Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD 3OD

Abstract

Doubly-protonated bradykinin (RPPGFSPFR) and an angiotensin III analogue (RVYIFPF) were subjected to hydrogen/deuterium (H/D) exchange with CD 3OD in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. A bimodal distribution of deuterium incorporation was present for bradykinin after H/D exchange for 90 s at a CD 3OD pressure of 4 × 10 −7 Torr, indicating the existence of at least two distinct populations. Bradykinin ion populations corresponding to 0–2 and 5–11 deuteriums (i.e., D 0, D 1, D 2, D 5, D 6, D 7, D 8, D 9, D 10, and D 11) were each monoisotopically selected and fragmented via sustained off-resonance irradiation (SORI) collision-induced dissociation (CID). The D 0–D 2 ion populations, which correspond to the slower exchanging population, consistently require lower SORI amplitude to achieve a similar precursor ion survival yield as the faster-reacting (D 5–D 11) populations. These results demonstrate that conformation/protonation motif has an effect on fragmentation efficiency for bradykinin. Also, the partitioning of the deuterium atoms into fragment ions suggests that the C-terminal arginine residue exchanges more rapidly than the N-terminal arginine. Total deuterium incorporation in the b 1/y 8 and b 2/y 7 ion pairs matches very closely the theoretical values for all ion populations studied, indicating that the ions of a complementary pair are likely formed during the same fragmentation event, or that no scrambling occurs upon SORI. Deuterium incorporation into the y 1/a 8 pseudo-ion pair does not closely match the expected theoretical values. The other peptide, doubly-protonated RVYIFPF, has a trimodal distribution of deuterium incorporation upon H/D exchange with CD 3OD at a pressure of 1 × 10 −7 Torr for 600 s, indicating at least three distinct ion populations. After 90 s of H/D exchange where at least two distinct populations are detected, the D 0–D 7 ion populations were monoisotopically selected and fragmented via SORI-CID over a range of SORI amplitudes. The precursor ion survival yield as a function of SORI amplitude falls into two distinct behaviors corresponding to slower- and faster-reacting ion populations. The slower-reacting population requires larger SORI amplitudes to achieve the same precursor ion survival yield as the faster exchanging population. Total deuterium incorporation into the y 2/b 5 ion pairs matches closely the theoretical values over all ion populations and SORI amplitudes studied. This result indicates the y 2 and b 5 ions are likely formed by the same mechanism over the SORI amplitudes studied.