The Methodology Of Plasma Membrane Proteomics Studise Of Rat Liver

Based on rat liver plasma membrane of the study is for thepurpose of optimizing the extraction, the purification as wellas the identification of membrane proteins. The two main areasof research: (1) purified rat liver plasma membrane proteomicsmethod research; (2) compare and optimization of stainingmethods in liver plasma membrane proteome analysis.First, we isolated plasma membrane from rat liver by twosteps of sucrose density gradient centrifugation. We usescanning electron microscopy to detect purity of the plasmamembrane, with 1DE, 2DE separation membrane proteins, thenanalyzes maps with PDQuest software. We identify 82 differentproteins, then digestion and mass spectrometry analysis, thefinal identification of a total of 56 proteins. Of them, 43 arein mapping software PDQuest defined as a 2-fold difference inpoints. Locationing information from the protein can be seenwith the 35 points difference is localized in the plasma Thereare 7 significantly reduce the difference points, including 5of mitochondrial proteins and two for peroxisome proteins. Inaddition 13 no significant differences in protein locationinformation is not clear, conjecture and ether subcellularorganelle membrane for a total of positioning proteins.Also using several new methods(two-phase, high salinity andhigh pH aqueous two-phase combination, aqueous two-phasecombination of sucrose density gradient) of the plasma membrane protein concentration. The results show : high salt with a highpH aqueous two-phase, and the two-phase combination of sucrosedensity gradient methods can better integrate enrichmentmembrane proteins, of which two-phase combination of sucrosedensity gradient method is better than the high-salt with a highpH aqueous two-phase treatment. The purified plasma membraneproteins be extracted and were separated by SDS-PAGE anddigested with trypsin. The digest peptides were identified byESI-Q-TOF MS/MS. The results show two-phase and sucrose densitygradient centrifugation as two different plasma membraneextraction and purification methods could combined, and willnot affect one another, but can be further purified plasmamembrane better.In order to improve the cell membrane purity, the study alsoexplored the use of a new affinity purification method. C57plasma membrane was immunoisolated by second antibodysuperparamagnetic beads. The plasma membrane of mouse liver wasenriched about 3-fold in comparison with the density gradientcentrifugation method, and contamination from mitochondrionwas reduced 2-fold. Meanwhile, four methods of immunoaffinitywere compared and the optimized quantities of sample, beads andantibodies suitable for proteome analysis were obtained. Theresult indicated that the ratio of plasma membran or plasmamembrane-related proteins was much higher than that obtained using the routine methods.In the second research, five kinds of staining methods werecompared through liver plasma membrane proteomics. Withcomparing, it was found that the accuracy of Sypro Rubystainings higher than two silver stainings, of which fastsilver staining(Blum silver staining) is better than generalsilver staining(PlusOne^TMsilver staining).The effect of G-250staining is better than that of R-350, with quick speed and lowbackground. Fast silver staining following coomassie bluestaining can not only improve the accuracy of proteinidentification, but also identify more low-abundance proteins.