Surface proteomics of Sahiwal spermatozoa: Unveiling key membrane proteins in fertilization and reproductive function.

Surface-associated proteins are critical plasma membrane entities (transporters, receptors, channels, and adhesion molecules) that manage cell-external communication; they are central in translationally silent mature sperm, where function relies on post-translational modifications. This study details the bovine (Sahiwal) sperm surface proteome using quantitative proteomics and targeted total membrane protein enrichment to identify key proteins associated with maturation, fertilization, and related reproductive processes. Proteomic profiling across biological replicates (n = 3 in each MP and TP) identified 2311 high-confidence proteins in the total protein (TP) fraction and 558 in the membrane-enriched protein (MP) fraction after stringent filtering (FDR ≤1%; combined q-value ≤0.01; at least one unique peptide; and proteins detected in at least 2 biological replicates). These, filtered protein sets were used for subsequent functional and comparative analyses. In silico topology and subcellular localisation (BUSCA, DeepLoc, TMHMM, WoLF PSORT) tools analysis identified 43 predicted surface-associated proteins in the MP fraction,and 116 in the TP fraction. Analysis of the surface landscape identified β-defensins, SPACA1, BSP-family members, ADAM-family members, and SLC family as prominent components involved in immune protection, adhesion, and fusion readiness. Functional annotation and protein-protein interaction mapping suggest a model of coordinated surface remodelling during maturation and capacitation. These findings identify potential biomarkers for bull fertility and underscore the requirement for targeted validation within bovine reproductive models.