Differential Analysis On Two-Dimensional Electrophresis Pattern Of Seminal Plasma Protein Of Varicocele Patients

Objective:(1)To establish and optimize the Two-dimensional polyacrylamide gel electrophoresis/ mass spectrometry technique for human seminal plasma proteomics(.2)Separated seminal plasma protein of varicocele patients and normal person with 2-DE,analyze and compared the maps to find out the significative discrepant proteins with PDQuest software.(3)Identified these proteins with MALDI-TOF-MS and search for the peptide mass fingerprints(PMFs),confirmed the disease-specific proteins(DSPs)of providing evidence for further exploring the molecular mechanism of infertility caused by varicocele.Methods:(1)Total soluble proteins of seminal plasma were extracted from five varicocele patients and five normal person,five postoperative patients.All samples harvest though the masturbation.(2)For purify and concentrate the sample proteins,spallation and precipitation have been executed.(3)Immobilized pH gradient(IPG)gels and even SDS polyacrylamide gels were selected for the electrophoresis,the gels were stained by silver nitrate after the electrophoresis were completed.Experimental conditions were adjusted to find out the best one for seminal plasma(.4)scanned 2-DE gels and analysis the protein spots in the 2-DE maps by PDQuest software to find out the significant discrepant protein spots.(5)Discrepant protein spots were extracted and been enzymolysis and progressing mass spectrogram identification , then according the results of PMFs to find the discrepant proteins by searching Internet using MS-Fit and MASCOT from NCBI database.Results:(1)By IPG(pH4-7)running isoelectric focusing in the first dimension and casting 12% SDS even gel in the second dimension,2-DE maps of seminal plasma is obtained,and repeatability of the maps is more satisfactied than which is obtained by IPG(pH3-10).(2)Comparing with different samples obtained on the maps,we think 300μg samples are the most appropriate for the 17cm IPG gel.(3)Compared the profiles of two groups , we found 12 significative discrepant protein spots through MALDI-TOF-MS,which 3 of them were homo-expression and 9 of them were hyp-expression in varicocele patients'seminal plasma.Conclusion:The male infertility might due to the expression and modification of some proteins in seminal plasma of varicocele patient.Further analysis on the different proteins' expression helps revealing molecule mechanism of varicocele causing male infertility.