Novel Micro-Separation Techniques and Their Couplings to Mass Spectrometry for the Analysis of Complex Peptides and Proteins

Mass spectrometry (MS)-based technology has evolved as a powerful tool for the analysis of a wide array of biomolecules. However, its application to complex peptidomics and proteomics samples, for example neuropeptides, has proven to be challenging due to their extreme chemical complexities and low abundances in biological tissue samples. This PhD dissertation is focused on addressing some of these challenges by developing novel micro-separation tools and coupling them with MS to achieve enhanced MS sensitivity and increased peptide and protein coverage with lower sample consumption and reduced experimental time. Both chromatography-based and electrophoresis-based separations as well as their couplings to MS have been explored in this work, including monolithic material-based chromatography, improved capillary electrophoresis (CE) separation and novel coupling of separations to mass spectrometric imaging. N-acryloxysuccinimide-based monolithic column has been fabricated with specific antibodies immobilized onto the column to enable the extraction of targeted neuropeptide family from biological matrices. Alternatively, enzymes were immobilized onto the column for highly efficient on-column enzymatic digestion for proteomics studies. A methacrylate-based monolithic column with C12 functional groups was developed and coupled to both electrospray ionization (ESI) MS and matrix-assisted laser desorption/ionization (MALDI) MS for reversed-phase liquid chromatography (RPLC). For capillary electrophoresis, two improved capillary isoelectric focusing (CIEF) systems have been developed, including membrane-assisted CIEF and immobilized pH-gradient CIEF to obtain reduced sample loss and enhanced MS signal for neuropeptide analysis. An improved sheathless pressure-assisted CE (PACE) system was also developed for coupling to MALDI MS. For novel separation-MS imaging coupling, three generations of interfaces have been designed to couple CE, LC and multi-dimensional separations to MS imaging (MSI) for quantitative analysis of complex peptides. Collectively, this work results in development of the new generation of micro-separation and sample preparation tools and their coupling to various mass spectrometric ionization and detection modes for sensitive and rapid analysis of complex peptides and proteins. The technological innovations have significantly expanded the toolbox for peptidomics/proteomics analysis and will enable more in-depth investigations on peptide function and regulation.