Capillary Electrophoresis-Based Immobilized Enzyme Microreactor Utilizing Pepsin for Bottom-Up Proteomics

Abstract

In this study, we introduced a novel method for pepsin immobilization through investigation into three different coupling reactions, aiming to simplify peptide mapping. We initially determined the optimal enzyme-to-substrate ratio to be 1:0.1 (mass:mass) for highest peptidic peak count via UV-detection. The study began with three coupling strategies, the triethoxysilylbutyraldehyde derivative, 4-triethoxysilylbutanoic acid (4-TESBA) approach proving most effective. The resultant coupled enzyme particles (CEPs) showcased robustness and fast digestion at varying temperatures. We streamlined the CEP wash procedure from our previous work, while maintaining the quality of digestions. The physical size of CEPs did not correlate to digestion efficiency, providing insights for potential cost-saving in enzyme utilization. Further optimization led to an immobilization efficiency of 50.8 ± 7.7% (SEM) as validated by Bradford’s assay. Adapting this method for in-situ immobilized enzyme microreactor (IMER) fabrication, we discovered that 4-TESBA could dual-serve by functionalizing the silica capillary’s inner wall while simultaneously acting as an enzyme coupler, plus being a safer alternative to 3-(Aminopropyl)triethoxysilane (APTES). A dipeptide was successfully fully cleaved, and a variety of proteins were digested with the IMER. The bovine serum albumin (BSA) digestion through the IMER, mirroring CEP digestion conditions, yielded a 33-40% primary sequence coverage per LC-MS/MS analysis in as short as 15 minutes. Our findings underscore the potential of our method in both CEP and IMER fabrication scenarios, paving the way for enhanced analysis and a reduction in enzyme usage, thereby contributing to more cost-effective and timely proteomic investigations.