Negative Ion Electron Capture Dissociation (niECD): A Novel Tandem Mass Spectrometric Technique.

Abstract

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) are powerful tandem mass spectrometry (MS/MS) techniques for biomolecular structural elucidation. However, one drawback of ECD/ETD is that they require multiply positively charged precursor ions, possibly precluding analysis of acidic molecules such as phospho-, sulfopeptides, and sialylated glycopeptides. Electron attachment to anions appears unlikely due to Coulomb repulsion. However, we found that such an intriguing reaction is indeed feasible within a narrow energy range (3.5-6.5 eV). The resulting charge-increased radicals further undergo dissociation analogous to ECD/ETD, thus constituting a novel MS/MS technique that we termed negative ion electron capture dissociation (niECD). niECD of phospho- and sulfopeptides yields predictable c’/z•-type backbone fragments without loss of phosphoric acid or sulfonate. Fragmentation pattern similarities between niECD and ECD indicate that niECD proceeds through a mechanism related to that of ECD. We proposed that gas-phase zwitterionic structures are necessary for successful niECD and that a positive charge is required to serve as the electron capture site, or to promote electron capture. N-terminal acetylation, which should reduce the probability of zwitterion formation, results in decreased niECD efficiency and introduction of fixed positive charge tags, which should promote zwitterion formation, enables niECD of peptides which could not undergo niECD in their unmodified forms. niECD efficiency also decreases with decreasing zwitterion propensity for five sets of synthetic peptides, further supporting the zwitterion mechanism. niECD was further applied to peptide chains bound by natural disulfide bonds or disulfide-containing cross-linkers. niECD of disulfide-linked peptides again results in very similar fragmentation patterns as those from ECD. S-S bond cleavage constitutes the preferred fragmentation pathway, producing characteristic fragments, which allow rapid detection of disulfide-linked peptides. Analogous to cation ECD, niECD of both N-linked and O-linked sialylated glycopeptides, which are readily deprotonated in negative ion mode, exhibits peptide backbone fragmentation with retention of labile glycans. Overall, the research presented in this thesis contributes to an increased understanding of the mechanism and utility of niECD, thereby allowing this unique approach to be developed into a valuable analytical tool for structural analysis of important biological samples.