Sperm functions may be influenced by seminal plasma (SP) proteins with affinity to zinc ions. The aim of the study was to isolate and characterise zinc-binding proteins (ZnBPs) from stallion SP using proteomic tools. Zinc-binding proteins were isolated from the SP of eight stallions by zinc-affinity chromatography. They were analysed in the SDS-PAGE system, and peptide extracts were prepared. Samples of ZnBPs isolated from stallion SP were injected onto the nLC-1000 nanoflow HPLC system coupled via a nano-electrospray ion source to the Orbitrap Elite FTMS mass spectrometer (Thermo Fisher Scientific). Raw MS data were analysed using MaxQuant software with label-free quantification (LFQ). Protein sequences were obtained from the UniProt database. Forty-seven proteins in ZnBPs were annotated in a gene ontology (GO) analysis. The LFQ intensity analysis of individual proteins revealed that ZnBPs comprised mainly clusterin (CLU, 27%), serin protease inhibitor F1 (SERPINF1, 13%), actin, cytoplasmic 1 (ACTB, 9%), nucleobindin 2 (NUCB2, 8%) and polymeric immunoglobulin receptor (PIGR, 6%). This is the first proteomic study to analyse ZnBPs in stallion SP. The present findings show that ZnBPs in stallion SP could play an important role in the regulation of sperm function.

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