Characterization of native protein structure with ion mobility mass spectrometry, multiplexed fragmentation strategies and multivariant analysis

Activated ion mobility measurements provide insights to the stability of tertiary and quaternary structures of proteins and when coupled with dissociation strategies can delineate how the fold is disrupted. In this work, we use 213 nm photodissociation coupled with ion mobility mass spectrometry and collisional activation to probe the conformational landscape of model proteins. UVPD experiments are performed on proteins following in source activation and on collisionally activated photoproducts post ion mobility separation. For all observed conformations of cytochrome c, there is a significant increase in the UVPD fragmentation yield with the addition of collisional activation post mobility. Similar strategies are deployed with the multimeric proteins, concanavalin a, and haemoglobin. For these complexes’ CID results in ‘classical’ asymmetric charge distribution in subunit products, which when preceded by UV irradiation, yields fragments from within sub-units. Combining these strategies provides complex multidimensional datasets, rich in information, which here we mine with multivariate analysis (MVA). This approach readily determines differences in UVPD and CID fragmentation patterns as a function of conformation and reveals diagnostic information about the precursor native structure without the limitations of current methods that only assign backbone cleavage sites.