mass spectrometric determination of protein structures. Eur. Mass Spectrom. 5, 123–131 (1999)

    Article  CAS  Google Scholar 

  15. Sullivan, D.M., Levine, R.L., Finkel, T.: Detection and affinity purification of oxidant-sensitive proteins using biotinylated glutathione ethyl ester. Methods Enzymol. 353, 101–113 (2002)

    Article  CAS  Google Scholar 

  16. Brennan, J.P., Miller, J.I.A., Fuller, W., Wait, R., Begum, S., Dunn, M.J., Eaton, P.: The utility of N,N-biotinyl glutathione disulfide in the study of protein S-glutathiolation. Mol. Cell. Proteomics 5(2), 215–225 (2006)

    Article  CAS  Google Scholar 

  17. Hamnell-Pamment, Y., Lind, C., Palmberg, C., Bergman, T., Cotgreave, I.A.: Determination of site-specificity of S-glutathionylated cellular proteins. Biochem. Biophys. Res. Commun. 332, 362–369 (2005)

    Article  CAS  Google Scholar 

  18. 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.J.: Proteomic identification and quantification of S-glutathionylation in mouse macrophages using resin-assisted enrichment and isobaric labeling. Free Radic. Biol. Med. 67, 460–470 (2014)

    Article  CAS  Google Scholar 

  19. Chiang, B.Y., Chou, C.C., Hsieh, F.T., Gao, S., Lin, J.C.Y., Lin, S.H., Chen, T.C., Khoo, K.H., Lin, C.H.: In vivo tagging and characterization of S-glutathionylated proteins by a chemoenzymatic method. Angew. Chem. Int. Ed. 51, 5871–5875 (2012)

    Article  CAS  Google Scholar 

  20. Samarasinghe, K.T.G., Godage, D.N.P.M., VanHecke, G.C., Ahn, Y.-H.: Metabolic synthesis of clickable glutathione for chemoselective detection of glutathionylation. J. Am. Chem. Soc. 136, 11566–11569 (2014)

    Article  CAS  Google Scholar 

  21. Srikanth, R., Wilson, J., Bridgewater, J.D., Numbers, J.R., Lim, J., Olbris, M.R., Kettani, A., Vachet, R.W.: Improved sequencing of oxidized cysteine and methionine containing peptides using electron transfer dissociation. J. Am. Soc. Mass Spectrom. 18, 1499–1506 (2007)

    Article  CAS  Google Scholar 

  22. Olsen, J.V., Macek, B., Lange, O., Makarov, A., Horning, S., Mann, M.: Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 4(9), 709–712 (2007)

    Article  CAS  Google Scholar 

  23. Syka, J.E., Coon, J.J., Schroeder, M.J., Shabanowitz, J., Hunt, D.F.: Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 101, 9528–9533 (2004)

    Article  CAS  Google Scholar 

  24. Chi, A., Huttenhower, C., Geer, L.Y., Coon, J.J., Syka, J.E.P., Bai, D.L., Shabanowitz, J., Burke, D.J., Troyanskaya, O.G., Hunt, D.F.: Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc. Natl. Acad. Sci. U. S. A. 104(7), 2193–2198 (2007)

    Article  CAS  Google Scholar 

  25. Good, D.M., Wirtala, M., McAlister, G.C., Coon, J.J.: Performance characteristics of electron transfer dissociation mass spectrometry. Mol. Cell. Proteomics 6(11), 1942–1951 (2007)

    Article  CAS  Google Scholar 

  26. Bean, M.F., Carr, S.A.: Characterization of disulfide bond position in proteins and sequence analysis of cysteine-bridged peptides by tandem mass spectrometry. Anal. Biochem. 201, 216–226 (1992)

    Article  CAS  Google Scholar 

  27. Rinna, A., Torres, M., Forman, H.J.: Stimulation of the alveolar macrophage respiratory burst by ADP causes selective glutathionylation of protein tyrosine phosphatase 1B. Free Radic. Biol. Med. 41, 86–91 (2006)

    Article  CAS  Google Scholar 

  28. Cross, J.V., Templeton, D.J.: Oxidative stress inhibits MEKK1 by site-specific glutathionylation in the ATP-binding domain. Biochem. J. 381, 675–683 (2004)

    Article  CAS  Google Scholar 

  29. Shelton, M.D., Kern, T.S., Mieyal, J.J.: Glutaredoxin regulates nuclear factor κ-B and intercellular adhesion molecule in Muller cells: model of diabetic retinopathy. J. Biol. Chem. 282, 12467–12474 (2007)

    Article  CAS  Google Scholar 

  30. Clavreul, N., Adachi, T., Pimental, D.R., Ido, Y., Schöneich, C., Cohen, R.A.: S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells. FASEB J. 20, 518–520 (2006)

    CAS  Google Scholar 

  31. Lander, H.M.: An essential role for free radicals and derived species in signal transduction. FASEB J. 11, 118–124 (1997)

    CAS  Google Scholar 

  32. Meissner, F., Molawi, K., Zychlinsky, A.: Superoxide dismutase 1 regulates caspase-1 and endotoxic shock. Nat. Immunol. 9, 866–872 (2008)

    Article  CAS  Google Scholar 

  33. Huang, Z., Pinto, J.T., Deng, H., Richie, J.P.J.: Inhibition of caspase-3 activity and activation by protein glutathionylation. Biochem. Pharmacol. 75, 2234–2244 (2008)

    Article  CAS  Google Scholar 

  34. Zheng, M., Åslund, F., Storz, G.: Activation of the OxyR transcription factor by reversible disulfide bond formation. Science 279, 1718–1721 (1998)

    Article  CAS  Google Scholar 

  35. Kim, S.O., Merchant, K., Nudelman, R., Beyer, W.F.J., Keng, T., DeAngelo, J., Hausladen, A., Stamler, J.S.: OxyR: a molecular code for redox-related signaling. Cell 109, 383–396 (2002)

    Article  CAS  Google Scholar 

  36. Prinarakis, E., Chantzoura, E., Thanos, D., Spyrou, G.: S-glutathionylation of IRF3 regulates IRF3-CBP interaction and activation of the IFN beta pathway. EMBO J. 27, 865–875 (2008)

    Article  CAS  Google Scholar 

  37. Dalle-Donne, I., Rossi, R., Colombo, G., Giustarini, D., Milzani, A.: Protein S-glutathionylation: a regulatory device from bacteria to humans. Trends Biochem. Sci. 34(2), 85–96 (2009)

    Article  CAS  Google Scholar 

  38. Barrett, W.C., DeGnore, J.P., Konig, S., Fales, H.M., Keng, Y.F., Zhang, Z.Y., Yim, M.B., Chock, P.B.: Regulation of PTP1B via glutathionylation of the active site cysteine 215. Biochemistry 38, 6699–6705 (1999)

    Article  CAS  Google Scholar 

  39. Wang, Z., Park, K., Comer, F., Hsieh-Wilson, L.C., Saudek, C.D., Hart, G.W.: Site-specific GlcNAcylation of human erythrocyte proteins. Diabetes 58, 309–317 (2009)

    Article  CAS  Google Scholar 

  40. Chong, C.M., Gao, S.J., Chiang, B.Y., Hsu, W.H., Lin, T.C., Chen, T.C., Lin, C.H.: An acyloxymethyl ketone-based probe to monitor the activity of glutathionylspermidine amidase in Escherichia coli. Chem. BioChem. 12(15), 2306–2309 (2011)

    CAS  Google Scholar 

  41. Olsen, J.V., de Godoy, L.M., Li, G., Macek, B., Mortensen, P., Pesch, R., Makarov, A., Lange, O., Horning, S., Mann, M.: Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4(12), 2010–2021 (2005)

    Article  CAS  Google Scholar 

  42. Chiang, B.Y., Chen, T.C., Pai, C.H., Chou, C.C., Chen, H.H., Ko, T.P., Hsu, W.H., Chang, C.Y., Wu, W.F., Wang, A.H.J., Lin, C.H.: Protein S-thiolation by glutathionylspermidine (Gsp): the role of Escherichia coli Gsp synthetase/amidase in redox regulation. J. Biol. Chem. 285(33), 25345–25353 (2010)

    Article  CAS  Google Scholar 

  43. Choi, S., Jeong, J., Na, S., Lee, H.S., Kim, H.Y., Lee, K.J., Paek, E.: New algorithm for the identification of intact disulfide linkages based on fragmentation characteristics in tandem mass spectra. J. Proteome Res. 9(1), 626–635 (2010)

    Article  CAS  Google Scholar 

  44. Wells, J.M., McLuckey, S.A.: Collision‐induced dissociation (CID) of peptides and proteins. Methods Enzymol. 402, 148–185 (2005)

    Article  CAS  Google Scholar 

  45. Chen, Y.Y., Chu, H.M., Pan, K.T., Teng, C.H., Wang, D.L., Wang, A.H., Khoo, K.H., Meng, T.C.: Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation. J. Biol. Chem. 283(50), 35265–35272 (2008)

    Article  CAS  Google Scholar 

  46. Pan, K.T., Chen, Y.Y., Pu, T.H., Chao, Y.S., Yang, C.Y., Bomgarden, R.D., Rogers, J.C., Meng, T.C., Khoo, K.H.: Mass spectrometry-based quantitative proteomics for dissecting multiplexed redox cysteine modifications in nitric oxide-protected cardiomyocyte under hypoxia. Antioxid. Redox Signal. 20(9), 1365–1381 (2014)

    Article  CAS  Google Scholar 

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Acknowledgments

The authors acknowledge support for this work by the Academia Sinica and a Taiwan National Science Council grant (NSC100-2325-B-001-029) to the Core Facilities for Protein Structural Analysis located at the Institute of Biological Chemistry (IBC), Academia Sinica. The authors also acknowledge the use of Academia Sinica Mass Spectrometry Facilities at IBC.

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Authors and Affiliations

  1. Core Facilities for Protein Structural Analysis, Academia Sinica, Taipei, 11529, Taiwan

    Chi-Chi Chou

  2. Institute of Biological Chemistry, Academia Sinica, Taipei, 11529, Taiwan

    Chi-Chi Chou, Bing-Yu Chiang, Jason Ching-Yao Lin, Kuan-Ting Pan, Chun-Hung Lin & Kay-Hooi Khoo

  3. Institute of Biochemical Sciences, National Taiwan University, Taipei, 10617, Taiwan

    Bing-Yu Chiang, Kuan-Ting Pan, Chun-Hung Lin & Kay-Hooi Khoo

  4. Department of Chemistry, National Tsing Hua University, Hsinchu, 30013, Taiwan

    Jason Ching-Yao Lin

  5. Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei, 11529, Taiwan

    Jason Ching-Yao Lin & Chun-Hung Lin

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  1. Chi-Chi Chou
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Chou, CC., Chiang, BY., Lin, J.CY. et al. Characteristic Tandem Mass Spectral Features Under Various Collision Chemistries for Site-Specific Identification of Protein S-Glutathionylation. J. Am. Soc. Mass Spectrom. 26, 120–132 (2015). https://doi.org/10.1007/s13361-014-1014-9

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