Enhancement Of Mass Spectrometry And Affinity Purification Methodologies Used To Acquire Novel Protein Identifications

Mass spectrometry (MS)-based proteomics has become a primary tool to study proteins underlying infection disorders, antibiotic resistance mechanisms, and identify significant new targets for drug discovery. Mass spectrometers must be capable of discriminating molecules of interest while maintaining a high level of robustness, sensitivity, and measurement accuracy. These specifications require a multi-faceted approach combining mass spectrometry and high performance analytical and biochemical separations. The first study enhanced performance for a common mass spectrometer design. Significant variability in this instrument impaired consistent accurate mass measurements and severely reduced molecular identification. Spectral averaging provided a simple and highly effective strategy to mitigate these instrument limitations. The second study improved consistency and reproducibility in Affinity Purification Mass Spectrometry (APMS), a powerful method to study proteins and protein interaction complexes. An optimized APMS method was developed through systematic evaluation of the most common resins and protein tag strategies, demonstrating that APMS experiments must consider the effect of solid-phase resin on the entire experimental design. These improved methods were applied in two biological experiments. The first experiment applied optimized APMS to isolate phosphoinositide 3-kinase (PI3K) within pancreatic cancer BxPC-3 cells treated with different tyrosine kinase inhibitors targeting the epidermal growth factor receptor (EGFR). This experiment revealed a signaling processes within the EGFR/PI3K/Akt and ERK pathways as a mechanism of EGFR inhibitor response and provide new insight into the molecular basis tyrosine kinase inhibition and potential resistance mechanisms. The second experiment utilized liquid-chromatography mass spectrometry (LC-MS) to study dormancy in Micrococcus luteus (M. luteus), a close relative of Mycobacterium tuberculosis (M. tuberculosis). Eighteen proteins upregulated in dormancy were identified, four of which linked to M. tuberculosis latency, and the remaining 14 novel. These proteins are future targets for molecular and therapeutic studies. These studies demonstrate improved methods for mass spectrometry-based proteomics and apply these methods to important molecular and drug-discovery systems, thereby progressing the field of systems biology.Biology and Biochemistry, Department o