A High-Efficiency and Automated Strategy for Rapid Pretreatment and Analysis of Low-Molecular-Weight Proteins Based on Capillary-Immobilized Enzyme Microreactors.

Low-molecular-weight proteins (LWPs, <30 kDa) are crucial in the identification of tumor markers and disease diagnosis. However, current methods for analyzing LWPs typically involve complex workflows, high sample consumption, poor automation, and prolonged reaction times. In this study, immobilized enzyme microreactors (IMERs) were prepared by immobilizing trypsin encapsulated in zeolitic imidazolate frameworks (ZIF-L) within a capillary, which was then integrated with capillary electrophoresis (CE) to develop a novel strategy for the pretreatment and assay of LWPs. Compared to traditional LWP analysis protocols, which typically involved a three-step process of sequential separation, denaturation, and enzymatic digestion, the proposed strategy achieved the LWP analysis in a single step within just 4 min in a 10 μL sample, significantly reducing sample pretreatment procedures and analytical time while offering advantages such as high efficiency, rapid processing, automation, and low consumption. The prepared trypsin@ZIF-L@IMER exhibited superior activity, enhanced affinity, remarkable stability, and excellent reusability when compared with free enzyme. In addition, the trypsin@ZIF-L@IMER demonstrated high selectivity toward LWPs after the pretreatment of single, binary, quaternary, and septenary model proteins. Additionally, the development method demonstrated ultrahigh sensitivity (0.05 nM cytochrome c (CYC)) and excellent anti-interference capability (CYC/bovine serum albumin (BSA) = 1:1000), outperforming previously reported methods. Finally, the developed strategy based on trypsin@ZIF-L@IMER was used for LWP analysis in human serum. The results showed that trypsin@ZIF-L@IMER possessed higher selectivity and pretreatment capability toward LWPs in human serum when compared with other methods and free trypsin, and the identified LWPs played critical functions and roles in serum-related biological processes by Gene Ontology analysis. The developed method provides a novel strategy for efficient protein pretreatment analysis, which can be further extended to the pretreatment analysis of other proteins.