Although structural isomers may yield indistinguishable ion mobility (IM) arrival times and similar fragment ions in tandem mass spectrometry (MS), it is demonstrated that post-IM/collision-induced dissociation MS (post-IM/CID MS) combined with chemometrics can enable independent study of the IM-overlapped isomers. The new approach allowed us to investigate the propensity of selected b type fragment ions from AlaAlaAlaHisAlaAlaAla-NH2 (AAA(His)AAA) heptapeptide to form different isomers. Principle component analysis (PCA) of the unresolved post-IM/CID profiles indicated the presence of two different isomer types for b4(+), b5(+), and b6(+) and a single isomer type for b7(+) fragments of AAA(His)AAA. We employed a simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) to calculate the total IM profiles and CID mass spectra of b fragment isomers. The deconvoluted CID mass spectra showed discernible fragmentation patterns for the two isomers of b4(+), b5(+), and b6(+) fragments. Under our experimental conditions, calculated percentages of the "cyclic" isomers (at the 95% confidence level for n = 3) for b4(+), b5(+), and b6(+) were 61 (± 5)%, 36 (± 5)%, and 48 (± 2)%, respectively. Results from the SIMPLISMA deconvolution of b5(+) species resembled the CID MS patterns of fully resolved IM profiles for the two b5(+) isomers. The "cyclic" isomers for each of the two-component b fragment ions were less susceptible to ion fragmentation than their "linear" counterparts.
It is shown that y-type ions, after losing C-terminal H2O or NH3, can lose an internal backbone carbonyl (CO) from different peptide positions and yield structurally different product fragment ions upon collision-induced dissociation (CID). Such CO losses from internal peptide backbones of y-fragment ions are not unique to a single peptide and were observed in four of five model peptides studied herein. Experimental details on examples of CO losses from y-type fragment ions for an isotopically labeled AAAAHAA-NH2 heptapeptide and des-acetylated-α-melanocyte-stimulating hormone (dα-MSH) (SYSMEHFRWGKPV-NH2) are reported. Results from isotope labeling, tandem mass spectrometry (MS(n)), and ion mobility-mass spectrometry (IM-MS) confirm that CO losses from different amino acids of m/z-isolated y-type ions yield structurally different ions. It is shown that losses of internal backbone carbonyls (as CID products of m/z-isolated y-type ions) are among intermediate steps towards formation of rearranged or permutated product fragment ions. Possible mechanisms for generation of the observed sequence-scrambled a-"like" ions, as intermediates in sequence-scrambling pathways of y-type ions, are proposed and discussed.
To date, only a limited number of reports are available on structural variants of multiply-charged b-fragment ions. We report on observed bimodal gas-phase hydrogen/deuterium exchange (HDX) reaction kinetics and patterns for substance P b10(2+) that point to presence of isomeric structures. We also compare HDX reactions, post-ion mobility/collision-induced dissociation (post-IM/CID), and sustained off-resonance irradiation-collision induced dissociation (SORI-CID) of substance P b10(2+) and a cyclic peptide with an identical amino acid (AA) sequence order to substance P b10. The observed HDX patterns and reaction kinetics and SORI-CID pattern for the doubly charged head-to-tail cyclized peptide were different from either of the presumed isomers of substance P b10(2+), suggesting that b10(2+) may not exist exclusively as a head-to-tail cyclized structure. Ultra-high mass measurement accuracy was used to assign identities of the observed SORI-CID fragment ions of substance P b10(2+); over 30% of the observed SORI-CID fragment ions from substance P b10(2+) had rearranged (scrambled) AA sequences. Moreover, post-IM/CID experiments revealed the presence of two conformer types for substance P b10(2+), whereas only one conformer type was observed for the head-to-tail cyclized peptide. We also show that AA sequence scrambling from CID of doubly-charged b-fragment ions is not unique to substance P b10(2+).
Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH2 (where "A" denotes carbon thirteen ((13)C1) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH2), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic "b-type" ions.
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