In the exploration of complex biological systems, integrated multi-omics analysis has emerged as a pivotal strategy for deciphering molecular interaction networks. However, traditional stepwise extraction methods are prone to technical biases introduced by tissue spatial heterogeneity and repeated processing, limiting the accuracy and comparability of results. Although existing co-extraction techniques enable efficient and sequential isolation of nucleic acids (DNA/RNA) and proteins from the same sample, a significant methodological gap remains in systematically evaluating their performance in integrated multi-omics studies. Addressing this critical issue, this study thoroughly investigated the performance of a co-extraction strategy (AllPrep) in multi-omics research using spatially heterogeneous mouse brain tissues.