Probing effects on protein charge state distributions by electrospray ionization mass spectrometry
All mass spectrometers combine ion formation, mass analysis and ion detection. The first chapter of this thesis introduces the fundamentals of some common ionization techniques, especially electrospray ionization source (ESI), and some common mass analyzers, along with their advantages and disadvantages. Since ESI is well recognized as a soft ionization method, ESI-MS has emerged as a powerful technique in modern bioanalytics. ESI charge state distribution provides an abundance of information on understanding the structures and dynamics of protein in solution, while it can be influenced by many factors. In this thesis, factors, such ESI sources, solution pH and redox reagents, were discussed. It is found that for identical protein sample ESI spectrum shows relative higher average charge state than that of nanoESI due to the size of the droplets produced, surface area-to-liquid volume ratio or protein conformation. As the pH of sprayed solution decreases, the average charge state increases. This is because at low pH, the protein unfolds and more ionizable basic sites are available so that it can accept a large number of protons, which lead to higher average charge states. When mixing with the denaturing reagents, protein charge state distributions shift to lower charge states with their increasing gas-phase basicities. Redox reagents play a very important role in protein charge state distributions. Acetaminophen was found to dramatically reduce protein charge state distributions and its effect is related to the protein/acetaminophen molar ratio. The average charge state of cytochrome c was observed to shift from 14.99 to 8.90 in the presence of acetaminophen (cyt c /acetaminophen molar ratio=1:4) by using ESI source and from 10.14 to 7.71 by using nanoESI source. The intermediate of N-acetyl-p-benzoquinone imine (NAPQ) is believed to play a key role in this charge reduction phenomenon.