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A Blue Native-PAGE Method and Application for Separation of Membrane Protein Complexes

Author: LiuNvYing
Tutor: ChenPing
School: Hunan Normal University
Course: Biochemistry and Molecular Biology
Keywords: plasma membrane proteomics synaptic membrane protein complex Blue native-PAGE SDS-PAGE mass spectrometry advanced glycation endproducts(AGEs)
CLC: R341
Type: Master's thesis
Year: 2011
Downloads: 122
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Abstract

Proteins are the main bearer of cell biological functions. However, most of the biological functions of cells are performed by protein complexes rather than a single protein. One of the most important ways for proteins to exert their functions in vivo is to form protein complexes. Plasma membrane is the place of material exchange and information communication, involving in the joint action of many proteins to accomplish these important physiological functions. Therefore, plasma membrane proteins form complexes more easily, which has become the research focus of protein complexomics. Identification and characterization of protein complexes especially plasma membrane protein complexes is an important step toward an integrative view of protein-protein interaction networks. The main limiting factor for identifying protein complexes is the method to their separation. Blue native polyacrylamide gel electrophoresis (Blue native-PAGE or BN-PAGE) is a powerful and effective tool for separating membrane protein complexes. In this technology, membrane proteins and protein complexes are solubilized by a mild detergent, charged with Coomassie brilliant blue G-250 and separated natively in accordance with molecular weights. After protein complexes were separated by the first-dimension BN-PAGE, it is possible to run a second-dimension SDS-PAGE to separate each protein complex into its subunits. This method can be used to study compositions of various protein complexes in membrane samples, however, the application is very actually few in the research of protein complexes of synaptic plasma membrane. In our experiment, we established and optimized a BN-PAGE method to separate synaptic plasma membrane protein complexes and this method was successfully used to screen the altered synaptic proteins of cerebral cortex in the D-galactose (D-gal) induced C57 BL/6 mice. Recent researches indicated that advanced glycation endproducts (AGEs) induced by continuous injection of D-gal in rodent led to deterioration of learning and memory functions. Advanced glycation endproducts (AGEs) have been implicated in the development of neuropathy. The deterioration of learning and memory functions or pathological changes of brain during neuropathy might be associated with the altered expression of proteins in synapse. To evaluate AGEs-induced protein network alterations in synapse, blue native/SDS-PAGE proteomic methods were used to compare the synaptosome protein expression patterns of cerebral cortex between D-galactose (D-gal) induced and normal C57 BL/6 mice. First we purified synaptic plasma membrane by discontinuous sucrose density gradient centrifugation. Then two-dimensional Blue native/SDS-PAGE in combination with LC-MS/MS was applied to separate and identify synaptic plasma membrane protein complexes. We also performed differential proteomic analysis between control and D-gal induced mice. Apart from the known protein complexes, such as syntaxin1B and synaptotagmin-1, our analysis also led to find a potential new synaptic protein complex, namely rab3A and synaptotagmin-1. In total, the amount of 43 proteins were changed during the D-gal induced process. These significantly altered proteins were mainly involved in neurotransmission, energy metabolism and signal transduction. Also we verified differentially expressed proteins which we were interested in by Western-Blotting and the results were consistent with identification results derived from mass spectrometry. In addition, in vivo activities of malondialdehyde (MDA) and superoxide dismutase (SOD) were tested. The average level of SOD was significantly reduced in the D-gal induced mice, while the MDA level was increased. These results indicated that AGEs accumulation in the brain led to the generation of reactive oxygen species (ROS), damaged the energy metabolism and attenuated neurotransmission in synapse. Therefore, elucidating the protein changes underlying the AGEs accumulation will benefit our further understanding the mechanism of learning and memory impairments in the neuropathy.As demonstrated here, this study can provide some methodological reference for purification and separation of plasma membrane proteins. BN-PAGE is a powerful tool for the separation of membrane hydrophobic proteins in general. Combination of BN-PAGE and MS will allow us to analyze protein-protein interactions at the protein complex level. Our findings on advanced glycation endproducts induced changes of synaptosome proteins in C57 BL/6 mice should shed light on understanding the physiology and pathology of neurosynapse and neuropathy about it.

A Blue Native-PAGE Method and Application for Separation of Membrane Protein Complexes

Abstract

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