Comprehensive Evaluation of Coextraction Workflows for Enhanced Multiomics Integration in Complex Biological Samples.

Integrative multiomics analysis offers valuable insights into complex biological systems, yet conventional stepwise extraction methods are often limited by tissue spatial heterogeneity and technical biases introduced through repeated processing, which may compromise cross-omics comparability. To address this, we evaluated a coextraction strategy for multiomics profiling of mouse brain tissue. This approach increased RNA yield per milligram of tissue by 32.55% compared with traditional methods while maintaining comparable sequence coverage (88.14%) and reproducibility (r > 0.98). At the proteome level, approximately 7100 proteins were identified, comparable to conventional protocols, with consistent representation of neural functional protein categories, including membrane proteins, kinases, ubiquitin ligase complexes, and transcription factors. Phosphoproteomic analysis revealed increased coverage with 4347 additional high-confidence phosphosites identified, enabling enhanced resolution of regulatory signaling pathways. Integrated multiomics analysis further showed higher RNA-protein correlations (median r value from 0.25 to 0.56) and strengthened enrichment of neural pathways such as synaptic transmission and nervous system development. Overall, this coextraction strategy provides a practical workflow for multiomics integration and may facilitate studies of complex molecular regulation in brain systems.