Supercharging methods for improving analysis and detection of proteins by electrospray ionization mass spectrometry
- Author(s): Going, Catherine Cassou
- Advisor(s): Williams, Evan R
- et al.
The characterization of mechanisms, analytical benefits, and applications of two different methods for producing high charge state protein ions in electrospray ionization (ESI) mass spectrometry (MS), or "supercharging", are presented in this dissertation. High charge state protein ions are desirable in tandem MS due to their higher fragmentation efficiency and thus greater amount of sequence information that can be obtained from them. The first supercharging method, supercharging with reagents (typically non-volatile organic molecules), is shown in this work to be able to produce such highly charged protein ions from denaturing solutions that about one in every three residues carries a charge. The high Coulomb repulsion in these ions results in these ions adopting near-linear gas-phase structures with little to no non-covalent interactions, making them ideal for efficient fragmentation in tandem MS experiments and for the minimization of gas phase HD scrambling during tandem MS. Supercharging with reagents from aqueous solutions typically produces much lower charge states as compared to that observed from a denaturing solution. However, two new reagents are presented in this work that increase protein ion charge past that from denaturing solutions when added to aqueous solutions at just 2% by volume. Increases in charge of up to 168% are reported in the presence of these reagents. The mechanism of the increases in protein ion charging with these reagents from aqueous solutions was investigated with fluorescence experiments and correlated to a destabilization of the protein structure by these reagents toward denaturation. The actual protein denaturation event likely occurs in the ESI droplet itself, consistent with previous studies of the mechanism of supercharging with reagents. Thus, efficient tandem MS of high charge states is possible from ESI of aqueous solutions in which a protein maintains its native or native-like structure and activity, enabling tandem MS analysis of protein modifications, ligand binding, or structural changes in real time. Interestingly, another application for supercharging reagents is protein desalting in the ESI droplet. Supercharging reagents bind to sodium ions, resulting in less non-specific sodium ion adduction to proteins, which can improve signal-to-noise ratios of protein ions, lower limits of detection, and enable the detection of bound ligands or specific binding of salts that might otherwise be obscured by sodium adduction. The second supercharging method, electrothermal supercharging (ETS), requires the presence of particular buffer salts rather than organic reagents to increase protein ion charge in the ESI droplet. An investigation of the effect of several different buffer salts on ETS is presented in this work, revealing that the choice of buffer salt is very important to obtaining effective ETS and that buffer salts likely stabilize or destabilize protein structure in the ESI droplet via Hofmeister effects. The application of ETS to tandem MS of proteins produced by ESI and its utility on proteins ranging in size over an order of magnitude (8.6 kDa to 83.0 kDa) is demonstrated. Hydrogen-deuterium exchange experiments can be performed in aqueous solutions and measured continuously with ETS coupled to tandem MS for protein structure analysis in real time with a spatial resolution of 1.3 residues and without gas phase hydrogen-deuterium scrambling. This work demonstrates the wide applicability of ETS for the study of primary and higher order protein structure for small and large proteins alike.