| Studies on protein-protein interactions are essential for understanding protein functions and mechanisms of cell process. Current methodologies of proteomics suffer from significant deficiencies for the analysis of protein-protein interactions in large scale. In this thesis, we first established unique interaction proteomics strategy based on “Nondenaturing micro two-dimensional gel electrophoresis-grid gel cutting-quantiative liquid chromatograpgy tandem mass spectrometry” and applied it to analyze high-density lipoprotein of human plasma.High density lipoprotein(HDL) is a high polymorphism plasma protein containing different proteins and lipids compositions, which shows wide distribution in two dimensions(p I 4.6-5.3, Mw 70-500 k Da) on the human plasma pattern obtained by nondenaturing micro two-dimensional electrophoresis as studied by immunochemical staining [1] and MALDI-MS PMF [2]. So this group of proteins is a proper model to validate our proteomics strategy. The human plasma sample was separated by nondenaturing micro 2DE and the gel area(5 mm ′ 18 mm) that included high-density lipoprotein(HDL) was cut into 90 gel pieces with the size of 1 mm ′1 mm. Each gel piece was treated by in-gel digestion, peptide extraction, standard addition and analyzed by quantitative LC-MS/MS(Lable-free). Grid-cutting of the gel was employed to;(i) ensure total analysis of the proteins in the area,(ii) standardize the conditions of analysis by LC-MS/MS, overcome the light stain area which was neglectd by stained spots cutting.(iii) reconstruct the distribution patterns of nondenaturing protein from the quantity data. In total 154 proteins(keratin removed) were identified in the 90 gel pieces and each protein had the quantity information in all gel grids. We programmed Excel Visual Basic(VB) macros to draw the corresponding grid(5 columns, 18 rows), and the quantity distribution of each protein in the grid cutting area was reconstructed as a color density pattern(a native protein map). In total 154 native protein maps were obtained.The map of Apolipoprotein(Apo) A-I showed a wide apparent mass distribution which is similar to HDL as studied by immunochemical staining. This result matched with Apo A-I is the major apolipoprotein of HDL. Compared the maps of Apo A-I with another 153 proteins maps, 11 proteins showed maps of wide distribution that overloapped with the map of Apo A-I(p I 4.8-5.1, Mw 80-200 k Da). It has been reported in the literature that these 11 proteins are the protein composition of HDL. Furthermore, 20 minor proteins were found in the main distribution area of Apo A-I, 7 secreted proteins(In total 9 secreted proteins) have been reported to interact with HDL.The originality of this method is that grid cutting of the nondenaturing 2DE gel facilitates the access of quantitative LC-MS/MS, and analyzes protein-protein interactions for the first time by reconstructing nondenaturing protein quantitation maps and comparing maps. These results for the first time visualized the distribution of the component of human plasma HDL and associated proteins on the nondenaturing gel, it is significant for understanding the mechanisms of hyperlipidemia diseases which are related to the distribution and metabolic of HDL and drug discovery. Currently, the method has been successfully applied to predict interaction of eukaryotic proteins in large scale [3].Finally, we discussed a plasma proteins database of Sprague Dawley(SD) rats by nondenaturing micro 2DE-CBB stained gel spots cutting-quantitative LC-MS/MS analysis. This work reported the species and distribution of plasma proteins on nondernaturing 2DE gel for the first time, which is important for SD rats as animal model. |
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