| Objective:①To Compare the differences of the substantia nigra and striatal protein between the animal model of Parkinson's disease which was long-term injected low dose 1-Methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) and control mice by proteomics technology and to select biomarkers to diagnose PD.②To cut and digest some of the different proteins by enzyme and to identify these proteins with MAILD-TOF-MS. To confirm the disease-specific proteins (DPs) of PD by the data searched.Methods:①This investigation adopted the mouse(C57BL) model of chronic parkinsonism which was induced by low dose MPTP injection. Set the test group which was received bake hypodermic injections of MPTP and the control group which was received bake hypodermic injections of physiology saline. Observed at different periods of time if there were parkinsonism symptoms on the test group after injection. Behavioral methods were used to determine the success or failure of the model.②Extracted protein from the treatment group and NC group mice's striatum and substantia nigra organizations.③Pured total protein with TCA/acetone or not.④Used them to run immobilized pH gradient isoelectic focusing (IPG-IEF), and then vertical flat sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).⑤The protein spots in gels were visualized by silver staining protocol.⑥Scanned gels and analyzed the images. Searched for the differentially displayed protein spots by comparing two groups of gel images.⑦Identified some of the differentially displayed protein spots with MS (mass spectrometry) followed digested with enzyme.Results:①Behavior observation results: compared with the control group, the pole climbing time of the test group was obviously lasting(p<0.05).The group treated with MPTP displayed markedly hypoactive behavior and the control group didn't display parkinsonism behavior.②Through the comparison of the substantia nigra 2-DE mappings(pH3-10 and pH4-7) of mouse between the PD and control groups, the resolution of 2-DE mappings with pH4-7 IPG DryStrip was better than those with pH3-10 IPG DryStrip.③Comparing two protein extraction methods by 2-DE mappings, we found the group with not any treatment was better.④Through the analysis of protein spots group with PDQuest8.0 analysis software, there were 812±21 protein spots on the substantia nigra 2-DE images of PD mouse in the range of pH4-7, and 775±38 on the controls. 18 protein spots were hyp-expression and 13 protein spots were homo-expression on the substantia nigra 2-DE mappings of PD by comparing the mappings of two groups,two protein spots were new. There were 912±21 protein spots on the striatum 2-DE images of PD patients in the range of pH3-10, and 875±38 on the controls. 6 protein spots were hyp-expression and 4 protein spots were homo-expression on the striatum 2-DE mappings of PD by comparing the mappings of two groups, two protein spots were new.⑤Through the MS identifition, the homo-expression proteins spot in substantia nigra wereα-enolase. The new protein spots were tumor necrosis factor ligand superfamily member 4 and cyclin-dependent kinase inhibitor 1B. The homo-expression protein spots in striatum were mitochondrial fission regulator 1 and Protein S100-A10. The new protein spots were Ubiquitin-like protein 3 precursor and Lin-7 homolog B.Conclusion: MPTP chronic parkinsonism model was able to represent natural couse of Parkinson's disease. The ideal substantia nigra 2-DE mappings could be obtained with pH4-7 IPG DryStrip, running IPG-IEF and then vertical flat SDS-PAGE.There were different protein expressions on the substantia nigra and striatum 2-DE mappings in PD and controls, some protein spots were homo-expression and others hyp-expression. Identifying and learning the function messages of them could establish foundation to further study the function of them in the pathogenesy and progression of PD.
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