High Mass Accuracy Mass Spectrometry as a Tool in Biomolecule Identification
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In this dissertation, three approaches were utilized for the characterization of biomolecules using high-resolution mass spectrometry (MS). First, the effect of metal oxide affinity chromatography (MOAC) was examined as an enrichment method for phosphopeptride identification. The results indicated that especially TiO 2 provides easier detection of phosphopeptides by eliminating the suppressing effects of non-phospohopeptides during mass spectrometric analysis. In addition, the possible effects of other parameters such as concentration, temperature, and the utilization of metal oxides in form of powder in the identification of phosphopeptides were studied. Of the parameters studied, only the temperature at which the metal oxides were prepared seemed to have an effect on their effectiveness in the enrichment process. Secondly, conventional matrix assisted laser desorption ionization (MALDI) and MALDI imaging were employed for identification of neuropeptide Y (NPY) to examine the effectiveness of NPY as a target in treatment of cocaine addiction. Both the exogenous and endogenous peptides were detected as well as cocaine itself in rat brain tissues with cocaine history. Finally, the application of top-down ECD in hydrogen-deuterium exchange (HDX) mass spectrometry in the study of the structure and dynamics of proteins was investigated, and compared with the bottom-up HDX MS using pepsin. We showed that top-down ECD provides much more detailed information about the structure and dynamics of proteins. Additionally, the effectiveness of immobilized pepsin microreactor coupled with H/D exchange MS in providing more rapid and complete digestion of protein was studied. The use of immobilized pepsin provides for improved results by eliminating the problem of back exchange during the process of in-solution digestion and reduces the requirement of using high amount of pepsin to reduce the digestion time, which in turn can result in plugging the columns used for high performance liquid chromatography (HPLC).