The fragmentation mechanisms of singlet oxygen [O2 (1Δg)]-derived oxidation products of tryptophan (W) were analyzed using collision-induced dissociation coupled with 18O-isotopic labeling experiments and accurate mass measurements. The five identified oxidized products, namely two isomeric alcohols (trans and cis WOH), two isomeric hydroperoxides (trans and cis WOOH), and N-formylkynurenine (FMK), were shown to share some common fragment ions and losses of small neutral molecules. Conversely, each oxidation product has its own fragmentation mechanism and intermediates, which were confirmed by 18O-labeling studies. Isomeric WOH lost mainly H2O + CO, while WOOH showed preferential elimination of C2H5NO3 by two distinct mechanisms. Differences in the spatial arrangement of the two isomeric WOHs led to differences in the intensities of the fragment ions. The same behavior was also found for trans and cis WOOH. FMK was shown to dissociate by a diverse range of mechanisms, with the loss of ammonia the most favored route. MS/MS analyses, 18O-labeling, and H218O experiments demonstrated the ability of FMK to exchange its oxygen atoms with water. Moreover, this approach also revealed that the carbonyl group has more pronounced oxygen exchange ability compared with the formyl group. The understanding of fragmentation mechanisms involved in O2 (1Δg)-mediated oxidation of W provides a useful step toward the structural characterization of oxidized peptides and proteins.
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