Maximizing Proteome Recovery and Digestion Efficiency for High-Throughput Bottom-up Mass Spectrometry

Abstract

With 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.