Mining public mass spectrometry data to map the bull sperm membrane-associated proteome

Abstract: The sperm plasma membrane is of the utmost importance to the structural integrity and fertilising ability of the spermatozoon. Without a functional membrane, a spermatozoon cannot be expected to survive the arduous journey from testes to oviduct, particularly when its journey is disrupted by assisted reproductive technology (ART). In this study, we endeavoured to characterise a more complete bull sperm membrane-associated proteome through reanalysing publicly available RAW mass spectrometer data files from three previously published studies. Individually, these studies have utilised differing software platforms and inclusion criteria for their identification of membrane-associated proteins. The reanalysis of these datasets with standardised search criteria against up-to-date databases has identified 742 proteins. Amongst the proteins identified, 378 overlap with those reported in earlier studies, whereas we describe 364 previously unreported bovine sperm membrane-associated proteins linked to key functions, including zona pellucida binding. Next, the bovine proteome was mapped to human homologues (humanised), with a 96.8% conversion rate, allowing us to perform a more advanced functional analysis of the identified membrane-associated proteins, highlighting roles in energy metabolism, sperm motility, actin polymerisation, and protein ubiquitination. This deeper understanding of the bull sperm membrane-associated proteome will inform biomarker discovery for evaluating bull fertility and guide strategies to preserve sperm integrity during storage and ART procedures. Lay summary: In this study, we combined and reanalysed publicly available data files from three previously published studies to generate an updated list of the proteins associated with the membrane of bull sperm cells. We identified 742 proteins in total, of which 364 were not reported previously in the original studies. Membrane proteins are important because they sit on the surface of the sperm and control how it recognises and communicates with the egg. Our functional analysis of these proteins revealed roles in sperm motility, energy production, and key fertilisation events, such as binding to the egg. This study highlights the value of depositing data in public repositories so that older datasets can be reanalysed as analytical tools improve. A greater understanding of sperm membrane-associated proteins will improve knowledge of bull fertility with important implications for breeding and livestock production industries.