Dynamic Atlas and Functional Regulatory Network of the Whole-Brain Proteome Across 24-Hour Temporal States in Vespertilio sinensis

We performed directDIA-based quantitative proteomics on whole-brain tissues of <i>Vespertilio sinensis</i> across four distinct 24-hour physiological states: Rest, Sleep, Wake, and Activity. Among the 7652 identified proteins, a total of 643 differentially expressed proteins (DEPs) were screened via pairwise comparisons across timepoints.

Time-series clustering further resolved two statistically significant temporal expression modules (Module 3 and Module 9). Combined with functional enrichment of DEPs and phase set enrichment analysis (PSEA) of 574 rhythmic proteins, our multi-layered omics results collectively uncovered stage-specific molecular adaptive patterns. The Active state upregulated oxidative phosphorylation and thermogenesis for high energy demands, the Rest state activated immune clearance and autophagy to eliminate flight-induced metabolic damage, the Sleep state suppressed global transcription, calcium signaling and DNA repair to reduce neural energy consumption, and the Wake state (pre-dusk) pre-activated sulfur biosynthesis, antioxidant defense, and energy metabolic pathways to prepare for upcoming nocturnal activity.