Capillary electrophoresis-mass spectrometry methods for advancements in proteomics

Abstract

Capillary electrophoresis -- mass spectrometry (CE-MS) is increasingly being used in proteomics research. In particular, the speed of analysis, high mass accuracy and sensitivity of many modern mass spectrometers make this technique appealing for the rapid identification of peptide and proteins mixtures. For protein digest mixtures, tandem mass spectrometry (MS/MS) of individual peptides is collected and a database search of the product ion spectra is conducted. While this method of analysis generally provides acceptable sequence coverage for most protein digests, certain protein digests that contain low abundance or high molecular weight proteins still suffer from low sequence coverage. While CE-MS has several notable advantages, such as low-volume sample requirements, short analysis time, and high separation efficiency, it also has its disadvantages such as protein/peptide-wall interactions, cumbersome fabrication of the CE-to-MS interface, concentration limited detection, and undersampling of complex protein digests. The effort of this thesis is to remedy several of these disadvantages using simple techniques. A self-coating background electrolyte (BGE) was introduced to reduce protein/peptide-wall interactions without requiring any pre-treatment of the capillary inner-wall, thus simplifying the analysis of peptides and proteins. An analytical technique called (CE-MS/MS)n was developed to address undersampling and to enhance the sequence coverage for better protein identification. Pre-concentration capillaries using sol-gel technology were evaluated to improve the concentration detection limit associated with CE-MS. These advances have simplified the CE-MS operation and have intensified the interest in CE-MS for high sequence coverage proteomics. In addition, a new technique for analyzing dilute samples and protein-metal ion complexes is also introduced.