Polydopamine-assisted immobilization of trypsin onto monolithic structures for protein digestion
Polydopamine-assisted immobilization of trypsin onto monolithic structures for protein digestion
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DOI：
10.1002/jssc.201200073
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发表时间：
2012-06-01
影响因子：
3.1
通讯作者：
Messersmith, Phillip B.
中科院分区：
文献类型：
--
作者：
Rivera, Jose G.;Messersmith, Phillip B.
受贻贝足部富含儿茶酚的粘附性蛋白的启发，我们报道了一种简单而通用的水相方法将胰酶固定在二氧化硅和二氧化钛整体载体上。该方法包括用由多巴胺在碱性pH下聚合得到的含儿茶酚的仿生聚合物(聚多巴胺)原位包覆整体底物，然后将胰酶偶联到聚多巴胺聚合物包衣上。考察了不同制备参数如多巴胺浓度和包被时间对固定化胰酶的影响。对固定化胰酶反应器的酶活性进行了评价，并通过对模型蛋白的消化进行了基于质谱学的蛋白质组学分析。由于多巴胺能够作为蛋白质共价固定化的引发剂，本手稿中提出的方法在将胰酶和其他酶固定到各种整体载体上具有广泛的潜力。
Inspired by the catechol-rich adhesive proteins of the mussel foot, we report a simple and versatile aqueous approach for the immobilization of trypsin onto silica and titania monolithic supports. The method involves in-situ coating of the monolithic substrates with a catechol-containing biomimetic polymer (polydopamine) derived from the polymerization of dopamine under alkaline pH, followed by conjugation of trypsin to the polydopamine polymer coating. The trypsin immobilization efficiency onto the monolithic materials was investigated as a function of different preparation parameters such as dopamine concentration and coating time. The enzymatic activity of the immobilized trypsin reactors was evaluated, and mass spectrometry based proteomic analysis was demonstrated by digestion of a model protein. The method presented in this manuscript has broad potential for immobilization of trypsin and other enzymes onto a wide variety of monolithic supports, due to the ability of polydopamine to act as a primer for covalent immobilization of proteins.