Proteomics is one of the most effective ways to study protein regulation including characterizing the changes in protein quantity as a result of biological perturbation. Liquid chromatography paired with tandem mass spectrometry (LC-MS/MS) is the most commonly used analytical technology in this field. After two decades of technological advancements, the quantitation of the total human proteome still precludes us. These advancements have allowed for high throughput quantitation of near tens of thousands of proteins in hours of analysis time. However, 30% of the proteome remain elusive in routine proteomic analysis. Current LC-MS/MS methods rely on increasing the number of peptides identified to yield more protein identifications. But it has been shown by Bekker-Jensen et al. that this is a logarithmic relationship with diminishing returns.

This diminishing return is because a majority of the peptides belong to high abundance or large proteins and are highly redundant. Prior peptide-centric approaches have shown promising improvements to proteome coverage by enriching a subset of peptides containing specific amino acids for analysis rather than analyzing the whole digested proteome. By reducing the number of peptides necessary to analyze and the overall protein identification redundancy, these approaches can potentially bypass the logarithmic relationship between peptide and protein identifications. We aim to develop a peptide-centric approach that can be generally applicable for a variety of sample types by enriching a subset of peptides that have a particular physicochemical property via chromatographic approaches. Therefore, our approach is named Proteome-Selective Isolation Chromatography (P-SLICY). In this thesis, we will describe the fundamental topics within proteomics, chromatography, and mass spectrometry and the innovations towards understanding peptide chromatography to develop P-SLICY.