Proteomics Study On Ubiquitination Of Spermatogenesis

More and more couples suffer from the difficulty of infertility. The basis to solve this problem is to interpret molecular mechanism of gametogenesis. Spermatogenesis is a highly complex process of cell division and differentiation which starting from the differentiation of spermatogonial stem cells and ends with the formation of mature sperm. Any disorders of the processes will cause arrest of spermatogenesis and eventually lead to infertility. As with other protein modifications, ubiquitination is essential for regulation of spermatogenesis. A growing number of evidence shows that various components of ubiquitin system involved in the process of spermatogenesis. The purpose of this project is to identify important ubiquitylation proteins, ubiquitinating and deubiquitinating enzymes of spermatogenesis in mouse and construct platforms for protein identification and quantification of ubiquitylation. Our study will provide insight into molecular mechanism of spermatogenesis and candidate biomarkers for the diagnosis and therapy of related clinical diseases.First, we identified ubiquitylation proteins and ubiquitinating regulatry enzymes by without enrichment, and compared the two different two-dimensional separation technologies. Results showed that high-pH RP(HP-RP) had better performance in peptide separation than SCX, and peptides in different fractions were evenly distributed. Furthermore, HP-RP produced more information for protein identification, which improved the confidence of proteins. HP-RP technology is compatible with the low-pH RP separation for large-scale identification of proteins. We constructed the proteomic profile in mouse testis based on the HP-RP platform. A total of 8511 proteins and 88207 peptides were identified in one experiment. We performed analysis about ubiquitylated proteins based on the proteomic profile of mouse testis and the result displayed that only 109 ubiquitylation sites on 76 proteins were found. And 274 ubiquitinating enzymes(E1, E2 and E3) and 59 deubiquitinating enzymes(DUBs) were included in our proteomic profile. Expression of large number of these regulatory enzymes indicates that ubiquitination modification may have a complex regulation in spermatogenesis.Therefore, we established and optimized the platform for large-scale identification of ubiquitination sites based on capturing K-GG peptides and successfully applied to investigate the endogenous ubiquitylation sites in mouse testis. Compared to data produced without enrichment, this method could significantly improve the efficiency of ubiquitylation peptides detected by mass spectrometry. Using this monoclonal antibody, we identified 13717 ubiquitylation sites corresponding to 4200 substrates in two biological duplicates. Bioinformatics analysis revealed that ubiquitylated proteins were involved in almost all of the biological processes such as RNA transport, metabolic pathways, ubiquitin mediated proteolysis, DNA repair, immune response, endocytosis, cell cycle and apoptosis. These results suggest that protein ubiquitination may be important for spermatogenesis.Finally, we investigated methods of quantificative proteomics. Two quantitation methods were studied, including TMT and 18 O labeling. Using TMT technology, we constructed a comparative proteomic profile related to SSC proliferation, migration and differentiation and validated the differentially expressed proteins by western_blot. Furthermore, we investigated the labeling efficiency and quantification accuracy of 18 O labeling, and performed quantitative analysis on ubiquitination sites combining of 18 O labeling and ubiquitylation enrichment, which may provide a basis for future quantification of ubiquitination proteomics in testis.Take together, the identification and analysis of expressed proteins and ubiquitylated proteins in mouse testis suggested that ubiquitination of proteins played an important role in spermatogenesis. Functional annotations based on bioinformatics showed possible important functions of ubiquitylated proteins, which may be rich resources for studying the mechanism in spermatogenesis regulated by ubiquitination. Furthermore, optimizing methods for quantification of in vivo ubiquitination will help identify key ubiquitylated proteins in spermatogenesis.