Design and optimization of porous polymer enzymatic digestors for proteomics

Effective protein characterization and identification are demanding and time-consuming operations in proteomics because of long-protein purification/separation procedures, and even longer enzymatic digestions. In this work, polymer-based monolithic enzyme reactors were fabricated in fused-silica capillaries, and performance was characterized through protein digestion and identification by MALDI-MS and ESI-MS. Reactors were prepared by fabricating a porous methacrylate base monolith followed by photografting with glycidyl methacrylate, and immobilization of the enzyme(s) with carbonyldiimidazole. Trypsin and Staphylococcus aureus V-8 protease (Glu-C) were used to produce three types of reactors: trypsin-based, Glu-C-based, and trypsin combined with Glu-C. Protein digestions, performed by perfusing protein solutions through the reactor under pressure, were evaluated based on the peptide map generated when directly coupled to an ESI mass spectrometer. Excellent digestion was observed over flow rates from 0.2 to 1 μL/min, which corresponds to reactor residence times of 0.24-1.4 min. As a proof of principle, chromatographic separation of model proteins followed by the digestion of specific fractions using these proteolytic enzyme reactors and ESI-MS is demonstrated.