Mapping disulfide bonds in insulin with the route 66 method: Selective cleavage of S-C bonds using alkali and alkaline earth metal enolate complexes

Articles

Abstract

Simple and fast identification of disulfide linkages in insulin is demonstrated with a peptic digest using the Route 66 method. This is accomplished by collisional activation of singly and doubly charged cationic Na+ and Ca2+ complexes generated using electrospray ionization mass spectrometry (ESI-MS). Collisional activation of doubly charged metal complexes of peptides with intermolecular disulfide linkages yields two sets of singly charged paired products separated by 66 mass units resulting from selective S-C bond cleavages. Highly selective elimination of 66 mass units, which corresponds to the molecular weight of hydrogen disulfide (H2S2), is observed from singly charged metal complexes of peptides with disulfide linkages. The mechanism proposed for these processes is initiated by formation of a metal-stabilized enolate at Cys, followed by cleavage of the S-C bond. Further activation of the products yields sequence information that facilitates locating the position of the disulfide linkages in the peptic digest fragments. For example, the doubly charged Ca2+ complex of the peptic digest product GIVEQCCASVCSL/FVNQHLCGSHL yields paired products separated by 66 mass units resulting from selective S-C bond cleavages at an intermolecular disulfide linkage under low-energy collision-induced dissociation. Further activation of the product comprising the A chain reveals the presence of a second disulfide bridge, an intramolecular linkage. Experimental and theoretical studies of the disulfide linked model peptides provide mechanistic details for the selective cleavage of the S-C bond.

References

  1. 1.
    Thornton, J. M. Disulfide Bridges in Globular-Proteins. J. Mol. Biol. 1981, 151, 261–287.CrossRefGoogle Scholar
  2. 2.
    Gorman, J. J.; Wallis, T. P.; Pitt, J. J. Protein Disulfide Bond Determination by Mass Spectrometry. Mass Spectrom. Rev. 2002, 21, 183–216.CrossRefGoogle Scholar
  3. 3.
    Chelius, D.; Wimer, M. E. H. Reversed-Phase Liquid Chromatography In-Line with Negative Ionization Electrospray Mass Spectrometry for the Characterization of the Disulfide-Linkages of an Immunoglobulin Gamma Antibody. J. Am. Soc. Mass Spectrom. 2006, 17, 1590–1598.CrossRefGoogle Scholar
  4. 4.
    Biemann, K.; Scoble, H. A. Characterization by Tandem Mass-Spectrometry of Structural Modifications in Proteins. Science 1987, 237, 992–998.CrossRefGoogle Scholar
  5. 5.
    Mann, M.; Jensen, O. N. Proteomic Analysis of Post-Translational Modifications. Nat. Biotechnol. 2003, 21, 255–261.CrossRefGoogle Scholar
  6. 6.
    Resing, K. A.; Johnson, R. S.; Walsh, K. A. Mass-Spectrometric Analysis of 21 Phosphorylation Sites in the Internal Repeat of Rat Profilaggrin, Precursor of an Intermediate Filament-Associated Protein. Biochemistry 1995, 34, 9477–9487.CrossRefGoogle Scholar
  7. 7.
    Gibson, B. W.; Cohen, P. Liquid Secondary-Ion Mass-Spectrometry of Phosphorylated and Sulfated Peptides and Proteins. Methods Enzymol. 1990, 193, 480–501.CrossRefGoogle Scholar
  8. 8.
    Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. Selective Detection of Phosphopeptides in Complex-Mixtures by Electrospray Liquid-Chromatography Mass-Spectrometry. J. Am. Soc. Mass Spectrom. 1993, 4, 710–717.CrossRefGoogle Scholar
  9. 9.
    Clauser, K. R.; Hall, S. C.; Smith, D. M.; Webb, J. W.; Andrews, L. E.; Tran, H. M.; Epstein, L. B.; Burlingame, A. L. Rapid Mass-Spectrometric Peptide Sequencing and Mass Matching for Characterization of Human-Melanoma Proteins Isolated by 2-Dimensional Page. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 5072–5076.CrossRefGoogle Scholar
  10. 10.
    Qin, J.; Chait, B. T. Identification and Characterization of Posttranslational Modifications of Proteins by Maldi Ion Trap Mass Spectrometry. Anal. Chem. 1997, 69, 4002–4009.CrossRefGoogle Scholar
  11. 11.
    Reid, G. E.; Roberts, K. D.; Kapp, E. A.; Simpson, R. J. Statistical and Mechanistic Approaches to Understanding the Gas-Phase Fragmentation Behavior of Methionine Sulfoxide Containing Peptides. J. Proteome Res. 2004, 3, 751–759.CrossRefGoogle Scholar
  12. 12.
    Kim, H. I.; Beauchamp, J. L. Identifying the Presence of a Disulfide Linkage in Peptides by the Selective Elimination of Hydrogen Disulfide from Collisionally Activated Alkali and Alkaline Earth Metal Complexes. J. Am. Chem. Soc. 2008, 130, 1245–1257.CrossRefGoogle Scholar
  13. 13.
    Gunawardena, H. P.; O’Hair, R. A. J.; McLuckey, S. A. Selective Disulfide Bond Cleavage in Gold(I) Cationized Polypeptide Ions Formed Via Gas-Phase Ion/Ion Cation Switching. J. Proteome Res. 2006, 5, 2087–2092.CrossRefGoogle Scholar
  14. 14.
    Kleinnijenhuis, A. J.; Mihalca, R.; Heeren, R. M. A.; Heck, A. J. R. Atypical Behavior in the Electron Capture Induced Dissociation of Biologically Relevant Transition Metal Ion Complexes of the Peptide Hormone Oxytocin. Int. J. Mass Spectrom. 2006, 253, 217–224.CrossRefGoogle Scholar
  15. 15.
    Mihalca, R.; van der Burgt, Y. E. M.; Heck, A. J. R.; Heeren, R. M. A. Disulfide Bond Cleavages Observed in Sori-Cid of Three Nonapeptides Complexed With Divalent Transition-Metal Cations. J. Mass Spectrom. 2007, 42, 450–458.CrossRefGoogle Scholar
  16. 16.
    Lioe, H.; Duan, M.; O’Hair, R. A. J. Can Metal Ions Be Used as Gas-Phase Disulfide Bond Cleavage Reagents?: A Survey of Coinage Metal Complexes of Model Peptides Containing an Intermolecular Disulfide Bond. Rapid Commun. Mass Spectrom. 2007, 21, 2727–2733.CrossRefGoogle Scholar
  17. 17.
    Morris, H. R.; Pucci, P. A New Method for Rapid Assignment of S-S Bridges in Proteins. Biochem. Biophys. Res. Commun. 1985, 126, 1122–1128.CrossRefGoogle Scholar
  18. 18.
    Sun, Y. P.; Smith, D. L. Identification of Disulfide-Containing Peptides by Performic Acid Oxidation and Mass-Spectrometry. Anal. Biochem. 1988, 172, 130–138.CrossRefGoogle Scholar
  19. 19.
    Wells, J. M.; Stephenson, J. L.; McLuckey, S. A. Charge Dependence of Protonated Insulin Decompositions. Int. J. Mass Spectrom. 2000, 203, A1-A9.CrossRefGoogle Scholar
  20. 20.
    Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. Electron Capture Dissociation of Gaseous Multiply-Charged Proteins Is Favored at Disulfide Bonds and Other Sites of High Hydrogen Atom Affinity. J. Am. Chem. Soc. 1999, 121, 2857–2862.CrossRefGoogle Scholar
  21. 21.
    Brown, R. S.; Lennon, J. J. Sequence-Specific Fragmentation of Matrix-Assisted Laser-Desorbed Protein Peptide Ions. Anal. Chem. 1995, 67, 3990–3999.CrossRefGoogle Scholar
  22. 22.
    Stephenson, J. L.; Cargile, B. J.; McLuckey, S. A. Ion Trap Collisional Activation of Disulfide Linkage Intact and Reduced Multiply Protonated Polypeptides. Rapid Commun. Mass Spectrom. 1999, 13, 2040–2048.CrossRefGoogle Scholar
  23. 23.
    Polfer, N. C.; Haselmann, K. F.; Zubarev, R. A.; Langridge-Smith, P. R. R. Electron Capture Dissociation of Polypeptides Using a 3 Tesla Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. Rapid Commun. Mass Spectrom. 2002, 16, 936–943.CrossRefGoogle Scholar
  24. 24.
    Becke, A. D. Density-Functional Thermochemistry. 3: The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648–5652.CrossRefGoogle Scholar
  25. 25.
    Lee, C. T.; Yang, W. T.; Parr, R. G. Development of the Colle-Salvetti Correlation-Energy Formula into a Functional of the Electron-Density. Phys. Rev. B 1988, 37, 785–789.CrossRefGoogle Scholar
  26. 26.
    Roepstorff, P.; Fohlman, J. Proposal for a Common Nomenclature for Sequence Ions in Mass-Spectra of Peptides. Biomed. Mass Spectrom. 1984, 11, 601–601.CrossRefGoogle Scholar
  27. 27.
    Pearson, R. G. Hard and Soft Acids and Bases. J. Am. Chem. Soc. 1963, 85, 3533–3539.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2009

Authors and Affiliations

  1. 1.Noyes Laboratory of Chemical Physics, Department of ChemistryCalifornia Institute of TechnologyPasadenaUSA
  2. 2.Jet Propulsion LaboratoryCalifornia Institute of TechnologyPasadena

Personalised recommendations