Development of mass spectrometric methods for membrane proteome analysis

While membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery, their analysis presents a special challenge largely due to their highly hydrophobic nature. Recently, the shotgun proteomics approach has emerged as a powerful technique for proteome identification and quantitation. It is based on the digestion of proteins from extracts of whole cells, organelles, or specific fractions of thereof, followed by liquid chromatography tandem mass spectrometry analysis of the resulting peptides.; In this thesis, the shotgun proteomics approach is further developed to tackle membrane protein analysis. Because membrane proteins are not easily dissolved in water, surfactants are employed for membrane protein solubilization. However, surfactants, especially sodium dodecyl sulfate (SDS), have been found to be detrimental to mass spectrometric signal by some of the researchers, whereas others have found that SDS did not deteriorate the mass spectrometric signal. This controversy was resolved from part of the work described in this thesis. It was found that for SDS-containing samples, the intensity of the MALDI signals can be affected by the conditions of sample preparation: on-probe washing, choice of matrix, deposition method, solvent system, and protein-to-SDS ratio. However, under appropriate conditions, the two-layer method gave reliable MALDI signals for samples with levels of SDS up to ∼1%. The mass resolution issue was also addressed and a method was developed to improve the resolution.; Furthermore, the surfactants' effects on protein digestion and mass spectrometric response were studied and a SDS-aided in-solution digestion for membrane protein identification method was developed. The developed method was validated by protein identification from a membrane protein fraction of a human cell line, with two shotgun approaches, LC/ESI and LC/MALDI MS/MS techniques. The developed in-solution method was also compared with the conventional gel electrophoresis approach in terms of protein identification. It was found that the three techniques are complementary to each other and a preferred proteomics studies should include all of them. The in-solution method was further applied to the analysis of lipid raft proteins and was optimized with multi-dimensional separation technique to obtain a more comprehensive proteome map.