Separation and Identification Techniques for Membrane Proteins using Ultra-High Pressure Liquid Chromatography coupled to Mass Spectrometry

Due to the importance of membrane proteins in biological pathways, the development of analytical techniques to improve membrane protein identifications is essential. For such complex mixtures, high resolution liquid chromatography (LC) is commonly utilized along with mass spectrometry (MS) for comprehensive proteomic analysis. However, commercial LC systems cannot provide the peak capacity required for such complex mixtures. With the advent of ultra-high pressure liquid chromatography (UHPLC) and multidimensional chromatography applications, peak capacities and protein identifications have increased. This dissertation will examine aspects of membrane protein sample preparation as well as instrumental analysis. Sample preparation techniques, specifically for membrane proteins, are crucial for proper protein analysis. Techniques involved with cell lysis, membrane protein extraction, solubilization, and digestion, are discussed (Chapter 2). Ultimately an optimized membrane protein sample preparation protocol was developed involving the use of high frequency sonication and the detergent sodium deoxycholate to improve solubilization. Peptides were ultimately analyzed on a modified (in-house) UHPLC constant pressure system. To improve proteomic separations, a new freeze/thaw valve and gradient storage loop were introduced to the UHPLC system, improving membrane protein identifications, instrumental reproducibility, and ruggedness (Chapter 3). To improve membrane protein digestion, an immobilized enzyme reactor (IMER) was introduced to the current multidimensional proteomic workflow. The IMER, placed after the protein separation, digested proteins as they eluted from the first dimension. This provided proteins with equivalent coverage to traditional in-solution digestions, yet avoided the 15 hr in-solution incubation process. Overall, the IMER produced high protein coverage (for both model and complex sample analysis) in a ~10 sec volumetric residence time on column (Chapter 4). Finally, a fully on-line two dimensional chromatographic system including IMER digestion was developed (Chapter 5). This system also provided equivalent coverage and protein identifications to in-solution off-line digestion, yet eliminated not only the overnight 15 hr digestion process as before, but also the ~4 hr fractionation and overnight lyophilization procedure from the workflow. Ultimately this saved the workflow about 35 hr and demonstrated the utility of the IMER.Doctor of Philosoph