A New Phosphorylated Protein Analysis Strategy based on Trypsin Encapsulated in Metal-Organic Frameworks with High Efficiency and a Simplified Workflow.

Phosphoproteomics research is crucial for clinical diagnosis. However, due to the self-hydrolysis of natural proteases and the complex typical pretreatment protocol, the traditional bottom-up method is not enough to achieve rapid analysis of phosphorylated proteins. In this work, we encapsulate trypsin (Try) in the ZIF-L(Co) to develop a new strategy that simplifies the phosphorylated protein analysis process and achieves rapid analysis. Try is encapsulated in the mesoporous ZIF-L(Co) to allow the proteins to be accessible to the enzymes. The hydrophobic ZIF-L(Co) can cause the unfolding of proteins and accelerates the digestion process. The Co(II) nodes enhance the affinity toward phosphorylated proteins and capture phosphopeptides selectively. Compared to the traditional denaturation, digestion, and enrichment method, which costs 20 h at least, our strategy simplifies the pretreatment workflow and yields phosphopeptides in just 3.4 h. This strategy is further applied in the analysis of phosphorylated proteins in biosamples such as nonfat milk, egg yolk, and human serum. The results show equivalent performance with the traditional method and exhibit great potential in bioanalysis. This new phosphorylated protein analysis strategy provides a powerful tool for proteomics analysis and promotes research in the field of biomedicine.