Protein analysis using enzymes immobilized to paramagnetic beads
Protein analysis using enzymes immobilized to paramagnetic beads
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DOI：
10.1006/abio.1999.4254
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发表时间：
1999-10-15
影响因子：
2.9
通讯作者：
Hojrup, P
中科院分区：
文献类型：
--
作者：
Krogh, TN;Berg, T;Hojrup, P
提出了一种将蛋白质化学与酶固定在顺磁珠上相结合的新方法。固定化酶可以在许多蛋白质化学方案中替代常规酶，从而加快反应速度，减少样品污染，并改进与现代程序的接口，如质谱仪。以胰酶为模型酶，用MALDI-MS和高效液相色谱(HPLC法)分析玻璃微珠上的蛋白质偶联情况和自消化程度。固定化胰酶可产生与以胎鼠蛋白为模型底物的标准溶液消化相当的消化量。此外，还利用胎球蛋白对酶包衣小球的稳定性进行了考察。经10次重复使用包被胰酶的小球后，酶活性没有明显的损失。将外切和内切糖苷酶固定在顺磁性小球上，可以提高灵敏度，加快糖苷酶对糖肽的顺序消化，并减少样品污染。以人肺蛋白沉积症表面活性蛋白A的胰酶糖肽为模型化合物，可在24 h内完成全部消化。(C)1999年学术出版社。
A new method combining protein chemistry with enzymes immobilized to paramagnetic beads is presented. The immobilized enzymes can substitute for regular enzymes in a number of protein chemistry protocols, resulting in faster reaction times, less sample contamination, and improved interfacing to modern procedures, like mass spectrometry. Trypsin was used as a model enzyme to test the amount of protein coupled to glass beads and the degree of autodigestion when analyzed by MALDI-MS and HPLC. Immobilization of trypsin resulted in digestions comparable with standard solution digestions using fetuin as a model substrate. Furthermore, fetuin was used to test the stability of the enzyme-coated beads. No apparent loss of enzyme activity was observed after 10 times reuse of trypsin-coated beads. Immobilization of exo- and endoglycosidases to paramagnetic beads resulted in high sensitivity, faster sequential glycosidase digestion of glycopeptides, and reduced sample contamination. All digestions could be performed in less than 24 h, when a tryptic glycopeptide from human lung proteinosis surfactant protein A was used as model compound. (C) 1999 Academic Press.