PBS- and urea-based dual extraction of Bombyx mori cocoon coat proteins: comparative analysis of yield, bioactivity and proteomic differences

To comprehensively characterize the bioactive components and proteomic composition of cocoon coat proteins, we compared phosphate-buffered saline (PBS) and 8 M urea as two extraction approaches for isolating proteins from the Bombyx mori cocoon coat, with particular emphasis on their effects on protein yield, bioactivity, and proteome composition. The results showed that the urea-based method produced nearly sixfold more total protein than PBS extraction; however, the PBS extract exhibited higher antioxidant activity in both the DPPH radical scavenging assay and the ABTS total antioxidant capacity assay, and it outperformed the urea extract in inhibiting the growth of two fungal strains. DIA-based proteomics identified 597 proteins across the two groups: the PBS group was enriched in immune defense–related sericins and protease inhibitors, whereas the urea group additionally yielded a substantial set of structural proteins and membrane-associated proteins. Further functional annotation, differential protein analysis, and structural biology validation indicated that the PBS system is more conducive to maintaining native protein conformations and preserving bioactivity, while the urea system is better suited for achieving broader proteome coverage. Moreover, integrating Western blotting with AlphaFold-based structure prediction revealed potential mechanisms underlying biases between proteomic quantification and functional activity, underscoring that extraction strategies should be selected according to study objectives and target proteins by balancing activity preservation against coverage depth. Overall, these findings provide methodological support and biological insights for functional interrogation and application-oriented development of silk proteins. The authors confirm that all data underlying the findings are fully available without restriction. The mass spectrometry proteomics data have been deposited to the iProx database (Integrated Proteome Resources, https://www.iprox.cn ) under the dataset identifier IPX0014663000.