ITRAQ-based Quantitative Proteomic Analysis Of Mouse Epididymal Luminal Proteins

ObjectiveAccording to the report of WHO(World Health Organization),infertility affects 10-15% of reproductive-aged couples worldwide,and half of the cases are due to male factors.Many factors contribute to male infertility,such as impaired spermatogenesis in the testes,and sperm maturational disorders in the epididymis.The luminal proteins of eoidiymis are essential for sperm maturation,but the protein compositions are still obscure,which seriously impact the studies on sperm maturation and male infertility.Thus,identifying the luminal proteins and the differentially enriched proteins between the caput and cauda is the main object of this study.MethodsFor the first time,isobaric tag for relative and absolute quantification(iTRAQ)coupled 2D-LC-MS/MS approach was applied to uncover proteins that were differentially enriched in the mouse epididymal fluid of caput and cauda.Biological information analysis was performed on the differentially abundant proteins through GO annotations,KEGG pathway and protein interaction.Two differentially enriched proteins were further studied by Western blotting and immunofluorescence.ResultFor the first time,as many as 2132 proteins were detected in the mouse epididymal fluids,395 proteins with dynamic expression were found,and 128 differentially enriched proteins were identified,of which,46 were caput fluid enriched and 82 were cauda enriched.GO annotations indicated that the cellular component of these proteins manly localized in the cytoplasm,membrane-bounded vesicles,extracellular exosome and extracellular membrane-bounded organelle.Molecular function and biological process of these proteins were very similar,binding activity and metabolism were the main functions enriched.Acyl and Mdh2 was the center of protein interaction networks in the caput and cauda enriched proteins,respectively.And the proteomic results were validated by Western blot and immunofluorescence.ConclusionsiTRAQ is a powerful method for the study of epididymal luminal proteins,compared with tradictional 2D-PAGE.We have first identified as many as 2132 proteins in the mouse epididymal fluid,largely increased the number of proteins have been identified.These results will provide the basic information for further exploring the mechanism of sperm maturation and male infertility.