Nickerson, Jessica2022-12-192022-12-192022-12-16http://hdl.handle.net/10222/82183With current mass spectrometry and bioinformatics platforms, proteome analysis is at the forefront of characterizing complex biological systems. Through deep qualitative coverage combined with precise quantitation, proteomics represents a powerful tool in the pursuit of understanding disease-driving mechanisms and elucidating precise therapeutic approaches. However, the stringency of proteomics output is limited by the coverage and precision afforded by front-end preparation strategies. Much of the current proteomics literature relies on the maximum potential of state-of-the-art MS acquisition technologies without leveraging optimal front-end processing. Furthermore, many of the existing sample preparation strategies (reviewed in Chapter 1 of this thesis) impart a trade-off between recovery, digestion efficiency, and precision. The present thesis aims to evaluate the factors limiting front-end workflows and propose practical alternatives that maximize coverage, quantitative precision, and throughput. Organic solvent-based precipitation as a means of proteome purification has often been overlooked based on conflicting reports of efficiency. Following previous work from this group, Chapter 2 of this thesis assesses the rate-limiting variables associated with protein precipitation and demonstrates a rapid and robust approach to precipitation-based proteome recovery. Chapter 3 provides an evaluation of the repeatability of a precipitation-based bottom-up proteome workflow on the basis of sample coverage and the precision of peptide quantitation. Chapter 4 evaluates the potential of the enhanced precipitation approach towards multi-omics preparations. Bottom-up proteome strategies rely on robust enzymatic digestion with trypsin. Many common proteomics additives, however, impede the enzyme’s stability. Chapter 5 of this thesis characterizes the effects of several denaturing additives, demonstrating that these solubilizing agents are included at the expense of proteolytic efficiency. A wide variety of alternative digestion approaches have been described towards improved throughput over the conventional overnight incubation, although the limited validation reduces their potential for precise quantitation. Chapter 6 of this thesis characterizes the effects of elevated temperature in combination with the stabilizing effects of calcium ions towards a rapid approach to complete digestion while demonstrating the implications for bottom-up proteome analysis. Future studies, summarized in Chapter 7, suggest the application of the described rapid precipitation and enzymatic digestion to the development of targeted assays in large-scale clinical settings.en-USproteomicssample preparationprotein precipitationtrypsin digestion