Sensitivity enhancement for capillary zone electrophoresis-mass spectrometry: Developments and applications.

摘要

Advances in mass spectrometry (MS), i.e., electrospray ionization and tandem mass spectrometry using triple quadrupole instruments, have greatly enhanced the analysis of biopolymers, such as proteins and glycoproteins. Electrospray ionization enables the direct coupling of liquid-based separations with mass spectrometric detection. Capillary zone electrophoresis (CZE) allows for high resolution separation of biomolecules, and its inherent low flow rate is ideally suited for coupling to electrospray mass spectrometry (ESMS). A major shortcoming of CZE is its high concentration detection limits relative to liquid chromatography. This thesis presents two approaches for the improvement of the sensitivity of CZE. These improved methods are applied to the characterization of proteins and glycoproteins by CZE-ESMS.; A nanoelectrospray mass spectrometry (nESMS) interface for CZE was constructed from metallized fused silica tips that were connected to the separation capillary. Several methods for connecting the tips to the separation capillary were evaluated. The tip geometry and capillary coating were optimized to produce a rugged and reliable interface. The optimized CZE-nESMS interface gave detection limits for peptides at least one order of magnitude lower than a conventional co-axial CZE-ESMS interface.; On-line chromatographic preconcentration was also used to enhance the sensitivity of CZE-ESMS. Several stationary phases were evaluated with respect to sample retention and elution for use in the preconcentrator. A sample loading and elution protocol for the reliable and reproducible use of the preconcentrator was developed. Detection limits were improved by a factor of 1000 (relative to a conventional coaxial interface without preconcentration) when the preconcentrator was coupled with the nanoelectrospray interface (PC-CZE-nESMS).; Several N-linked and O-linked glycoproteins were analyzed by CZE-nESMS. The high resolution separation provided by CZE gave glycoform population information, not typically generated by the more conventional HPLC analysis of glycoproteins. CZE with on-line tandem mass spectrometry was used to study the size and composition of the oligosaccharides attached to the proteins. New scanning methods were developed to investigate the peptide sequence of the glycopeptides. First generation fragment ions produced in the orifice/skimmer region of the ion source were analyzed by tandem mass spectrometry in the collision cell to provide peptide sequence ions. The sequence allowed the assignment of oligosaccharide linkage for the N-linked glycopeptides.; The PC-CZE-nESMS method was capable of analyzing a protein tryptic digest at the femtomole/{dollar}mu{dollar}L, equivalent to approximately 5 picoomoles of protein injected. The data generated permitted the exclusive identification of the protein from the search of a database of 32000 proteins.