Date of Award
Doctor of Philosophy
Cheng S. Lee
Robert S. Houk
On-line coupling of capillary isoelectric focusing (CIEF) and liquid chromatography (LC) with electrospray ionization mass spectrometry (ESIMS) is utilized for protein characterization;A microdialysis junction, based on a microdialysis membrane connecting both a separation capillary and a short, sharply tapered microelectrospray emitter capillary, is demonstrated for CIEF-ESIMS. Besides the electrical connection across the microdialysis junction, post-run acidification via the dialysis membrane is demonstrated for enhancing the protonation and the ionization efficiency of focused proteins while maintaining separation efficiency, resolution, and sensitivity in CIEF-ESIMS;On-line combination of CIEF with ESI-FTICRMS is demonstrated for high resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins. The acquisition of high-resolution mass spectra of hemoglobin [beta] chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 dalton. Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICRMS;An integrated approach, involving the combination of exoglycosidase enzyme array with LC/MS/MS, is demonstrated for structural analysis of carbohydrate moieties of glycopeptides from a glycoprotein digest. The molecular mass information from a series of controlled digestions together with specific compositions of exoglycosidases in the enzyme array provide the sequence and linkage of individual glycan species attached to glycopeptides and glycoproteins.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Yang, Liyu, "On-line coupling of capillary electrophoresis and liquid chromatography with electrospray ionization mass spectrometry for protein characterization " (1998). Retrospective Theses and Dissertations. 11827.