Charging of Proteins and Protein Complexes in Native Mass Spectrometry
- Author(s): Susa, Anna Christine
- Advisor(s): Williams, Evan R
- et al.
Electrospray ionization (ESI) mass spectrometry is a powerful analytical tool for investigating the identities, structures, functions, and energetics of biomolecules. ESI transfers intact molecules from buffered aqueous solutions in which they are folded or in native conformations into the gas phase for mass spectral analysis. ESI is widely used for the analysis of proteins in mass spectrometry, but the factors that influence charging of protein ions formed by ESI are not well understood. Higher charge states of protein ions are desirable because they fragment more easily in tandem mass spectrometry methods, leading to more sequence coverage than lower charge protein ions. Lower charge protein ions are advantageous in native mass spectrometry when preservation of native protein conformation is desirable. The work described in this dissertation explores factors that control charging of macromolecular ions in native mass spectrometry. This work provides evidence that the charging of macromolecular ions is not significantly limited by ion evaporation of cations, such as alkylammonium or alkali metal ions, or proton transfer to salts that are commonly added to aqueous buffered ESI solutions. In addition, a novel and simple method for ESI of proteins directly from buffers commonly used in biochemistry laboratories is demonstrated. In native mass spectrometry, protein solutions are typically desalted and buffer exchanged into volatile ammonium salt buffers to prevent salt adduction to the protein ions because salt adduction significantly hinders detection and sensitivity of mass spectral analysis. However, some salts are essential for protein structure and function, and this new method of ion desalting allows formation of protein and protein complex ions directly from buffers that contain high ionic strengths of nonvolatile salts to mimic the intracellular and extracellular environments. This technique greatly impacts the way native mass spectrometry is performed because it eliminates the need to reinvestigate properties of the proteins and protein complexes in traditional ammonium salt buffers used in mass spectrometry. Therefore, biochemists will no longer need to adapt their protein solutions to make them suitable for mass spectrometry.