Abstract
The ratio between reduced and oxidized thiols, mainly glutathione and oxidized glutathione, is one of the biomarkers for the evaluation of oxidative stress. The accurate measurement of thiol concentrations is challenging because reduced thiols are easily oxidized during sample manipulation. Derivatization is commonly used to protect thiols from oxidation. The objective of this work was to systematically compare two cell-permeable derivatizing agents: N-ethyl maleimide (NEM) and (R)-(+)-N-(1-phenylethyl)maleimide (NPEM) in terms of derivatization efficiency, ionization enhancement, side product formation, reaction selectivity for thiols, pH dependence of the reaction, and derivative stability. All thiol measurements and the characterization of side products were performed using a biphenyl reversed phase liquid chromatography–high-resolution mass spectrometry (LC-HRMS). Four thiols, cysteine (CYS), homocysteine, N-acetylcysteine (NAC), and glutathione (GSH), were used for the evaluation. Using 1:10 ratio of thiol:derivatizing agent, complete derivatization was obtained within 30 min for both agents tested with the exception of CYS-NEM, where 97% efficiency was obtained. The more hydrophobic NPEM provided better ionization of the thiols, with enhancement ranging from 2.1x for GSH to 5.7x for CYS in comparison to NEM. NPEM derivatization led to more extensive side reactions, such as double derivatization and ring opening, which hindered the accurate measurement of the thiol concentrations. Both NEM and NPEM also showed poor stability of CYS derivative due to its time-dependent conversion to cyclic cysteine-maleimide derivative. Both reagents also showed significant reactivity with amine-containing metabolites depending on the pH used during derivatization, but overall NEM was found to be more selective towards thiol group than NPEM. Taking into account all evaluation criteria, NEM was selected as a more suitable reagent for the thiol protection and derivatization, but strict control of pH 7.0 is recommended to minimize the side reactions. This work illustrates the importance of the characterization of side products and derivative stability during the evaluation of thiol derivatizing agents and contributes fundamental understanding to improve the accuracy of thiol determinations. The key sources of errors during maleimide derivatization include the derivatization of amine-containing metabolites, poor derivative stability of certain thiols (CYS and NAC), and the side reactions especially if ring opening of the reagent is not minimized.
Similar content being viewed by others
References
Dalle-Donne I, Rossi R. Analysis of thiols. J Chromatogr B Anal Technol Biomed Life Sci. 2009;877:3271–3. https://doi.org/10.1016/j.jchromb.2009.08.034.
Pastore A, Federici G, Bertini E, Piemonte F. Analysis of glutathione: implication in redox and detoxification. Clin Chim Acta. 2003;333:19–39. https://doi.org/10.1016/S0009-8981(03)00200-6.
Toyo’oka T. Recent advances in separation and detection methods for thiol compounds in biological samples. J Chromatogr B Anal Technol Biomed Life Sci. 2009;877:3318–30. https://doi.org/10.1016/j.jchromb.2009.03.034.
Ortmayr K, Schwaiger M, Hann S, Koellensperger G. An integrated metabolomics workflow for the quantification of sulfur pathway intermediates employing thiol protection with N-ethyl maleimide and hydrophilic interaction liquid chromatography tandem mass spectrometry. Analyst. 2015;140:7687–95. https://doi.org/10.1039/C5AN01629K.
Whillier S, Raftos JE, Chapman B, Kuchel PW. Role of N-acetylcysteine and cystine in glutathione synthesis in human erythrocytes. Redox Rep. 2009;14:115–24. https://doi.org/10.1179/135100009X392539.
Bouligand J, Deroussent A, Paci A, Morizet J, Vassal G. Liquid chromatography-tandem mass spectrometry assay of reduced and oxidized glutathione and main precursors in mice liver. J Chromatogr B Anal Technol Biomed Life Sci. 2006;832:67–74. https://doi.org/10.1016/j.jchromb.2005.12.037.
Sentellas S, Morales-Ibanez O, Zanuy M, Albertí JJ. GSSG/GSH ratios in cryopreserved rat and human hepatocytes as a biomarker for drug induced oxidative stress. Toxicol in Vitro. 2014;28:1006–15. https://doi.org/10.1016/j.tiv.2014.04.017.
Giustarini D, Tsikas D, Colombo G, Milzani A, Dalle-Donne I, Fanti P, et al. Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: an elephant in the room. J Chromatogr B. 2016;1019:21–8. https://doi.org/10.1016/j.jchromb.2016.02.015.
Giustarini D, Colombo G, Garavaglia ML, Astori E, Portinaro NM, Reggiani F, et al. Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells. Free Radic Biol Med. 2017;112:360–75. https://doi.org/10.1016/j.freeradbiomed.2017.08.008.
Hansen RE, Winther JR. An introduction to methods for analyzing thiols and disulfides: reactions, reagents, and practical considerations. Anal Biochem. 2009;394:147–58. https://doi.org/10.1016/j.ab.2009.07.051.
Giustarini D, Dalle-Donne I, Milzani A, Fanti P, Rossi R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat Protoc. 2013;8:1660–9. https://doi.org/10.1038/nprot.2013.095.
Winters RA, Zukowski J, Ercal N, Matthews RH, Spitz DR. Analysis of glutathione, glutathione disulfide, cysteine, homocysteine, and other biological thiols by high-performance liquid chromatography following derivatization by N-(1-pyrenyl)maleimide. Anal Biochem. 1995;227:14–21. https://doi.org/10.1006/abio.1995.1246.
Wang W, Rusin O, Xu X, Kim KK, Escobedo JO, Fakayode SO, et al. Detection of homocysteine and cysteine. J Am Chem Soc. 2005;127:15949–58. https://doi.org/10.1021/ja054962n.
Camera E, Picardo M. Analytical methods to investigate glutathione and related compounds in biological and pathological processes. J Chromatogr B. 2002;781:181–206. https://doi.org/10.1016/S1570-0232(02)00618-9.
Paroni R, De Vecchi E, Cighetti G, Arcelloni C, Fermo I, Grossi A, et al. HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue. Clin Chem. 1995;41:448–54.
Asensi M, Sastre J, Pallardo FV, Estrela JM, Vina J. Determination of oxidized glutathione in blood: high-performance liquid chromatography. Methods Enzymol. 1994;234:367–71. https://doi.org/10.1016/0076-6879(94)34106-0.
Seiwert B, Karst U. Simultaneous LC/MS/MS determination of thiols and disulfides in urine samples based on differential labeling with ferrocene-based maleimides. Anal Chem. 2007;79:7131–8. https://doi.org/10.1021/ac071016b.
D’Agostino LA, Lam KP, Lee R, Britz-McKibbin P. Comprehensive plasma thiol redox status determination for metabolomics. J Proteome Res. 2011;10:592–603. https://doi.org/10.1021/pr100771g.
Welch L, Dong X, Hewitt D, Irwin M, McCarty L, Tsai C, et al. Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization. J Chromatogr B Anal Technol Biomed Life Sci. 2018;1092:158–67. https://doi.org/10.1016/j.jchromb.2018.05.039.
Wang J, Zhou L, Lei H, Hao F, Liu X, Wang Y, et al. Simultaneous quantification of amino metabolites in multiple metabolic pathways using ultra-high performance liquid chromatography with tandem-mass spectrometry. Sci Rep. 2017;7:1423. https://doi.org/10.1038/s41598-017-01435-7.
Liu P, Huang YQ, Cai WJ, Yuan BF, Feng YQ. Profiling of thiol-containing compounds by stable isotope labeling double precursor ion scan mass spectrometry. Anal Chem. 2014;86:9765–73. https://doi.org/10.1021/ac5023315.
Gori SS, Lorkiewicz P, Ehringer DS, Belshoff AC, Higashi RM, Fan TWM, et al. Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry. Anal Bioanal Chem. 2014;406:4371–9. https://doi.org/10.1007/s00216-014-7810-z.
Smyth DG, Nagamatsu A, Fruton JS. Some reactions of N-Ethylmaleimide. J Am Chem Soc. 1960;82:4600–4. https://doi.org/10.1021/ja01502a039.
Sutton TR, Minnion M, Barbarino F, Koster G, Fernandez BO, Cumpstey AF, et al. A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome. Redox Biol. 2018;16:359–80. https://doi.org/10.1016/j.redox.2018.02.012.
Ates B, Ercal BC, Manda K, Abraham L, Ercal N. Determination of glutathione disulfide levels in biological samples using thiol-disulfide exchanging agent, dithiothreitol. Biomed Chromatogr. 2009;23:119–23. https://doi.org/10.1002/bmc.1083.
Majima E, Goto S, Hori H, Shinohara Y, Hong YM, Terada H. Stabilities of the fluorescent SH-reagent eosin-5-maleimide and its adducts with sulfhydryl compounds. BBA - Gen Subj. 1995;1243:336–42. https://doi.org/10.1016/0304-4165(94)00159-U.
Jemal M, Hawthorne D. High performance liquid chromatography/ionspray mass spectrometry of N-ethylmaleimide and acrylic acid ester derivatives for bioanalysis of thiol compounds. Rapid Commun Mass Spectrom. 1994;8:854–7. https://doi.org/10.1002/rcm.1290081012.
Giustarini D, Dalle-Donne I, Milzani A, Rossi R. Detection of glutathione in whole blood after stabilization with N-ethylmaleimide. Anal Biochem. 2011;415:81–3. https://doi.org/10.1016/j.ab.2011.04.013.
Asensi M, Sastre J, Pallardo FV, de la Asuncion JG, Estrela JM, Viña J. A high-performance liquid chromatography method for measurement of oxidized glutathione in biological samples. Anal Biochem. 1994;217:323–8.
Santori G, Domenicotti C, Bellocchio A, Pronzato MA, Marinari UM, Cottalasso D. Different efficacy of iodoacetic acid and N-ethylmaleimide in high-performance liquid chromatographic measurement of liver glutathione. J Chromatogr B Biomed Appl. 1997;695:427–33. https://doi.org/10.1016/S0378-4347(97)00159-X.
Winther JR, Thorpe C. Quantification of thiols and disulfides. Biochim Biophys Acta, Gen Subj. 2014;1840:838–46. https://doi.org/10.1016/j.bbagen.2013.03.031.
Fontana A, Toniolo C. Detection and determination of thiols. In: Patai S, editor. The chemistry of the thiol group. Bristol: Wiley; 1974. p. 294–8.
Paulech J, Solis N, Cordwell SJ. Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry. Biochim Biophys Acta, Proteins Proteomics. 2013;1834:372–9. https://doi.org/10.1016/j.bbapap.2012.08.002.
Funding
This work was supported by the Natural Sciences and Engineering Research Council of Canada (Grant RGPIN 435814-20013) and Concordia University (Grant J00126). The funding agencies had no role in study design, the collection, analysis, and interpretation of data, manuscript writing, or the decision to submit the article for publication.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 1635 kb)
Rights and permissions
About this article
Cite this article
Russo, M.S.T., Napylov, A., Paquet, A. et al. Comparison of N-ethyl maleimide and N-(1-phenylethyl) maleimide for derivatization of biological thiols using liquid chromatography-mass spectrometry. Anal Bioanal Chem 412, 1639–1652 (2020). https://doi.org/10.1007/s00216-020-02398-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-020-02398-x