Optimization Of Conditions On Proteome Analysis And The Comparative Proteome Study On Seminal Fluid Of Human Asthenospermia

Objective: To optimize conditions of analysis of proteome by MALDI-TOF-MS and investigate the differentially-expressed protein in seminal fluid between anthenospermia and healthy male, and obtain some informations about sterility reason that male was anthenospermia.Method: We selected some key research parameters to optimize the conditions of proteome analysis, such as analysis conditions of MALDI-TOF-MS, accuracy of MS data, search engine, database type and the main parameters of set of database. In search, data of BSA was used to establish standard model and the data of CAH2_BOVIN and Hsp70s as sample to test optimized parameters and we obtained relatively sutiable search conditions in analysis of proteome.In compared proteome of seminal fluid between anthenospermia and healthy male, we collected seminal fluid of anthenospermia and healthy male that confirmed by clinical test, and prepared sperm and seminal plasma follow standard experiment procedure in respecticely, the differentially-expressed protein to carry out enzymolysis process with trypsin after separated by 2-DE and detected by silver staining, and enzymolysised peptides mixture analysized with MALDI-TOF-MS, then put the data of PMF into search engine MS-Fit or MASCOT to get some informations of protein.Result: 1.We adopted data of BSA as research target, inspected the influence of analysis conditions and accuracy data of MS on research result of protein database when obtained PMF with MALDI-TOF-MS. 2.we optimized the bioinformatics annlysis conditions through PMF to identify proteins under data of CAH2_BOVIN was used to set up model and tested by data of human Hsp70s. 3. we set up the differentially-expressed proteins'2-DE map of sperm and seminal plasma between anthenosperm and healthy male by 2-DE. 4.After compared study on sperm protein between anthenospermia and healthy male, we found 27 proteins highly expressed in sperm of anthenospermia which weakly expressed or no existence in healthy male, in contrast, 37 proteins highly expressed in sperm of healthy male which weakly expressed or no existence in anthenospermia patients. Through PMF search with MALDI-TOF-MS, we gained 47 possible proteins. 5. In process of compared study on seminal plasma protein between anthenospermia and healthy male, we found 18 proteins highly expressed in sperm of anthenospermia which weakly expressed or no existence in healthy male, at the same time, 24 proteins highly expressed in sperm of healthy male which weakly expressed or no existence in anthenospermia patients, obtained 36 possible protein informations by PMF search, and confirmed EHD1, PSMA, KLC1 and CDC25A with western blot.Conclusion: The ionization of MALDI-TOF-MS was a process of matrix mediated and complicated solid and gaseous phase, the selection of analysis conditions had greatly effect on the posterior protein research, and suitable analysis conditions would bring better reliability to experiment result. In order to get the correct PMF, we must attach great importance to mass spectrum analysis and optimization of the conditions of choice, which including the control of mass spectrum accuracy, the select of laser intensity, the set of accelerating voltage, the choice of delay extraction time and number of laser strike and so on.The proteomics research was different from the classical protein investigate, which mainly through the obtained mass spectrum data compared with standard protein database to identify protein, if the obtained mass spectrum data existed error or the installation of parameters weren't rational under compared with database of protein, which should produce severe effect. Our investigate result show that the database of Swissprot is the best database for PMF research, and both use two research engines of MASCOT and MS-Fit to scan candidate proteins in aspect of protein research, which would avoid the leak and reduce the mistake probability of select candidate proteins, at the same time, we must optimize the set of missed cleavage and mass tolerance based on research request.Some functional sperm proteins of anthenospermia patient emerged up or dowm in aspect of protein transcription, synthesis, transform, transport and so on, the major of differentially-expressed proteins were active proteins with spermiogenesis and maturation process in seminal plasma , in meanwhile, we found some unknown functional proteins in sperm and seminal plasma, these results played ground for posterior research and created conditions for infertility study of anthenospermia from proteomics.