Structural heterogeneity of doubly-charged peptide b-ions.

Performing collisionally activated dissociation (CAD) and electron capture dissociation (ECD) in tandem has shown great promise in providing comprehensive sequence information that was otherwise unobtainable by using either fragmentation method alone or in duet. However, the general applicability of this MS(3) approach in peptide sequencing may be undermined by the formation of non-direct sequence ions, as sometimes observed under CAD, particularly when multiple stages of CAD are involved. In this study, varied-sized doubly-charged b-ions from three tachykinin peptides were investigated by ECD. Sequence scrambling was observed in ECD of all b-ions from neurokinin A (HKTDSFVGLM-NH(2)), suggesting the presence of N- and C-termini linked macro-cyclic conformers. On the contrary, none of the b-ions from eledoisin (pEPSKDAFIGLM-NH(2)) produced non-direct sequence ions under ECD, as it does not contain a free N-terminal amino group. ECD of several b-ions from Substance P (RPKPQQFFGLM-NH(2)) showed series of c(m)-Lys fragment ions which suggested that the macro-cyclic structure may also be formed by connecting the C-terminal carbonyl group and the ε-amino group of the lysine side chain. Theoretical investigation of selected Substance P b-ions revealed several low energy conformers, including both linear oxazolones and macro-ring structures, in corroboration with the experimental observation. This study showed that a b-ion may exist as a mixture of several forms, with their propensities influenced by its N-terminus, length, and certain side-chain groups. Further, the presence of several macro-cyclic structures may result in erroneous sequence assignment when the combined CAD and ECD methods are used in peptide sequencing. © American Society for Mass Spectrometry, 2011