Development Of New Proteomics Methods And Their Applications To The Analysis Of Membrane Proteome

Proteomics is dependent on three major technologies:protein separation technology, protein identification technology and bioinformatics technology that analyzes and predicts the structure and function of identified proteins. The development of proteomics is not only impulsed by technology, but also limited by it. The achievement of proteomics researches depends on the methodology to a great extent. Therefore, development of newer and more effective methods for proteins analysis is still one of the main tasks in proteomics studies now and in a rather long period of time in the future. In the present thesis, we have made a series of exploratory researches on the methodology aiming at the analysis of membrane proteome.Membranes are critical components of cellular structure and play important roles in partitioning of organelles, providing defense against foreign molecules and external conditions that may damage or destroy the cell and so on. The functions of membranes are essentially carried out by membrane proteins. It has been reported that membrane proteins constitute roughly 30% of cellular proteins. They are integrated into lipid bilayer or associated with lipid membranes. More than half of all membrane proteins are predicted to be pharmacological targets. Of the membrane proteins, plasma membrane (PM) proteins are of particular importance because they act as "doorbells" and "doorways" playing a crucial role in the fundamental biological process of the cell, including the exchange of the material and energy between the cell and its environment, cell-cell interactions, and signal transport. However, despite the biological importance of membrane proteins, their analysis has lagged behind that of soluble proteins and still presents a great challenge mainly because of their high hydrophobic nature and low abundance. Poor solubility of most of membrane proteins in water or aqueous buffers has limited their solubilization, extraction and enzymolysis. Therefore, how to prepare membrane proteins for mass spectrometry analysis is a subject worthy of investigation.Sodium deoxycholate (SDC) is originally a native strong ionic detergent and is found in mammalian bile at high concentrations. Our previous work demonstrated that SDC was compatible with the activity of trypsin based on MALDI-TOF MS analysis, and, compared with SDS, SDC could more effectively enhance the reliability of membrane protein identification. In this study, the application of SDC to the solubilization, tryptic digestion and identification of proteolytically resistant myoglobin and integral membrane proteins were systematically investigated. The results showed that little decrease in the trypsin activity was observed in 1% SDC solution,2-5% SDC only decreased the enzyme activity by about 13.6%, and even in the presence of 10% SDC, trypsin still retained 77.4% of its activity. SDC was removed from sample solution with acid treatment followed by centrifugation, and MALDI-TOF MS analysis showed that the remaining SDC, if any, could hardly make interference with MS analysis in terms of the number and S/N of ions in the mass spectra. Compared with urea and methanol, another two commonly used additives besides SDS in proteomic analysis, SDC improved more efficiently the denaturation, solubilization and tryptic digestion of proteins particularly proteolytically resistant compact myoglobin and highly hydrophobic integral membrane proteins, thereby enhancing the efficiency of their identification in terms of the number of identified proteins and unique peptides and the sequence coverage of matched proteins, which demonstrated that SDC had an attractive prospect in shotgun analysis of membrane proteome.Unlike many other detergents, SDC does not interfere with subsequent LC-MS/MS analysis because it can be easily removed from the sample solution by acidification followed by centrifugation (i.e. acid precipitation, AP) or by extraction with organic solvents such as ethyl acetate (i.e. phase transfer, PT). For further evaluating the application value of SDC in membrane proteome analysis, the effects of the two strategies for SDC removal on the tryptic peptide recovery and protein identification were comparatively studied. The results of the study demonstrated that both of the AP and PT strategies led to a certain amounts of tryptic peptides to be lost, and in PT strategy even more peptides were lost during SDC removal process. However, the lost peptides could be mostly recovered by washing the pellet and solid content produced during acid precipitation and phase transfer, respectively. By recovery of the lost peptides, the identification efficiency of membrane proteins especially transmembrane proteins and low-abundance proteins was significantly improved. Comparatively, after optimized by recovery of lost peptides, AP strategy was superior over PT strategy because the former not only could achieve better identification efficiency than the latter, but also was more economical, safer and easier to operation than the latter.Our previous studies had demonstrated that SDC was suited for shotgun analysis of membrane proteome. However, in our experiments we also found it had limitations in solubilizing membranes and extracting membrane proteins, because there was always a little of residual membranes left after solubilization with SDC. The reason is that SDC is a steroidal compound, having a polar face and a nonpolar face rather than a typical linear head and tail structure. This face amphipathicity of SDC leads to certain limitations in the solubilization, extraction and denaturation of membrane proteins. Hence, it is a significant work to seek a more suitable detergent that not only has a strong ability to extract and solubilize membrane proteins, but also is compatible with enzyme activity and mass spectrometric analysis. Starting with the comparatively analyzing the structures of SDS and SDC, we have made a series of detergent screenings and speculated that sodium laurate (SL) should have strong potential to improve the shotgun analysis of membrane proteome, because it has a typical linear head and tail structure with a long hydrophobic hydrocarbon chain similar to that of SDS and a hydrophilic head (a carboxyl group) similar to that of SDC, which is favourable for the removal of it from the sample by acidification before LC-MS/MS. Through a series of comparative studies, we found that SL not only had strong ability to solubilize membranes and extract membrane proteins, but also could enhance enzyme activity when its concentration was 0.1%. Furthermore, it was found that SL could be efficiently removed by phase transfer method from sample, thus ensuring the efficiency of MS analysis of peptides. Finally, SL was applied to the digestion and identification of standard membrane protein and the proteins in rat liver plasma membrane-enriched sample, and results showed that, compared with ALS- and SDC-assisted methods, SL-assisted method was more suitable for the analysis and identification of membrane proteins particularly those with strong hydrophobicity and multiple transmembrane domains.Because majority of current proteomic strategies have limitations in the direct analysis of intact proteins, solubilization of proteins and generation of detectable peptides are crucial to proteins identification. Although in-gel enzymatic digestion of proteins following gel electrophoresis is most commonly used in the proteomic researches, problems with incomplete protein digestion and low peptide recovery are common, especially for hydrophobic membrane proteins, which would affect the achievement ratio and confidence of proteins identification. Here, we present a new method for efficient protein digestion and tryptic peptide recovery, which involved electroblotting gel-separated proteins onto a PVDF membrane, excising the PVDF bands containing protein of interest, and dissolving the bands with pure DMF (>99.8%). Before tryptic digestion, NH4HCO3 buffer was added to moderately adjust the DMF concentration (to 40%) in order for trypsin to exert its activity. Experimental results using protein standards showed that, due to actions of DMF in dissolving PVDF membrane and the membrane-bound substances, the proteins were virtually in-solution digested in DMF-containing buffer. This protocol allowed more efficient digestion and peptide recovery, thereby increasing the sequence coverage and the confidence of protein identification. The comparative study using rat hippocampal membrane-enriched sample showed that the method was superior to the reported on-membrane tryptic digestion for further protein identification, including low abundant and/or highly hydrophobic membrane proteins.