MULTIDIMENSIONAL SEPARATIONS WITH ULTRAHIGH PRESSURE LIQUID CHROMATOGRAPHY - MASS SPECTROMETRY FOR THE PROTEOMICS ANALYSIS OF SACCHAROMYCES CEREVISIAE

Many biological pathways are controlled by proteins. For proteomics analysis, the peak capacity of one-dimensional separations is routinely inadequate for the number of components in a sample. Advances in mass spectrometry (MS) and liquid chromatography (LC) have improved the limits of detection and sensitivity problems associated with co-elution. However, the pressure capabilities of the pump on a standard ultrahigh performance LC (UPLC) limit the dimensions of commercial columns resulting in a maximum peak capacity of 200 in 90 minutes. Various multidimensional strategies have been developed to further increase the peak capacity. This dissertation will show the effects of 2DLC prefractionation method and frequency on proteome coverage. New ultrahigh pressure LC instrumentation with a constant pressure, high temperature approach for peptide separations is introduced. The system modified a standard UPLC with a pneumatic amplifier through a configuration of tubing and valves for separations up to 45000 psi. The modified UHPLC, coupled to a qTOF Premier, produced a peak capacity of 500 in 90 minutes on a meter-long microcapillary column packed with sub-2 micron particles. Peak capacity plateaued above 800 in 12 hours. The improved prefractionation methodology and modified UHPLC were coupled for the separation of a model proteome, S. cerevisiae. The number of protein identifications and coverage improved two-fold as compared to an analogous separation on the standard UPLC with a commercial column.Doctor of Philosoph