Advances in Peptide Analysis by Capillary Electrophoresis coupled with Laser Induced Fluorescence and Mass Spectrometry

Capillary electrophoresis (CE) is the fastest and the most efficient liquid separation method for biomolecule analysis. However, it has not been widely applied in proteomic applications, mostly due it small loading capacity and difficulties in hyphenation with electrospray (ESI) mass spectrometry.;The first part of this thesis describes a new design of CE-ESI-MS interface, that addresses some issues encountered with traditional interfaces, including spray stability, sensitivity and versatility. The performance of the interface is characterized both experimentally and with the help of modeling by Comsol Multiphysics Software. The interface is also evaluated in the context of shotgun proteomic applications.;Diagonal separations are used for targeted analysis of complex mixtures. They employ identical separation conditions in both dimensions and rely on chemical or enzymatic analyte modification in between the separation dimensions to cause a shift in its mobility.;The second part of the thesis describes fully automated two dimensional diagonal capillary electrophoresis, incorporating a replaceable enzymatic microreactor in the end of the first dimension separation. The technique is applied to the analysis of phosphopeptides, which are dephosphorylated as they pass through online microreactor containing alkaline phosphatase immobilized to paramagnetic beads. Loss of phosphate group(s) causes a shift in peptide mobility in the second separation dimension. The technique is demonstrated with both laser induced fluorescence and mass spectrometry as modes of detection.