Cyclization and Rearrangement Reactions of a_n Fragment Ions of Protonated Peptides

摘要

a_n ions are frequently formed in collision-induced dissociation (CID) of protonated peptides in tandem mass spectrometry (MS/MS) based sequencing experiments. These ions have generally been assumed to exist as immonium derivatives (-HN~+=CHR). Using a quadrupole ion trap mass spectrometer, MS/MS experiments have been performed and the structure of a_n ions formed from oligoglycines was probed by infrared spectroscopy. The structure and isomerization reactions of the same ions were studied using density functional theory. Overall, theory and infrared spectroscopy provide compelling evidence that a_n ions undergo cyclization and/or rearrangement reactions, and the resulting structure(s) observed under our experimental conditions depends on the size (η). The a_2 ion (GG sequence) undergoes cyclization to form a 5-membered ring isomer. The a_3 ion (GGG sequence) undergoes cyclization initiated by nucleophilic attack of the carbonyl oxygen of the N-terminal glycine residue on the carbon center of the C-terminal immonium group forming a 7-membered ring isomer. The barrier to this reaction is comparatively low at 10.5 kcal mol(-1), and the resulting cyclic isomer (-5.4 kcal mol~(-1)) is more energetically favorable than the linear form. The a_4 ion with the GGGG sequence undergoes head-to-tail cyclization via nucleophilic attack of the N-terminal amino group on the carbon center of the C-terminal immonium ion, forming an 11-membered macroring which contains a secondary amine and three trans amide bonds. Then an intermolecular proton transfer isomerizes the initially formed secondary amine moiety (-CH_2-NH_2+-CH_2-NH-CO-) to form a new -CH_2-NH-CH_2-NH_2+-CO- form. This structure is readily cleaved at the -CH_2-NH_2+- bond, leading to opening of the macrocycle and formation of a rearranged linear isomer with the H_2C=NH~+-CH_2- moiety at the N terminus and the -CO-NH_2 amide bond at the C terminus. This rearranged linear structure is much more energetically favorable (-14.0 kcal mol~(-1)) than the initially formed imine-protonated linear a_4, ion structure. Furthermore, the barriers to these cyclization and ring-opening reactions are low (8-11 kcal mol(-1)), allowing facile formation of the rearranged linear species in the mass spectrometer. This finding is not limited to 'simple' glycine-containing systems, as evidenced by the IRMPD spectrum of the a_3 ion generated from protonated AAAAA, which shows a stronger tendency toward formation of the energetically favorable (-12.3 kcal mol(-1)) rearranged linear structure with the MeHC=NH~+-CHMe- moiety at the N terminus and the -CO-NH_2 amide bond at the C terminus. Our results indicate that one needs to consider a complex variety of cyclization and rearrangement reactions in order to decipher the structure and fragmentation pathways of peptide a_n ions. The implications this potentially has for peptide sequencing are also discussed.