A systematic study on the effect of histidine position and fragment ion size on the formation of bn ions

In this study, we utilized gas-phase hydrogen/deuterium (H/D) exchange reactions and ion mobility-mass spectrometry (IM-MS) to examine the effect of: (i) histidine (His) amino acid position and (ii) fragment ion size on the structures of bn (n = 4–7) ions generated from nozzle-skimmer fragmentation. Both H/D exchange patterns and semi-log temporal plots of histidine-containing bn fragment ions generated from seven isobaric model heptapeptides {i.e., (His)AAAAAA-NH2, A(His)AAAAA-NH2, AA(His)AAAA-NH2, AAA(His)AAA-NH2, AAAA(His)AA-NH2, AAAAA(His)A-NH2, and AAAAAA(His)-NH2} suggested the presence of at least two structures for all b fragment ions: “fast” and “slow” H/D-exchanging ion populations. The observed H/D exchange rate constants (for disappearance of isolated 12Call or D0) for b4+ and b5+ fragments were higher than those observed for b7+ fragment ions, suggesting more compact and/or stable structures for b7+ fragment ions. Among the studied histidine-containing bn+ fragments, b4+ and b5+ showed the most variation in H/D exchange reactivity as a function of histidine position in the original peptide sequence. Ion mobility arrival time distributions (ATDs) for histidine-containing b5+ fragments from AA(His)AAAA-NH2, AAA(His)AAA-NH2, and AAAA(His)AA-NH2 showed two ion populations. H/D exchange and ion mobility results both imply the potential presence of a mixture of compact and open structures for b5+. ATDs for b4+ fragments generated from A(His)AAAAA-NH2 and AA(His)AAAA-NH2 (compared to ATDs of the other model heptapeptides) suggest the appearance or increase in the percentage of a more compact ion population. ATDs of histidine-containing b6+ and b7+ fragments varied significantly as a function of histidine position in the original heptapeptides.

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► We examine the effect of His position and fragment size on b4–b7 ion structures.
► b4–b7 have two conformers with different H/D exchange reactivities.
► b4–b7 fragment ions exhibit two ion populations in H/D exchange reactions.
► The presence of two conformers for b4–b7 is independent of His position.
► IMS of b5 from peptides with His in positions 3–5 show two ion populations.