UC Riverside

The continuing development of mass spectrometry as a tool for structural biology requires further elucidation of protein behavior in the gas-phase. Recent years have brought about numerous studies on protein stability in the gas-phase, without a clear consensus as to whether the non-covalent interactions defining protein structure are stable and therefore ultimately amenable to evaluation in the absence of solvent. Despite this, native mass spectrometry has already become a technique of significant interest for the structural examination of large proteins and protein complexes in the gas-phase. Many high molecular weight biomolecules sprayed from native-like aqueous environments contain moderate amounts of net charge and are stabilized by extensive numbers of intramolecular interactions such as hydrogen bonding. As proteins become smaller, however, there are fewer of these stabilizing interactions. Additionally, as protein size diminishes, the ratio of highly charged solvent-accessible surface to the protein interior also increases, creating an environment which experiences enhanced coulombic interactions. For these and other reasons, careful consideration of each factor involved in the stabilization or destabilization of biomolecular structure in the gas-phase is needed.

Specific factors scrutinized herein include a detailed understanding of participating electrostatic interactions within gas-phase peptides and proteins, as these are the most influential factors for the structures in the absence of intermolecular interactions. The exquisite precision afforded by action excitation energy transfer paired with molecular dynamics is used to probe local ion-dipole and ion-ion behavior in the absence of solvent versus partial solvation. Utilizing a newly developed technique termed photoelectron transfer dissociation, the prevalence of gaseous zwitterions are investigated. This particular coulombic interaction was chosen due to important structural implications being tied to their existence and the fact that little is currently known about their propensity to exist in the gas-phase. Efforts are also made to facilitate crosslinking analysis used for structural elucidation by incorporating 213 nm UVPD into the MS/MS workflow. Crosslink-specific fragmentation produces reporter ions able to drastically reduce search space allowing for confident identification and further insights into molecular structure. These and other experiments serve to better our understanding of protein structure and its stability outside of a native context.