Development of on-line capillary electrophoresis and monolithic capillary HPLC interfaced with mass spectrometry for analysis of human cancer proteomes

The development of several high-throughput methods using liquid separations of low flow rates interfaced on-line for mass spectrometric analysis of various human cancer proteomes is described in this thesis. Two liquid separation methods, including capillary electrophoresis (CE) and monolithic capillary HPLC, have been developed for on-line interface to both ESI- and MALDI-based mass spectrometric analyses. Each of these separation methods provides highly efficient separations for protein digests completed within several minutes by generating peak widths of only several seconds to result in improved peak capacities. Combined with protein fractionation from two-dimensional liquid phase separations based on pI and hydrophobicity, both methods have proven ideally suited for high-throughput analysis. Polymer-based monolithic capillary HPLC separation, in particular, effectively solves several limitations posed by CE and typically resulted in a theoretical peak capacity of up to 100 in a separation window of 10 min due to the lack of interstitial space inside the separation medium to minimize diffusion. As analyte does not dwell inside the column, a higher recovery is also expected for enhanced sensitivity. Excellent separation qualities were obtained using monolithic LC/MS and its versatility has been successfully demonstrated using various types of human cancer samples.; The usefulness of intact protein MW value has been demonstrated in pH fraction digest analysis, where the proteins whose identification were suggested only by a single sequenced peptide can be more confidently obtained with closely matching intact protein MW. The proteins rapidly identified through monolith-based LC/MALDI were also analyzed for intact MW values. In particular, with successful demonstration of automation of all liquid handling procedures for the LC/MALDI platform for protein analysis, the method appears to have great potential for a high-throughput analysis to study large numbers of proteins, where monolith-based separation of proteins pre-fractionated according to pI and enzymatic digestion altogether requires less than one hour. Given a very low sample consumption required by this experimental scheme that requires minimal sample transfers, a thorough examination of proteins in the biological mixtures available in very low concentrations can be achieved.