Characterization of the 36kDa sperminogen by MALDI-MS and peptide sequence analyses in boar spermatozoa

Abstract

Thirty six kDa sperminogen from boar spermatozoa has been characterized by MALDI-MS and partial peptide sequence analyses. The purified 36 kDa sperminogen from the acid extracts of boar spermatozoa was subjected to in-gel digestion by trypsin. The cleaved peptides were then characterized for their mass map by MALDI-MS analysis. Among the peptides generated by trypsin-digestion, the best-resolved peptide by HPLC was subjected to amino acid sequence analysis. In addition, the gel-purified 36 kDa sperminogen was subjected to CNBr-digestion and then analyzed for their internal amino acid sequences. The mass map of 36 kDa sperminogen demonstrated that 4 peptides matched perfectly with the 9 registered peptides of the zona pellucida binding protein, Sp38 precursor. Furthermore, amino acid sequencing of the HPLC-separated peptide showed it has 82% homology with that of Sp38, which strongly implies that 36 kDa sperminogen is the same protein as Sp38. These results were substantiated by the resolved amino acid sequence from the peptides generated by CNBr-digestion, which showed 74% match with that of Sp38 precursor. In conclusion, the 36 kDa sperminogen could be a novel protease that is reported as the zona pellucida binding protein, Sp38. And although collectively called perminogen, the 36 kDa sperminogen may have different origin and different function from the 32 kDa sperminogen which was reported as a part of proacrosin/acrosin family.