Shotgun Proteomics-based distinctive fingerprint of buffalo sperm surface unravels over-representation of proteins driving spermatogenesis, motility, and fertility

The mammalian sperm surface is radically modified in its biomolecular composition and structure during the transit of sperm through the male and female reproductive tract (MRT, FRT). Numerous distinct secretagogues such as (glyco)proteins and many GPI-anchored proteins (GPI-APs) are applied as peripheral coats on the sperm surface in the MRT, which is critical for the maturation, survival and function of the spermatozoa. This study aimed to characterize the buffalo sperm surface proteins by extracting them either by using elevated salt (NaCl) or Phosphatidylinositol-specific phospholipase-C (for GPI-APs) or by inducing in vitro capacitation. A buffalo sperm surface-specific proteomic fingerprint was subsequently generated using the protein informatics platform, Proteome Discoverer (v2.2) for protein identification. Overall, 1695 unique proteins isoforms (proteoforms) (≥ 1 high-quality PSM/s, minimum 2 peptides, P < 0.05, FDR < 0.01) that exhibited remarkable heterogeneity in their molecular weight, pI, distribution of expression from the genome and their functional roles in the MRT and the FRT were identified. We characterized the identified including 200 orphan proteins by extrapolation of function from their sequence orthologs using BLAST2GO software. The extensive, yet unexplored, reproduction-specific buffalo sperm-surface proteomic repertoire discovered in this study is unparalleled vis-à-vis the depth identification of fertility-related and reproduction-specific cell-surface proteins.