Astral-DIA Proteomics Deciphers the Molecular Landscape of Human Spermatozoa: A Comprehensive Quantitative Proteomic Characterization of Sperm Function and Fertility Potential

Male infertility is fundamentally rooted in developmental defects of germ cells and associated molecular dysregulation, yet the underlying mechanisms remain poorly understood. Data-independent acquisition (DIA) has emerged as a powerful tool in discovery proteomics, enabling the identification of disease biomarkers, therapeutic targets, and molecular pathways with high precision and reproducibility. To analyze the aberrant regulatory events in human sperm proteins associated with male reproductive disorders in a high-throughput, reproducible, and reliable manner, we employed the Orbitrap Astral mass spectrometer, which independently operates Orbitrap Full Scan and Astral MS/MS to generate high-resolution full-scan spectra and high-quality secondary maps. Leveraging the principles of data-independent acquisition (DIA) technology, we constructed the most comprehensive human sperm proteomic expression profile reported to date, encompassing 10,464 proteins, 200,951 precursors, 156,670 modified peptides, and 147,879 peptides.