Symmetry of Charge Partitioning in Collisional and UV Photon Induced Dissociation of Protein Assemblies

Tandem mass spectrometry can provide structural information on intact protein assemblies, generating mass fingerprints indicative of the stoichiometry and quaternary arrangement of the subunits. However, in such experiments, collision induced dissociation yields restricted information due to simultaneous subunit single subunit. Alternative fragmentation strategies can potentially overcome this and unfolding, charge rearrangement, and subsequent ejection of a highly charged unfolded supply a deeper level of structural detail. Here, we implemented ultraviolet photodissociation (UVPD) on an Orbitrap mass spectrometer optimized for native MS and benchmark its performance to HCD fragmentation using various protein oligomers. We investigated dimeric beta-lactoglobulin, dimeric superoxide dismutase, dimeric and tetrameric concanavalin A, and heptameric GroES and Gp313 ranging in molecular weight from 32 to 102 kDa. We find that, for the investigated systems, UVPD produces more symmetric charge partitioning than HCD. While HCD spectra show sporadic fragmentation over the full protein backbone sequence of the subunits with a bias toward fragmenting labile bonds, UVPD 'spectra provided higher sequence coverage. Taken together, we conclude that UVPD is a strong addition to the toolbox of fragmentation methods for top-down proteomics experiments, especially for native protein assemblies.