Investigating the Relationship Between the Gas-Phase Structures of Protein Ions and Their Charge States

Abstract

This dissertation explores the utility of cation-to-anion proton transfer reactions (CAPTR) in native mass spectrometry, and investigates the relationship between the charge state (z) and collision cross section (Ω) of gas-phase protein ions from native-like and denaturing conditions. In CAPTR, protein cations are generated via nano-electrospray ionization (nESI) and reacted with stored anions via proton transfers. The products of those reactions are then studied using ion mobility (IM), which measures the Ω of an ion-neutral pair, and mass spectrometry (MS), which measures the mass-to-charge ratio (m/z) of an ion. Chapter 1 discusses those reactions involving native-like protein and protein complex ions. Those results indicate that CAPTR may be used to aid in z determination, and separate mixtures of ions in m/z. The following several chapters then explore the relationship between z and Ω of denatured and native-like proteins and native-like protein complexes. Charge states of ubiquitin from denaturing conditions were m/z selected for CAPTR. The results indicate that the ions compact following each reaction, and that the Ω of the product ions depends on the product ion charge state (C) and is independent of the precursor charge state (P). Energy-dependent experiments indicate that product ions of the same C, but from different P, have different subpopulations of ions. Similar experiments were performed with cytochrome C from native-like and denaturing conditions, which showed that the lowest C ions from those experiments have similar Ω. Additionally, the Ω of native-like cytochrome C CAPTR product ions depend more weakly on C than their denatured counterparts. The Ω of CAPTR products of lysozyme from native-like, denaturing/reducing and denaturing/disulfide intact solution conditions are also similar at the lowest C measured. Energy-dependent experiments reveal the importance of disulfide bonds on the gas-phase structure of lysozyme ions. Ω of the CAPTR products of native-like protein and protein complexes were also studied using IM-MS, energy-dependent pre-CAPTR collisional activation, and energy-dependent post-CAPTR collisional activation experiments. Those studies reveal the stabilities of the product ions, and that their Ω weakly depends on C compared to protein ions from denaturing conditions. CAPTR was also performed on poly-ubiquitin ions from native-like and denaturing solution conditions. The Ω of poly-ubiquitin ions from native-like solutions were used to develop coarse-grained models to interpret the domain structure of the CAPTR products of poly-ubiquitin ions from denaturing conditions. Further, figures of merit were used to quantitate the Ω of poly-ubiquitin ions from denaturing conditions as a function of C. Lastly, trajectory method calculations were performed to investigate how ion-induced dipole interactions may affect Ω of native-like and denatured protein ions.