Methods development and application of two-dimensional chromatography and tandem mass spectrometry in proteomics

In recent years, proteomics has gained considerable attention as a complimentary discipline to genomic research. The ability to identify, quantify, and characterize complex mixtures of proteins expressed in an organism provides a better understanding of the physiological processes taking place. As such, some thought has recently been given to two-dimensional (2-D) LC-MS/MS as a "shotgun" approach to proteomics. By coupling 2-D chromatographic separations to present-day quadrupole ion traps, one gains the chromatographic peak capacity to resolve complex mixtures of peptides/proteins while performing "on-the-fly" MS/MS analysis.; This work describes one such 2-D LC-MS/MS method for proteomics. The first three chapters describe the background and methods development of fabricating 2-D chromatographic capillary columns and interfacing them with an ion-trap mass spectrometer. A scheme for packing 2-D LC capillary columns is described, and their ability to resolve complex mixtures is assessed. In addition, scan parameters of a Finnigan LCQ Deca ion trap mass analyzer were evaluated for proteomics applications. The effect of parameters, such as the number of averaged scans and ion injection times, on the ability to analyze the maximum number of closely eluting peptides while maintaining high quality MS/MS spectra is described.; The latter portion of this dissertation describes the application of 2-D LC-MS/MS to two separate proteomic studies of human ventricular cerebrospinal fluid (CSF). The first study was a qualitative investigation of CSF from 10 cognitively normal, elderly subjects. A cumulative 249 CSF proteins were identified, while only 20 were found to be common among all subjects. These results indicate extensive subject to subject variability in the CSF proteome. However, variability associated with the 2-D LC MS/MS method itself is also addressed.; The last study describes a quantitative application of 2-D LC-MS/MS through the use of isotope-coded affinity tags (ICAT). Here, ventricular CSF from two populations of control and Alzheimer's disease (AD) subjects was investigated in search of quantitative proteomic differences. A total of 106 unique proteins were cumulatively identified as a result of this study. However, only uracil DNA glycosylase exhibited a statistically significant decrease in the AD subjects (Wilcoxon rank-sum, P = 0.02).