Electrospray ionization of trypsinized proteins is typically performed in positive ion mode, producing protonated peptides that on average have at least two charges, i.e. 'extra' protons. A generic structure for such a peptide is shown above. One of the charges is 'sequestered' by a C-terminal lysine or arginine due to their high proton affinity,ǂ and is a spectator to the subsequent fragmentation chemistry. The location of the second charge is not well defined, but for convenience, the charge is shown here on the N-terminus. Upon “slow” collisional heating, peptide ions undergo ‘isomerization’ reactions that move the extra proton to sites that are thermodynamically less stable. In other words, the non-sequestered proton becomes “mobile”. 1
Among the potential locations of the mobile proton, N-protonation of an amide bond is the most relevant for fragmentation. Interestingly, the neutral amide bond is very strong due to resonance stabilization between the carbonyl and nitrogen lone pair. However, N-protonation abolishes the resonance stabilization,2 leading to a much weaker amide bond, which cleaves to produce sequence-informative fragment ions. Of course, amide N-protonation and cleavage can theoretically occur between any two adjacent residues. Stochastic (random) cleavage of the backbone produces a mass spectrum with peaks that are separated by m/z ratios that correspond to amino acid masses (as shown in overview).
The majority of the peptide fragments observed in typical MS2 experiments are satisfactorily explained by the ‘mobile proton’ model alone. The most important exceptions are preferential fragmentations at acidic residues3 and proline.4 Selective cleavage at acidic residues occurs most often in singly protonated peptides. The trend in selectivity for proline is just the opposite, typically increasing with higher charge states.
1 Dongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V.H. Influence of peptide composition, gas-phase basicity, and chemical modification on fragmentation efficiency: Evidence for the mobile proton model. J. Am. Chem. Soc. 2006, 118, 8365-8374.
2 Somogyi, A.; Wysocki, V. H.; Mayer, I. The Effect of Protonation Site on Bond Strengths in Simple Peptides: Application of Ab Initio and Modified Neglect of Differential Overlap Bond Orders and Modified Neglect of Differential Overlap Energy Partitioning. J. Am. Soc. Mass Spectrom. 1994, 5, 704-717.
3 Gu, C. G.; Tsaprailis, G.; Breci, L.; Wysocki, V. H. Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides. Anal. Chem. 2000, 72, 5804-5813.
4 Vaisar, T., Urban, J. Probing the proline effect in CID of protonated peptides. J. Mass Spectrom., 1996, 31, 1185-1187.