| Proteomics---which is the analysis of the full complement of proteins produced by an organism---plays a crucial part in a variety of fields of research, including basic biological studies, pharmaceutical development, and clinical diagnostics. Separations are a key element of proteomic analyses. The traditional means of separating protein mixtures is two-dimensional gel electrophoresis, which offers very high resolution. Over the past several decades, alternative methods for protein separations based on liquid chromatography have been developed. These methods have advantages over gel-based analyses, including reduced bias against certain classes of proteins, straightforward automation, and easier coupling to mass spectrometry. However, in order to effectively manage the complexity of a proteome, the separation technique must be able to resolve a very large number of components. Multidimensional liquid chromatography (MDLC) is well-suited to this task. Chapter 1 of this dissertation introduces the theoretical framework behind MDLC. Previous work involving protein separations using comprehensive two-dimensional liquid chromatography (LC x LC), which is a form of MDLC, is also discussed.; Chapters 2 and 3 of the dissertation focus on the development of methods for intact protein separations using liquid chromatography. Several separation modes were evaluated, including size exclusion, ion exchange, and reversed phase. Two-dimensional separations of E. coli proteins were performed which use anion exchange in the first dimension and ultra-high pressure reversed-phase LC in the second dimension. Although high peak capacities were demonstrated, the technique was limited in that proteins could not be identified solely based on the intact protein molecular weight data which was obtained.; The research presented in Chapters 4 and 5 use the same intact-protein LC x LC separations, but also incorporates enzymatic digestion of proteins followed by LC-MS analysis of the resulting peptides. The resulting technique is a hybrid of "top-down" and "bottom-up" proteomics methods. It allows proteins to be identified on the basis of tandem mass spectra of peptides, but retains the information gained from intact protein MS. In Chapter 6, this technique was applied to study differential protein expression in yeast cultures grown under different conditions. |
