Characterization of membrane protein primary structure and protein covalent modification by MALDI-TOF-MS

The subjects of this thesis were to illustrate membrane protein primary structure determination, protein covalent modification and protein-protein interaction by using matrix assisted laser desorptiodionization time-of-flight mass spectrometry (MALDITOF- MS). First, in one mode of sample preparation referred to as wall-less sample preparation, an increase in sequence coverage of a model membrane protein, bacteriorhodopsin, increased from 8 1 % using traditional sample methodology to 89%. Second, chemical modification sites of proteins, pheromone binding protein 2 (PBP2) and cytochrome P45OCam (P45OCam), were characterized by MALDI-MS. This finding contributed to further studies of protein-ligand interaction of PBP2, and also provided information about native conformation of P45OC,,. Third, co-imrnunoprecipitation and MALDI-MS was used to identify the interaction of synapse-associated protein (SAP97) with potassium voltage-gated ion channel Kv1.5. This study provided the first direct mass spectral evidence to support their protein-protein interaction. In addition, to better understand one of MALDI ionization pathways, a simple two-plate method was used to examine the MALDI gas-phase cationization.