Abstract
The gas-phase fragmentation mechanisms of small models for peptides containing intermolecular disulfide links have been studied using a combination of tandem mass spectrometry experiments, isotopic labeling, structural labeling, accurate mass measurements of product ions, and theoretical calculations (at the MP2/6-311 + G(2d,p)//B3LYP/3-21G(d) level of theory). Cystine and its C-terminal derivatives were observed to fragment via a range of pathways, including loss of neutral molecules, amide bond cleavage, and S—S and C—S bond cleavages. Various mechanisms were considered to rationalize S—S and C—S bond cleavage processes, including charge directed neighboring group processes and nonmobile proton salt bridge mechanism. Three low-energy fragmentation pathways were identified from theoretical calculations on cystine N-methyl amide: (1) S—S bond cleavage dominated by a neighboring group process involving the C-terminal amide N to form either a protonated cysteine derivative or protonated sulfenyl amide product ion (44.3 kcal mol−1); (2) C—S bond cleavage via a salt bridge mechanism, involving abstraction of the α-hydrogen by the N-terminal amino group to form a protonated thiocysteine derivative (35.0 kcal mol−1); and (3) C—S bond cleavage via a Grob-like fragmentation process in which the nucleophilic N-terminal amino group forms a protonated dithiazolidine (57.9 kcal mol−1). Interestingly, C—S bond cleavage by neighboring group processes have high activation barriers (63.1 kcal mol−1) and are thus not expected to be accessible during low-energy CID experiments. In comparison to the energetics of simple amide bond cleavage, these S—S and C—S bond cleavage reactions are higher in energy, which helps rationalize why bond cleavage processes involving the disulfide bond are rarely observed for low-energy CID of peptides with mobile proton(s) containing intermolecular disulfide bonds. On the other hand, the absence of a mobile proton appears to “switch on” disulfide bond cleavage reactions, which can be rationalized by the salt bridge mechanism. This potentially has important ramifications in explaining the prevalence of disulfide bond cleavage in singly protonated peptides under MALDI conditions.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
O’Hair, R. A. J. The Role of Nucleophile-Electrophile Interactions in the Unimolecular and Bimolecular Gas-Phase Ion Chemistry of Peptides and Related Systems. J. Mass Spectrom. 2000, 35, 1377–1381.
Paizs, B.; Suhai, S. Fragmentation Pathways of Protonated Peptides. Mass Spectrom. Rev. 2005, 24, 508–548.
Bowie, J. H.; Brinkworth, C. S.; Dua, S. Collision-Induced Fragmentations of the [M − H]t- Parent Anions of Underivatized Peptides: An Aid to Structure Determination and Some Unusual Negative Ion Cleavages. Mass Spectrom. Rev. 2002, 21, 87–107.
Jensen, O. N. Modification-Specific Proteomics: Characterization of Post-Translational Modifications by Mass Spectrometry. Curr. Op. Chem. Biol. 2004, 8, 33–41.
Mann, M.; Jensen, O. N. Proteomic Analysis of Post-Translational Modifications. Nat. Biotech. 2003, 21, 255–261.
O’Hair, R. A. J.; Reid, G. E. Neighboring Group Versus cis-Elimination Mechanisms for Side Chain Loss from Protonated Methionine, Methionine Sulfoxide, and Their Peptides. Eur. J. Mass Spectrom. 1999, 5, 325–334.
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.
Reid, G. E.; Simpson, R. J.; O’Hair, R. A. J. Leaving Group and Gas Phase Neighboring Group Effects in the Side Chain Losses from Protonated Serine and Its Derivatives. J. Am. Soc. Mass Spectrom. 2000, 11, 1047–1060.
Gorman, J. J.; Wallis, T. P.; Pitt, J. J. Protein Disulfide Bond Determination by Mass Spectrometry. Mass Spectrom. Rev. 2002, 21, 183–216.
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.
Fung, Y. M. E.; Kjeldsen, F.; Silivra, O. A.; Chan, T. W. D.; Zubarev, R. A. Facile Disulfide Bond Cleavage in Gaseous Peptide and Protein Cations by Ultraviolet Photodissociation at 157 nm. Angew. Chem. Int. Ed. 2005, 44, 6399–6403.
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.
Loo, J. A.; Edmonds, C. G.; Udseth, H. R.; Smith, R. D. Effect of Reducing Disulfide-Containing Proteins on Electrospray Ionization Mass Spectra. Anal. Chem. 1990, 62, 693–698.
Wu, J.; Watson, J. T. A Novel Methodology for Assignment of Disulfide Bond Pairings in Proteins. Prot. Sci. 1997, 6, 391–398.
Jones, M. D.; Patterson, S. D.; Lu, H. S. Determination of Disulfide Bonds in Highly Bridged Disulfide-Linked Peptides by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry with Postsource Decay. Anal. Chem. 1998, 70, 136–143.
Wells, J. M.; Stephenson, J. L.; McLuckey, S. A. Charge Dependence of Protonated Insulin Decompositions. Int. J. Mass Spectrom. 2000, 203, A1-A9.
Wysocki, V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. Mobile and Localized Protons: A Framework for Understanding Peptide Dissociation. J. Mass Spectrom. 2000, 35, 1399–1406.
Kapp, E. A.; Schütz, F.; Reid, G. E.; Eddes, J. S.; Moritz, R. L.; O’Hair, R. A. J.; Speed, T. P.; Simpson, R. J. Mining a Tandem Mass Spectrometry Database To Determine the Trends and Global Factors Influencing Peptide Fragmentation. Anal. Chem. 2003, 75, 6251–6264.
Rubino, F. M.; Verduci, C.; Giampiccolo, R.; Pulvirenti, S.; Brambilla, G.; Colombi, A. Characterization of the Disulfides of Biothiols by Electrospray Ionization and Triple-Quadrupole Tandem Mass Spectrometry. J. Mass Spectrom. 2004, 39, 1408–1416.
de Moraes, P. R. P.; Linnert, H. V.; Aschi, M.; Riveros, J. M. Experimental and Theoretical Characterization of Long-Lived Triplet State CH3CH2S+ Cations. J. Am. Chem. Soc. 2000, 122, 10133–10142.
Freitas, M. A.; O’Hair, R. A. J.; Williams, T. D. Gas-Phase Reactions of Cysteine with Charged Electrophiles: Regioselectivities of the Dimethylchlorinium Ion and the Methoxymethyl Cation. J. Org. Chem. 1997, 62, 6112–6120.
Reid, G. E.; Simpson, R. J.; O’Hair, R. A. J. A Mass Spectrometric and ab Initio Study of the Pathways for Dehydration of Simple Glycine and Cysteine-Containing Peptide [M + H]+ Ions. J. Am. Soc. Mass Spectrom. 1998, 9, 945–956.
Feenstra, R. W.; Stokkingreef, E. H. M.; Reichwein, A. M.; Lousberg, W. B. H.; Ottenheijm, H. C. J. Oxidative Preparation of Optically Active N-Hydroxy-α-Amino Acid Amides. Tetrahedron. 1990, 46, 1745–1756.
Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A. Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; oth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03 (Rev B.04); Gaussian, Inc: Pittsburgh PA, 2003.
Turecek, F. Proton Affinity of Dimethyl Sulfoxide and Relative Stabilities of C2H6OS Molecules and C2H7OS+ Ions: A Comparative G2(MP2) ab Initio and Density Functional Theory Study. J. Phys. Chem. A. 1998, 102, 4703–4713.
Keith, T.; Millam, J.; Eppinnett, K.; Hovell, W. L.; Gilliland, R. GaussView (Ver 3.0) Dennington II, R.; Semichem, Inc: Shawnee Mission, KS, 2003.
Lioe, H.; O’Hair, R. A. J.; Reid, G. E. Gas-Phase Reactions of Protonated Tryptophan. J. Am. Soc. Mass Spectrom. 2004, 15, 65–76.
Lioe, H.; O’Hair, R. A. J. Neighboring Group Processes in the Deamination of Protonated Phenylalanine Derivatives. Org. Biomol. Chem. 2005, 3, 3618–3628.
Bowen, R. D. Ion-Neutral Complexes. Acc. Chem. Res. 1991, 24, 364–371.
Morgan, D. G.; Bursey, M. M. A Linear Free-Energy Correlation in the Low-Energy Tandem Mass Spectra of Protonated Tripeptides Gly-Gly-Xxx. Org. Mass Spectrom. 1994, 29, 354–359.
Grob, C. A. Mechanisms and Stereochemistry of Heterolytic Fragmentation. Angew. Chem. Int. Ed. 1969, 8, 535–546.
Kurti, L.; Czako, B. Strategic Applications of Named Reactions in Organic Synthesis: Background and Detailed Mechanisms; Elsevier Academic: Burlington, 2005; p 190.
Hunter, E. P.; Lias, S. G. Proton Affinity Evaluation. In NIST Chemistry WebBook, NIST Standard Reference Database Number 69; Linstrom, P. J., Mallard, W. G.; National Institute of Standards and Technology: Gaithersburg, MD, 20899 (http://webbook.nist.gov/cgi/cbook.cgi?Contrib-), June 2005.
Salmeen, A.; Andersen Jannik, N.; Myers Michael, P.; Meng, T.-C.; Hinks John, A.; Tonks Nicholas, K.; Barford, D. Redox Regulation of Protein Tyrosine Phosphatase 1B Involves a Sulphenyl-Amide Intermediate. Nature. 2003, 423, 769–773.
van Montfort-Rob, L. M.; Congreve, M.; Tisi, D.; Carr, R.; Jhoti, H. Oxidation State of the Active-Site Cysteine in Protein Tyrosine Phosphatase 1B. Nature. 2003, 423, 773–777.
Bohme, D. K. Proton Transport in the Catalyzed Gas-Phase Isomerization of Protonated Molecules. Int. J. Mass Spectrom. Ion Processes. 1992, 115, 95–110.
Amunugama, M.; Roberts, K. D.; Reid, G. E. Mechanisms for the Selective Gas-Phase Fragmentation Reactions of Methionine Side Chain Fixed Charge Sulfonium Ion Containing Peptides. J. Am. Soc. Mass Spectrom. 2006, 17, 1631–1642.
Zhou, J.; Ens, W.; Poppeschriemer, N.; Standing, K. G.; Westmore, J. B. Cleavage of Interchain Disulfide Bonds Following Matrix-Assisted Laser-Desorption. Int. J. Mass Spectrom. Ion Processes. 1993, 126, 115–122.
Chrisman, P. A.; McLuckey, S. A. Dissociations of Disulfide-Linked Gaseous Polypeptide/Protein Anions: Ion Chemistry with Implications for Protein Identification and Characterization. J. Proteome Res. 2002, 1, 549–557.
Bilusich, D.; Maselli, V. M.; Brinkworth, C. S.; Samguina, T.; Lebedev, A. T.; Bowie, J. H. Direct Identification of Intramolecular Disulfide Links in Peptides Using Negative Ion Electrospray Mass Spectra of Underivatized Peptides: A Joint Experimental and Theoretical Study. Rapid Commun. Mass Spectrom. 2005, 19, 3063–3074.
Bilusich, D.; Bowie, J. H. Identification of Intermolecular Disulfide Linkages in Underivatized Peptides Using Negative Ion Electrospray Mass Spectrometry: A Joint Experimental and Theoretical Study. Rapid Commun. Mass Spectrom. 2007, 21, 619–628.
Farrugia, J. M.; O’Hair, R. A. J. Involvement of Salt Bridges in a Novel Gas-Phase Rearrangement of Protonated Arginine-Containing Dipeptides which Precedes Fragmentation. Int. J. Mass Spectrom. 2003, 222, 229–242.
Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. Influence of Secondary Structure on the Fragmentation of Protonated Peptides. J. Am. Chem. Soc. 1999, 121, 5142–5154.
Bailey, T. H.; Laskin, J.; Futrell, J. H. Energetics of Selective Cleavage at Acidic Residues Studied by Time- and Energy-Resolved Surface-Induced Dissociation in FT-ICR MS. Int. J. Mass Spectrom. 2003, 222, 313–327.
Author information
Authors and Affiliations
Corresponding author
Additional information
This article is Part 54 of the series “Gas Phase Ion Chemistry of Biomolecules.”
Published online April 26, 2007
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Lioe, H., OrsHair, R.A.J. A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation. J Am Soc Mass Spectrom 18, 1109–1123 (2007). https://doi.org/10.1016/j.jasms.2007.03.003
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2007.03.003