Innovative Approaches and Tool Development for Proteomics Data Analysis: Applications Across Diverse Biological Systems

Bottum up proteomics (BUP) is a powerful analytical technique that involves digesting complex protein mixtures into peptides and analyzing them with liquid chromatography and tandem mass spectrometry to identify and quantify many proteins simultaneously. This produces massive multidimensional datasets which require informatics tools to analyze. The landscape of software tools for BUP analysis is vast and complex, and often custom programs and scripts are required to answer biological questions of interest in any given experiment. This dissertation introduces novel methods and tools for analyzing BUP experiments and applies those methods to new samples. First, PrIntMap-R, a custom application for intraprotein intensity mapping, is developed and validated. This application is the first open-source tool to allow for statistical comparisons of peptides within a protein sequence along with quantitative sequence coverage visualization. Next, innovative sample preparation techniques and informatics methods are applied to characterize MUC16, a key ovarian cancer biomarker. This includes the proteomic validation of a novel model of MUC16 differing from the dominant isoform reported in literature. Shifting to bacterial studies, custom differential expression workflows are employed to investigate the role of virulence lipids in mycobacterial protein secretion by analyzing mutant strains of mycobacteria. This work links lipid presence and virulence factor secretion for the first time. Building on these efforts, OnePotN??TA, a labeling technique enabling quantification of N-terminal acetylation in mycobacterial samples, introduced. This method is the first technique to simultaneously quantify protein and N-terminal acetylation abundance using bottom-up proteomics, advancing the field of post-translational modification quantification. This project resulted in the identification of 37 new putative substrates for an N-acetyltransferase, three of which have since been validated biochemically. These tools and methodologies are further applied to various biological research areas, including breast cancer drug characterization and insect saliva analysis to perform the first proteomic studies of their kind with these respective treatments and samples. Additionally, a project focused on teaching programming skills relevant to analytical chemistry is presented. Collectively, this work enhances the analytical capabilities of bottom-up proteomics, providing novel tools and methodologies that advance protein characterization, post-translational modification analysis, and biological discovery across diverse research areas.