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Quantitation of Glutathione, Glutathione Disulphide, and Protein-Glutathione Mixed Disulphides by High-Performance Liquid Chromatography-Tandem Mass Spectrometry

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Functional Disulphide Bonds

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1967))

Abstract

Protein-glutathione mixed disulphides (PSSG) are an important redox-sensitive posttranslational modification. Quantitation of protein-glutathione mixed disulphides (PSSG) is achieved by the reduction of the disulphide bond to liberate glutathione (GSH); however, this method leaves the assay susceptible to contamination by cytosolic GSH and glutathione disulphide (GSSG) captured during protein precipitation. The method herein describes a workflow in which protein from mouse liver is precipitated and adventitious GSH contamination is removed by reaction with N-ethylmaleimide. The sample is divided into two equal aliquots, a control aliquot that allows for direct quantitation of adventitious GSSG and a chemically reduced aliquot that contains GSH from both the GSSG and PSSG disulphides. Determining the concentration of adventitious GSSG allows for correction of the latter value to provide an accurate assay of PSSG. This assay also provides quantitation of cytosolic GSH and GSSG.

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References

  1. Mailloux RJ, Treberg JR (2016) Protein S-glutathionylation links energy metabolism to redox signaling in mitochondria. Redox Biol 8:110–118. https://doi.org/10.1016/j.redox.2015.12.010

    Article  CAS  PubMed  Google Scholar 

  2. Mailloux RJ, Fu A, Robson-Doucette C, Allister EM, Wheeler MB, Screaton R, Harper M-E (2012) Glutathionylation state of uncoupling protein-2 and the control of glucose-stimulated insulin secretion. J Biol Chem 287:39673–39685. https://doi.org/10.1074/jbc.M112.393538

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mailloux RJ, Craig Ayre D, Christian SL (2016) Induction of mitochondrial reactive oxygen species production by GSH mediated S-glutathionylation of 2-oxoglutarate dehydrogenase. Redox Biol 8:285–297. https://doi.org/10.1016/j.redox.2016.02.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. O’Brien M, Chalker J, Slade L, Gardiner D, Mailloux RJ (2017) Protein S-glutathionylation alters superoxide/hydrogen peroxide emission from pyruvate dehydrogenase complex. Free Radic Biol Med 106:302–314. https://doi.org/10.1016/j.freeradbiomed.2017.02.046

    Article  CAS  PubMed  Google Scholar 

  5. Barinova KV, Serebryakova MV, Muronetz VI, Schmalhausen EV (2017) S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme. Biochim Biophys Acta Gen Subj 1861:3167–3177. https://doi.org/10.1016/j.bbagen.2017.09.008

    Article  CAS  PubMed  Google Scholar 

  6. Reynaert NL, Van Der VA, Guala AS, Mcgovern T, Hristova M, Pantano C, Heintz NH, Heim J, Ho Y, Matthews DE, Wouters EFM, Janssen-heininger YMW (2006) Dynamic redox control of NF-κB through glutaredoxin-regulated S-glutathionylation of inhibitory κB kinase β. Proc Natl Acad Sci U S A 103:13086–13091

    Article  CAS  Google Scholar 

  7. Ullevig SL, Kim HS, Short JD, Tavakoli S, Weintraub ST, Downs K, Asmis R (2016) Protein S -Glutathionylation mediates macrophage responses to metabolic cues from the extracellular environment. Antioxid Redox Signal 25:836–851. https://doi.org/10.1089/ars.2015.6531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nagarkoti S, Dubey M, Awasthi D, Kumar V, Chandra T, Kumar S, Dikshit M (2018) S-Glutathionylation of p47phox sustains superoxide generation in activated neutrophils. Biochim Biophys Acta, Mol Cell Res 1865:444–454. https://doi.org/10.1016/j.bbamcr.2017.11.014

    Article  CAS  Google Scholar 

  9. Beer SM, Taylor ER, Brown SE, Dahm CC, Costa NJ, Runswick MJ, Murphy MP (2004) Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant defense. J Biol Chem 279:47939–47951. https://doi.org/10.1074/jbc.M408011200

    Article  CAS  PubMed  Google Scholar 

  10. Picklo MJ, Idso JP, Jackson MI (2013) S-Glutathionylation of hepatic and visceral adipose proteins decreases in obese rats. Obesity 21:297–305. https://doi.org/10.1002/oby.20002

    Article  CAS  PubMed  Google Scholar 

  11. Hill BG, Ramana KV, Cai J, Bhatnagar A, Srivastava SK (2010) Measurement and identification of S-glutathiolated proteins. In: Methods enzymology, 1st edn. Elsevier Inc., Amsterdam, pp 179–197

    Google Scholar 

  12. Tipple TE, Rogers LK (2012) Methods for the determination of plasma or tissue glutathione levels. Methods Mol Biol 889:315–324. https://doi.org/10.1007/978-1-61779-867-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Aesif SW, Anathy V, Kuipers I, Guala AS, Reiss JN, Ho Y-S, Janssen-Heininger YMW (2011) Ablation of glutaredoxin-1 attenuates lipopolysaccharide-induced lung inflammation and alveolar macrophage activation. Am J Respir Cell Mol Biol 44:491–499. https://doi.org/10.1165/rcmb.2009-0136OC

    Article  CAS  PubMed  Google Scholar 

  14. Menon D, Board PG (2013) A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3-naphthalenedicarboxaldehyde. Anal Biochem 433:132–136. https://doi.org/10.1016/j.ab.2012.10.009

    Article  CAS  PubMed  Google Scholar 

  15. Zhang C, Rodriguez C, Circu ML, Aw TY, Feng J (2011) S-Glutathionyl quantification in the attomole range using glutaredoxin-3-catalyzed cysteine derivatization and capillary gel electrophoresis with laser-induced fluorescence detection. Anal Bioanal Chem 401:2165–2175. https://doi.org/10.1007/s00216-011-5311-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lou FM, Mckellar R, Chyan O (1986) Quantitation of lens protein mixed disulfides by ion-exchange chromatography. Exp Eye Res 42:607–616

    Article  CAS  Google Scholar 

  17. Rodriguez-Ariza A, Toribio F, López-Barea J (1994) Rapid determination of glutathione status in fish liver using high-performance liquid chromatography and electrochemical detection. J Chromatogr B Biomed Appl 656:311–318

    Article  CAS  Google Scholar 

  18. Squellerio I, Caruso D, Porro B, Veglia F, Tremoli E, Cavalca V (2012) Direct glutathione quantification in human blood by LC-MS/MS: comparison with HPLC with electrochemical detection. J Pharm Biomed Anal 71:111–118. https://doi.org/10.1016/j.jpba.2012.08.013

    Article  CAS  PubMed  Google Scholar 

  19. Smith NC, Dunnett M, Mills PC (1995) Simultaneous quantitation of oxidised and reduced glutathione in equine biological fluids by reversed-phase high-performance liquid chromatography using electrochemical detection. J Chromatogr B Biomed Appl 673:35–41

    Article  CAS  Google Scholar 

  20. Richie JP, Lang CA (1987) The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection. Anal Biochem 163:9–15

    Article  CAS  Google Scholar 

  21. Vandeberg PJ, Johnson DC (1993) Pulsed electrochemical detection of cysteine, cystine, methionine, and glutathione at gold electrodes following their separation by liquid chromatography. Anal Chem 65:2713–2718. https://doi.org/10.1021/ac00068a002

    Article  CAS  Google Scholar 

  22. Hansen RE, Roth D, Winther JR (2009) Quantifying the global cellular thiol-disulfide status. Proc Natl Acad Sci U S A 106:422–427. https://doi.org/10.1073/pnas.0812149106

    Article  PubMed  PubMed Central  Google Scholar 

  23. Bukowski MR, Bucklin C, Picklo MJ (2015) Quantitation of protein S-glutathionylation by liquid chromatography-tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide. Anal Biochem 469:54–64. https://doi.org/10.1016/j.ab.2014.10.002

    Article  CAS  PubMed  Google Scholar 

  24. Webb K, Bristow T, Sargent M (2004) Methodology for accurate mass measurement of small molecules best practice guide. LCG, Teddington

    Google Scholar 

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Acknowledgments

The authors thank Joseph Idso and Christopher Bucklin for their technical assistance. Funding was provided through USDA–ARS project 3062-51000-053-00D. The US Department of Agriculture–Agricultural Research Service, Plains Area, is an equal opportunity/affirmative action employer, and all agency services are available without discrimination. Mention of trade names or commercial products in this chapter is solely for providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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Authors and Affiliations

  1. USDA-ARS Grand Forks Human Nutrition Research Center, University of North Dakota, Grand Forks, ND, USA

    Michael R. Bukowski & Matthew J. Picklo Sr.

  2. Department of Chemistry, University of North Dakota, Grand Forks, ND, USA

    Michael R. Bukowski & Matthew J. Picklo Sr.

Authors
  1. Michael R. Bukowski
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  2. Matthew J. Picklo Sr.
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Correspondence to Michael R. Bukowski .

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Editors and Affiliations

  1. The Centenary Institute, NHMRC Clinical Trials Centre, Sydney Medical School, University of Sydney, Sydney, NSW, Australia

    Philip Hogg

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Bukowski, M.R., Picklo, M.J. (2019). Quantitation of Glutathione, Glutathione Disulphide, and Protein-Glutathione Mixed Disulphides by High-Performance Liquid Chromatography-Tandem Mass Spectrometry. In: Hogg, P. (eds) Functional Disulphide Bonds. Methods in Molecular Biology, vol 1967. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9187-7_12

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  • DOI: https://doi.org/10.1007/978-1-4939-9187-7_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9186-0

  • Online ISBN: 978-1-4939-9187-7

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