4D-DIA proteomics unveils the dual engines of human sperm capacitation

The systemic molecular regulatory landscape of sperm capacitation remains incompletely understood. This study utilized Four-dimensional independent data acquisition (4D-DIA) high-throughput quantitative proteomics to systematically analyze the global proteomic changes in healthy human sperm before and after in vitro capacitation. A total of 5812 quantifiable proteins were identified, of which 201 were significantly differentially expressed (Fold Change > 1.5, P < 0.05). Functional analysis revealed that upregulated proteins were highly enriched in core energy metabolism pathways, such as oxidative phosphorylation and the mitochondrial respiratory chain, confirming the driving role of an "energy burst" in capacitation. Furthermore, processes including RNA processing and translation, as well as post-translational modifications, were significantly activated, and the interaction network demonstrated a tight coupling between energy metabolism and translational machinery components. Concurrently, this study identified a group of potential novel regulators (e.g., TMEM89, TEX29) with high testis-specific expression that are upregulated post-capacitation. This study provides the first systematic mapping of the multidimensional proteomic dynamics during sperm capacitation, offering novel molecular biomarkers and potential targets for the etiological diagnosis of male infertility and assisted reproductive interventions.