用于MALDI-TOF MS分析的毛细管层析柱制备方法研究

研究了一种用于MALDI-TOF MS分析中样品预处理毛细管亲和层析柱的制备方法。首先采用硫酸和双氧水氧化法将羟基引入到石英毛细管内表面,进一步使用氨基丙基三乙氧基硅烷（APTES）对毛细管进行修饰以将氨基偶联到毛细管内表面;采用1,4-丁二醇二缩水甘油醚（BDDE）将魔芋葡甘聚糖（KGM）进行活化,将活化后的KGM与毛细管上的氨基进行了偶联,在此基础上将模型蛋白（胰蛋白酶）偶联到毛细管内KGM,成功制备出毛细管亲和层析柱,考察了KGM和环氧基含量对模型蛋白偶联量及其样品预处理效果的影响。结果表明,KGM分子量是影响毛细管层析柱上蛋白偶联量的关键因素,以胰蛋白酶抑制剂为目标物结合MALDI-TOF MS对亲和层析柱的分离效果进行了评价,证明了偶联胰蛋白酶的毛细管层析柱可有效实现胰蛋白酶抑制剂的分离和浓缩。基于表面处理和KGM衍生的毛细管亲和层析柱制备技术具有可行性,并可用于MALDI-TOF MS分析的样品预处理。

Preparation of capillary affinity column used for MALDI-TOF MS

A method for preparation of capillary affinity column used for MALDI-TOF MS was investigated. The hydroxyl group was introduced on the inner-face using＂Piranha＂solution. Amino group was coupled on the capillary by the reaction between 3-aminopropyltriethoxysilane（ APTES） and hydroxyl group. Konjac glucomannan（ KGM）,activated with 1,4-butanediol diglycidyl ether（ BDDE）,was conjugated on the capillary by the reaction between the epoxy group on the KGM and-NH2 on the capillary,then model protein（ trypsin） was conjugated on the activated KGM. The effects of KGM molecular weight and the ratio of epoxy group to the KGM on the trypsin conjugation were determined. The results indicated that the KGM molecular weight was the key factor affecting the quantity of conjugated trypsin. With the trypsin inhibitor as target protein,the capillary affinity column could be used for desalting or sample concentrating. The strategy for preparation of capillary affinity column,to be used for sample pretreatment prior to MALDI-TOF MS analysis,was confirmed.