Proteomic Analysis Of Novel Post-translational Modifications In Human And Mouse Testes

With the development of proteomics, proteomes of numerouskinds of tissue and cell have been mapped, producing numerous proteomics raw data. However,these data might not be sufficiently analyzed and utilized,leading to the accumulation and waste of data. Thus, integration and reanalysis of these data is urging, and may discover novel ideas that have been missed. For some modifications, like glycosylation, acetylation, ubiquitination and phosphorylation, efficient enrichment and identification methods have been developed to analyze them. And quite a few studies have been conducted to uncover their roles in male reproduction. Others, like palmitoylation, mono-ADP ribosylation and crontonylation, have not been systemically studied in male reproduction. However, Palmitoylation regulate many biological processes such as signal transduction and apoptosis. Besides, it has been reported to regulate the function of Hedgehog(Hh) in embryonic development, testicular development and spermatogenesis. ADP ribosylation plays an important role in DNA replication and repair, genome stability, and cell cycle regulation. It has been reported to regulate the function of ATM and ATR in spermatogenesis. Moreover, crontonylation has a role in histone replacement and Meiotic Sex Chromatin Inactivation(MSCI). Thus, they might have a significant role in male reproduction, and identification of these three modifications in male reproductive system would provide molecular basis for further study their functions in spermatogenesis and sperm function.To uncover their functions in male reproduction, we applied MaxQuant software to reprocess the raw MS dataof human testis and mouse testis proteomics published by our lab, and identify palmitoylated, mono-ADP ribosylated and crontonylated sites and proteins. Then we applied bioinformatics analysis to systematically annotate the functions specific to male reproduction of the modified proteins and provide a data resource for further study of these modifications. Most importantly, this study can discover novel research directions from raw MS data and provide new ideas for integration and recycling of accumulated proteomic data in the further.In human testis, we identified 122 palmitoylated sites from 85 proteins, and 107 crontonylated sites from 55 proteins. Sabrina Laing et al.,(2011) have proved that the enrichment method of phosphopeptides is also available for the enrichment of ADP-ribosylated peptides. Thus, we applied the phosphoproteomics of adult mouse testis and identified 216 mono-ADP ribosylated sites from 166 proteins.Then we analyzed these identified proteins with bioinformatics analysis, such as Gene Ontology annotation, KEGG pathway analysis, phenotype annotationspecific tomale reproduction, to uncover their potential function in male reproduction. For ADP-ribosylated proteins, GO annotation revealed significant enrichment in several Biological Processes, including “Phospholipase activity " and " lipase activity”, and Cellular Component, including " Cytoskeleton" and "Nucleus part". Pathway studio revealed that ADP-ribosylated proteins were associated with “Spermatogenesis”, “Spermatid development”, “DNArecombination”, “The assembly of cytoskeleton”, etc. For palmitoylated proteins, GO annotation revealed significant enrichment in "intermediate filament", "keratin filament" and " NF-kappaB complex". Pathway studio revealed that they were related to “infertility”,“cell cycle” and “DNA recombination”. Moreover, Pathway studio revealed that crontonylated proteins were related to “meiosis”, “DNA recombination” and “cell cycle”.According to these results, we uncovered that ADP ribosylation might regulate the function of targeted proteins and influence several key events in spermatogenesis, including the proliferation of SSCs and spermatogonia, DSBs repair and spermiogenesis. Palmitoylation might regulate the structure of BTB, intracellular bridge and apical ES through regulation the transport and localization of proteins. Crontonylation might have a role in the regulation of meiotic cell cycle and the dynamic renewal of BTB.