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In-Source Reduction of Disulfide-Bonded Peptides Monitored by Ion Mobility Mass Spectrometry

  • Bradley B. Stocks
  • Jeremy E. Melanson
Research Article

Abstract

Many peptides with antimicrobial activity and/or therapeutic potential contain disulfide bonds as a means to enhance stability, and their quantitation is often performed using electrospray ionization mass spectrometry (ESI-MS). Disulfides can be reduced during ESI under commonly used instrument conditions, which has the potential to hinder accurate peptide quantitation. We demonstrate that this in-source reduction (ISR) is predominantly observed for peptides infused from acidic solutions and subjected to elevated ESI voltages (3–4 kV). ISR is readily apparent in the mass spectrum of oxytocin—a small, single disulfide-containing peptide. However, subtle m/z shifts due to partial ISR of highly charged (z ≥ 3) peptides with multiple disulfide linkages may proceed unnoticed. Ion mobility (IM)-MS separates ions on the basis of charge and shape in the gas phase, and using insulin as a model system, we show that IM-MS arrival time distributions (ATDs) are particularly sensitive to partial ISR of large peptides. Isotope modeling allows for the relative quantitation of disulfide-intact and partially reduced states of the mobility-separated peptide conformers. Interestingly, hepcidin peptides ionized from acidic solutions at elevated ESI voltages undergo gas-phase compaction, ostensibly due to partial disulfide ISR. Our IM-MS results lead us to propose that residual acid is the likely cause of disparate ATDs recently measured for hepcidin from different suppliers [Anal. Bioanal. Chem. 409, 2559–2567 (2017)]. Overall, our results demonstrate the utility of IM-MS to detect partial ISR of disulfide-bonded peptides and reinforce the notion that peptide/protein measurements should be carried out using minimally activating instrument conditions.

Graphical Abstract

Keywords

Peptide Disulfide bond In-source reduction Ion mobility mass spectrometry 

Supplementary material

13361_2018_1894_MOESM1_ESM.pdf (633 kb)
ESM 1 (PDF 633 kb)

References

  1. 1.
    Fosgerau, K., Hoffmann, T.: Peptide therapeutics: current status and future directions. Drug Discovery Today. 20, 122–128 (2015)CrossRefGoogle Scholar
  2. 2.
    Bird, G.H., Madani, N., Perry, A.F., Princiotto, A.M., Supko, J.G., He, X., Gavathiotis, E., Sodroski, J.G., Walensky, L.D.: Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic. Proc. Natl. Acad. Sci. U. S. A. 107, 14093–14098 (2010)CrossRefGoogle Scholar
  3. 3.
    Betz, S.F.: Disulfide bonds and the stability of globular proteins. Protein Sci. 2, 1551–1558 (1993)CrossRefGoogle Scholar
  4. 4.
    Liu, T., Wang, Y., Luo, X., Li, J., Reed, S.A., Xiao, H., Young, T.S., Schultz, P.G.: Enhancing protein stability with extended disulfide bonds. Proc. Natl. Acad. Sci. U. S. A. 113, 5910–5915 (2016)CrossRefGoogle Scholar
  5. 5.
    Li, Y., Li, X., Zheng, X., Tang, L., Xu, W., Gong, M.: Disulfide bond prolongs the half-life of therapeutic peptide-GLP-1. Peptides. 32, 1400–1407 (2011)CrossRefGoogle Scholar
  6. 6.
    Góngora-Benítez, M., Tulla-Puche, J., Albericio, F.: Multifaceted roles of disulfide bonds. Peptides as therapeutics. Chem. Rev. 114, 901–926 (2014)CrossRefGoogle Scholar
  7. 7.
    Moore, S.J., Leung, C.L., Cochran, J.R.: Knottins: disulfide-bonded therapeutic and diagnostic peptides. Drug Discovery Today: Technol. 9, e1–e11 (2012)Google Scholar
  8. 8.
    Gorman, J.J., Wallis, T.P., Pitt, J.J.: Protein disulfide bond determination by mass spectrometry. Mass Spectrom. Rev. 21, 183–216 (2002)CrossRefGoogle Scholar
  9. 9.
    Xia, Y., Cooks, R.G.: Plasma induced oxidative cleavage of disulfide bonds in polypeptides during nanoelectrospray ionization. Anal. Chem. 82, 2856–2864 (2010)CrossRefGoogle Scholar
  10. 10.
    Stinson, C.A., Xia, Y.: Radical induced disulfide bond cleavage within peptides via ultraviolet irradiation of an electrospray plume. Analyst. 138, 2840–2846 (2013)CrossRefGoogle Scholar
  11. 11.
    Mentinova, M., McLuckey, S.A.: Cleavage of multiple disulfide bonds in insulin via gold cationization and collision-induced dissociation. Int. J. Mass Spectrom. 308, 133–136 (2011)CrossRefGoogle Scholar
  12. 12.
    Kraj, A., Brouwer, H.-J., Reinhoud, N., Chervet, J.-P.: A novel electrochemical method for efficient reduction of disulfide bonds in peptides and proteins prior to MS detection. Anal. Bioanal. Chem. 405, 9311–9320 (2013)CrossRefGoogle Scholar
  13. 13.
    Nicolardi, S., Deelder, A.M., Palmblad, M., van der Burgt, Y.E.: Structural analysis of an intact monoclonal antibody by online electrochemical reduction of disulfide bonds and Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 86, 5376–5382 (2014)CrossRefGoogle Scholar
  14. 14.
    Trabjerg, E., Jakobsen, R.U., Mysling, S., Christensen, S., Jørgensen, T.J.D., Rand, K.D.: Conformational analysis of large and highly disulfide-stabilized proteins by integrating online electrochemical reduction into an optimized H/D exchange mass spectrometry workflow. Anal. Chem. 87, 8880–8888 (2015)CrossRefGoogle Scholar
  15. 15.
    Cramer, C.N., Kelstrup, C.D., Olsen, J.V., Haselmann, K.F., Nielsen, P.K.: Complete mapping of complex disulfide patterns with closely-spaced cysteines by in-source reduction and data-dependent mass spectrometry. Anal. Chem. 89, 5949–5957 (2017)CrossRefGoogle Scholar
  16. 16.
    Morand, K., Talbo, G., Mann, M.: Oxidation of peptides during electrospray ionization. Rapid Commun. Mass Spectrom. 7, 738–743 (1993)CrossRefGoogle Scholar
  17. 17.
    Boys, B.L., Kuprowski, M.C., Konermann, L.: Protein oxidative modifications during electrospray ionization: solution phase electrochemistry or corona discharge-induced radical attack? Anal. Chem. 81, 4027–4034 (2009)CrossRefGoogle Scholar
  18. 18.
    Koeniger, S.L., Merenbloom, S.I., Clemmer, D.E.: Evidence for many resolvable structures within conformation types of electrosprayed ubiquitin ions. J. Phys. Chem. B. 110, 7017–7021 (2006)CrossRefGoogle Scholar
  19. 19.
    Scarff, C.A., Patel, V.J., Thalassinos, K., Scrivens, J.H.: Probing hemoglobin structure by means of traveling-wave ion mobility mass spectrometry. J. Am. Soc. Mass Spectrom. 20, 625–631 (2009)CrossRefGoogle Scholar
  20. 20.
    Knapman, T.W., Morton, V.L., Stonehouse, N.J., Stockley, P.G., Ashcroft, A.E.: Determining the topology of virus assembly intermediates using ion mobility spectrometry-mass spectrometry. Rapid Commun. Mass Spectrom. 24, 3033–3042 (2010)CrossRefGoogle Scholar
  21. 21.
    Bleiholder, C., Dupuis, N.F., Wyttenbach, T., Bowers, M.T.: Ion mobility-mass spectrometry reveals a conformational conversion from random assembly to β-sheet in amyloid fibril formation. Nat. Chem. 3, 172–177 (2011)CrossRefGoogle Scholar
  22. 22.
    Seo, J., Hoffman, W., Warnke, S., Bowers, M.T., Pagel, K., von Helden, G.: Retention of native protein structures in the absence of solvent: a coupled ion mobility and spectroscopic study. Angew. Chem. Int. Ed. 55, 14173–14176 (2016)CrossRefGoogle Scholar
  23. 23.
    Göth, M., Pagel, K.: Ion mobility-mass spectrometry as a tool to investigate protein-ligand interactions. Anal. Bioanal. Chem. 409, 4305–4310 (2017)CrossRefGoogle Scholar
  24. 24.
    Pacholarz, K.J., Burnley, R.J., Jowitt, T.A., Ordsmith, V., Pisco, J.P., Porrini, M., Larrouy-Maumus, G., Garlish, R.A., Taylor, R.J., de Carvalho, L.P., Barran, P.E.: Hybrid mass spectrometry approaches to determine how L-histidine feedback regulates the enzyzme MtATP-phosphoribosyltransferase. Structure. 25, 730–738 (2017)CrossRefGoogle Scholar
  25. 25.
    Ruotolo, B.T., Robinson, C.V.: Aspects of native proteins are retained in vacuum. Curr. Opin. Chem. Biol. 10, 402–408 (2006)CrossRefGoogle Scholar
  26. 26.
    Devine, P.W.A., Fisher, H.C., Calabrese, A.N., Whelan, F., Higazi, D.R., Potts, J.R., Lowe, D.C., Radford, S.E., Ashcroft, A.E.: Investigating the structural compaction of biomolecules upon transition to the gas-phase using ESI-TWIMS-MS. J. Am. Soc. Mass Spectrom. 28, 1855–1862 (2017)CrossRefGoogle Scholar
  27. 27.
    Vahidi, S., Stocks, B.B., Konermann, L.: Partially disordered proteins studied by ion mobility-mass spectrometry: implications for the preservation of solution phase structure in the gas phase. Anal. Chem. 85, 10471–10478 (2013)CrossRefGoogle Scholar
  28. 28.
    Distler, U., Kuharev, J., Navarro, P., Levin, Y., Schild, H., Tenzer, S.: Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics. Nat. Methods. 11, 167–170 (2014)CrossRefGoogle Scholar
  29. 29.
    Pierson, N.A., Chen, L., Valentine, S.J., Russell, D.H., Clemmer, D.E.: Number of solution states of bradykinin from ion mobility and mass spectrometry measurements. J. Am. Chem. Soc. 133, 13810–13813 (2011)CrossRefGoogle Scholar
  30. 30.
    Pierson, N.A., Chen, L., Russell, D.H., Clemmer, D.E.: Cis-trans isomerizations of proline residues are key to bradykinin conformations. J. Am. Chem. Soc. 135, 3186–3192 (2013)CrossRefGoogle Scholar
  31. 31.
    Massonnet, P., Haler, J.R.N., Upert, G., Degueldre, M., Morsa, D., Smargiasso, N., Mourier, G., Gilles, N., Quinton, L., De Pauw, E.: Ion mobility-mass spectrometry as a tool for the structural characterization of peptides bearing intramolecular disulfide bond(s). J. Am. Soc. Mass Spectrom. 27, 1637–1646 (2016)CrossRefGoogle Scholar
  32. 32.
    Pritchard, C., O’Connor, G., Ashcroft, A.E.: The role of ion mobility spectrometry-mass spectrometry in the analysis of protein reference standards. Anal. Chem. 85, 7205–7212 (2013)CrossRefGoogle Scholar
  33. 33.
    Bros, P., Josephs, R.D., Stoppacher, N., Cazals, G., Lehmann, S., Hirtz, C., Wielgosz, R.I., Delatour, V.: Impurity determination for hepcidin by liquid chromatography-high resolution and ion mobility mass spectrometry for the value assignment of candidate primary calibrators. Anal. Bioanal. Chem. 409, 2559–2567 (2017)CrossRefGoogle Scholar
  34. 34.
    Jordan, J.B., Poppe, L., Haniu, M., Arvedson, T., Syed, R., Li, V., Kohno, H., Kim, H., Schnier, P.D., Harvey, T.S., Miranda, L.P., Cheetham, J., Sasu, B.J.: Hepcidin revisited, disulfide connectivity, dynamics, and structure. J. Biol. Chem. 284, 24155–24167 (2009)CrossRefGoogle Scholar
  35. 35.
    Bush, M.F., Campuzano, I.D.G., Robinson, C.V.: Ion mobility mass spectrometry of peptide ions: effects of drift gas and calibration strategies. Anal. Chem. 84, 7124–7130 (2012)CrossRefGoogle Scholar
  36. 36.
    Chalkley, R.J., Baker, P.R., Medzihradszky, K.F., Lynn, A.J., Burlingame, A.L.: In-depth analysis of tandem mass spectrometry data from disparate instrument types. Mol. Cell. Proteomics. 7, 2386–2398 (2008)CrossRefGoogle Scholar
  37. 37.
    Koehbach, J., O'Brien, M., Muttenthaler, M., Miazzo, M., Akcan, M., Elliott, A.G., Daly, N.L., Harvey, P.J., Arrowsmith, S., Gunasekera, S., Smith, T.J., Wray, S., Göransson, U., Dawson, P.E., Craik, D.J., Freissmuth, M., Gruber, C.W.: Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design. Proc. Natl. Acad. Sci. U. S. A. 110, 21183–21188 (2013)CrossRefGoogle Scholar
  38. 38.
    Fuller, D.R., Glover, M.S., Pierson, N.A., Kim, D., Russell, D.H., Clemmer, D.E.: Cis-->trans isomerization of Pro(7) in oxytocin regulates Zn(2+) binding. J. Am. Soc. Mass Spectrom. 27, 1376–1382 (2016).CrossRefGoogle Scholar
  39. 39.
    Ryle, A.P., Sanger, F., Smith, L.F., Kitai, R.: The disulphide bonds of insulin. Biochem. J. 60, 541–556 (1955)CrossRefGoogle Scholar
  40. 40.
    Snijder, J., van de Waterbeemd, M., Glover, M.S., Shi, L., Clemmer, D.E., Heck, A.J.: Conformational landscape and pathway of disulfide bond reduction of human alpha defensin. Protein Sci. 24, 1264–1271 (2015)CrossRefGoogle Scholar
  41. 41.
    Li, G., Pei, J., Yin, Y., Huang, G.: Direct sequencing of a disulfide-linked peptide with electrospray ionization tandem mass spectrometry. Analyst. 140, 2623–2627 (2015)CrossRefGoogle Scholar
  42. 42.
    Gianelli, L., Amendola, V., Fabbrizzi, L., Pallavicini, P., Mellerio, G.G.: Investigation of reduction of Cu(II) complexes in positive-ion mode electrospray mass spectrometry. Rapid Commun. Mass Spectrom. 15, 2347–2353 (2001)CrossRefGoogle Scholar
  43. 43.
    Nemeth, E., Tuttle, M.S., Powelson, J., Vaughn, M.B., Donovan, A., McVey Ward, D., Ganz, T., Kaplan, J.: Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 306, 2090–2093 (2004)CrossRefGoogle Scholar
  44. 44.
    Krause, A., Neitz, S., Mägert, H.-J., Schulz, A., Forssmann, W.-G., Schulz-Knappe, P., Adermann, K.: LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett. 480, 147–150 (2000)CrossRefGoogle Scholar
  45. 45.
    Guaratini, T., Gates, P.J., Pinto, E., Colepicolo, P., Lopes, N.P.: Differential ionisation of natural antioxidant polyenes in electrospray and nanospray mass spectrometry. Rapid Commun. Mass Spectrom. 21, 3842–3848 (2007)CrossRefGoogle Scholar
  46. 46.
    Pagel, K., Natan, E., Hall, Z., Fersht, A.R., Robinson, C.V.: Intrinsically disordered p53 and its complexes populate compact conformations in the gas phase. Angew. Chem. Int. Ed. 52, 361–365 (2013)CrossRefGoogle Scholar
  47. 47.
    Abonnenc, M., Qiao, L., Liu, B., Girault, H.H.: Electrochemical aspects of electrospray and laser desorption/ionization for mass spectrometry. Annu. Rev. Anal. Chem. 3, 231–254 (2010)CrossRefGoogle Scholar
  48. 48.
    Zinck, N., Stark, A.K., Wilson, D.J., Sharon, M.: An improved rapid mixing device for time-resolved electrospray mass spectrometry measurements. Chemistry Open. 3, 109–114 (2014)Google Scholar
  49. 49.
    Stoppacher, N., Josephs, R.D., Daireaux, A., Choteau, T., Westwood, S., Wielgosz, R.I.: Accurate quantification of impurities in pure peptide material—angiotensin I: comparison of calibration requirements and method performance characteristics of liquid chromatography coupled to hybrid tandem mass spectrometry and linear ion trap high-resolution mass spectrometry. Rapid Commun. Mass Spectrom. 29, 1651–1660 (2015)CrossRefGoogle Scholar

Copyright information

© UK Crown 2018

Authors and Affiliations

  1. 1.National Research Council of Canada, Measurement Science and StandardsOttawaCanada

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