Negative-Ion Electron Capture Dissociation of MALDI-Generated Peptide Anions

Negative-ion electron capture dissociation (niECD) is an anion MS/MS technique that provides fragmentation analogous to conventional ECD, including high peptide sequence coverage and retention of labile post-translational modifications (PTMs). niECD has been proposed to be the most efficient for salt-bridged zwitterionic precursor ion structures. Several important PTMs, e.g., sulfation and phosphorylation, are acidic and can, therefore, be challenging to characterize in the positive-ion mode. Furthermore, PTM-friendly techniques, such as ECD, require multiple precursor ion-positive charges. By contrast, singly charged ions, refractory to ECD, are most compatible with niECD. Because electrospray ionization (ESI) typically yields multiply charged ions, we sought to explore matrix-assisted laser desorption/ionization (MALDI) in combination with niECD. However, the requirement for zwitterionic gaseous structures may preclude efficient niECD of MALDI-generated anions. Unexpectedly, we found that niECD of anions from MALDI is not only possible but proceeds with similar or higher efficiency compared with ESI-generated anions. Matrix selection did not appear to have a major effect. With MALDI, niECD is demonstrated up to m/z ∼4300. For such larger analytes, multiple electron captures are observed, resulting in triply charged fragments from singly charged precursor ions. Such charge-increased fragments show improved detectability. Furthermore, significantly improved (∼20-fold signal-to-noise increase) niECD spectral quality is achieved with equivalent sample amounts from MALDI vs ESI. Overall, the reported combination with MALDI significantly boosts the analytical utility of niECD.