固定化胰蛋白酶整体小柱的制备及其用于微量蛋白质的快速酶解

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

发展了一种光引发聚合法制备固定化胰蛋白酶整体小柱的方法,以用于微量蛋白质的快速酶解。整体小柱由功能单体4-戊烯酸琥珀酰亚胺酯、甲基丙烯酸羟乙酯,交联剂季戊四醇三丙烯酸酯和三元致孔剂二甲基亚砜、N,N-二甲基甲酰胺、十二醇在20μL的移液器吸头尖端原位聚合而成。形成整体柱后,胰蛋白酶分子通过氨基与琥珀酰亚胺酯反应实现固定化。系统研究了聚合溶液中活性酯含量与柱床体积对胰蛋白酶固载量的影响,评价了固定化酶整体小柱对标准蛋白细胞色素C和牛血清白蛋白的酶解效率,以及整体小柱的稳定性和重复性。结果表明,在离心辅助下,酶解过程可在10 min内完成,批次间具有良好的重复性。最后将固定化酶整体小柱应用于1×10^5个人急性早幼粒白血病(NB4)细胞与人急性T细胞白血病(Jurkat T)细胞的快速酶解,经纳升级液相色谱与高分辨质谱联用分析后鉴定得到2 489个和2 572个蛋白质。相比于溶液状态下的酶解,分别提高了2.2%和6.1%的蛋白鉴定数量,展现了其在蛋白组学研究中的应用潜力。

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 1×10^5 human acute promyelocytic leukemia(NB4) cells and human acute T-cell leukemia(Jurkat T) cells. A total of 2 489 and 2 572 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.