Phosphoproteomics Identification And Protein Interaction Network Analysis Of Boar Sperm

To become fertilization competent, mammalian spermatozoa mustundergo a series of molecular event of physiology and biochemicaltransformations in the female reproductive tract termed capacitation. Maturespermatozoa are mostly highly compartmentalized, transcriptionally inactiveand unable to synthesize a new protein. Phosphorylation, as one of the mostwidespread post-translational modification, is very important for thefertilization process, such as sperm capacitation, hyperactivated motility andacrosome reaction. Therefore more and more reseachers pay attention to themammalian research. So far, there’s no such report about boar spermphosphoproteome. Using two-dimensional electrophoresis, western blotting,protein immunoprecipitation, phosphopeptide enrichment and LTQ-MS/MSMS identification technology, we separate and identify proteinphosphorylation and phosphorylation sites of boar fresh and capacitatedspermatozoa. The reseach is vital to find out the important biomarkers offertilization and get further understanding of the molecular mechanism ofsperm capacitation process. Using bioinformatics research methods we construct the phosphorylation proteins regulatory networks of boar spermand predict biological functions of the key phosphorylation protein. Thisresult will provide scientific basis fordeveloping the importantphosphorylated proteins signal molecular regulatory pathways ofmammalian sperm and the study of molecular mechanism of fertilization.This paper includes three parts:First, employing the two-dimensional gel electrophoresis (TD), proteinwesternblotting (TD-WB) and (MALDI-TOF-MS) mass spectrometrycombined technique we identify the protein substrates of tyrosinephosphorylation of boar sperm during capacitation. Exploring the conditionof protein extracting, two-dimensional gel electrophoresis isoelectricfocusing and concentration of antibody, we establish the method model.Using this method model we have identified11tyrosine phosphorylationproteins. Acrosin binding protein (ACRBP), Voltage-dependent anionchannel2(VDAC2) and Tubulin alpha-1C (TUBAC) have been reportedin mouse and human sperm. And8tyrosine phosphorylation of the proteinsin mammalian sperm are newly identified, such as Tropomyosin3(TPM1)and Whirlin.Second, using antibody (α-PY) immuneprecipitation we enrichedtyrosine phosphorylated proteins of capacitation boar sperm. With tandemmass spectrometry (LC-MS/MS) identification, a total of120pigcapacitation sperm tyrosine phosphorylated proteins were discovered. Thefunctional classifications of30kinds of known proteins show thatmitochondrial proteins, enzymes, sperm-egg fusion proteins and cytoskeletalproteins account for high proportion. Mitochondrial proteins are themitochondrial inner membrane transporter family(MSCF), involved in metabolites transmembrane transportation; most enzymes are phosphataseand glucose metabolism related enzymes; sperm egg fusion proteins wereprimarily involved in sperm-egg recognition, binding, fusion, adhesion otherassociated proteins; the other cell cytoskeletal proteins such astubulin(TUBB2A), outer dense fiber protein (ODF2), a kinase-anchoringprotein (AKAP-3, AKAP-4)were associated with sperm flagellar motility.In order to identify the tyrosine phosphorylation sites, we use TiO2enrichment technology to isolate phosphorylated peptide, then using themass spectrometry (LTQ-MS/MS) for detection, a total of9tyrosinephosphorylation sites were idendified. The identification results show that aspecial nuclear protein LRRFIP1, containing the coiled-coil domain, is aclassic Wnt signaling pathway activation factor and a critical molecule forthe signal transduction.Third, the TiO2enrichment technology of boar capacitation spermphosphorylated peptides and high precision LTQ-MS/MS MS identificationtechnology were used for phosphoproteome analysis of capacitatated boarspermatozoa.375phosphorylated proteins,612phosphorylated peptide and1067phosphorylation sites (775Ser sites,207Thr sites,85Tyr sites),Ser/Thr/Tyr phosphorylation distribution ratio of72.6:19.4:8.0(%)waspresent. The statistics show that the phosphorylation proteins with bindingand metabolic are the large proportions. Using the phosphoproteomicsanalysis combined with signal transduction research strategy wesuccessfully established and improved the level of boar spermphosphorylated proteins identification technology platform. A new batch ofphosphorylated proteins are found and a boar sperm phosphorylated peptidelibrary is built. Through the interactions between proteins of sperm motility and enzyme activity, two signaling networks are created. Both of them arecovered with100kinds of proteins. These two diagrams provided aadvantageous way to find the new key involved protein in the signaltransduction pathway of sperm capacitation process. This study is the first toprovide the phosphoproteome data of boar capacitation sperm and is thefoundation of signal pathway research of capacitation process in mammaliansperm.