Proteomic Analysis Of Mouse/rat Liver Plasma Membrane

Plasma membrane (PM) mediates the exchange of substance, energy and information between cell and its environment, as well as between cells themselves. PM proteins are of great importance in drug design. Liver is one of the most important organs in the body, displays the main digestive function for the metabolism. Therefore, the proteomic analysis of liver PM proteins is of outstanding significance.First, we isolated PM from mouse liver by sucrose density gradient centrifugation and optimized lysis buffer for 2DE. We found that the optimized lysis buffer containing 4% CHAPS and 1% NP-40 was able to extract more PM proteins. Several treatments such as chloroform/methanol, sodium carbonate and Triton X—114 were tested to enrich low abundant and strong hydrophobic membrane proteins. The result showed that chloroform/methanol and sodium carbonate treatments can enrich more integral PM proteins, and chloroform/methanol treatment is better than sodium carbonate. Through 1DE and 2DE combining Mass Spectrometry (MS), 175 non-redundant proteins were identified, of which four proteins may be related with limb-girdle muscular dystrophy 2B and so on.The identification of low abundant and strong hydrophobic membrane proteins is still the main challenge of membrane proteomics.In the second research, PM was purified from rat liver through twice sucrose density grade centrifugation. Judged by electron microscope and western blot, the PM obtained after two sucrose density centrifugations was about 6 times purer than one. PM was treated with sodium carbonate, chloroform/methanol (C/M) and Triton X-100. Then, the Triton X-100 treated PM was centrifugated to separate three micro-domain fractions. 457 non-redundant proteins were identified, of which 46% were from microdomain strategy, 23% were integral membrane proteins with one or more transmembrane domains (TM). 53 proteins involved in cell communication pathway. The result indicated that Na₂CO₃ treatment is better for the extracting of basic proteins, C/M better for proteins with high transmembrane domains (TMs), and microdomain strategy better for low copy proteins extraction.The purity of the PM is another main challenge. In the third research, C57 PM was immunoisolated by second antibody superparamagnetic beads. The PM of mouse liver was enriched about 3-fold in comparison with the density gradient centrifugation method, and contamination from mitochondrion was reduced 2-fold. Meanwhile, four methods of immunoaffinity were compared and the optimized quantities of sample, beads and antibodies suitable for proteome analysis were obtained. The result indicated that the ratio of PM or PM-related proteins was much higher than that obtained using the routine methods.