[Preparation of immobilized trypsin micro monolithic column and its application to rapid digestion of trace proteins]

A method for the preparation of a micro monolithic column with immobilized trypsin was developed for the rapid and efficient digestion of proteins. The micro monolithic column was prepared by photo-polymerization inside the tip of 20 μL pipette. The polymerization solution was composed of the functional monomers 4-pentenoic succinimide ester (PAS) and 2-hydroxyethyl methacrylate (HEMA), crosslinker pentaerythritol triacrylate (PETA) dissolved in a ternary porogenic system comprising dimethyl sulfoxide (DMSO), formyldimethylamine (DMF) and 1-dodecanol. Immobilization of trypsin was achieved by a chemical reaction between the amino group and succinimide. The effects of the active ester content in the polymerization mixture and the volumes of the monolithic bed on the column capacities of the immobilized trypsin were systematically investigated. The digestion efficiency as well as the stability and repeatability of the immobilized trypsin were systematically investigated by using standard proteins cytochrome C and bovine serum albumin. The experimental results indicated that high digestion efficiency with great reproducibility between batches and the digestion procedure could be obtained within 10 min. The trypsin immobilized columns were applied to the digestion of proteins extracted from 1×10⁵ human acute promyelocytic leukemia (NB4) cells and human acute T-cell leukemia (Jurkat T) cells. A total of 2489 and 2572 proteins were readily identified by Nano-LC-MS/MS analysis. The quantity ratio of the identified proteins increased 2.2% and 6.1%, respectively, compared to the case of in-solution digestion, demonstrating the robustness of the trypsin immobilized micro column and its potential application to proteome studies.