Chen X, Eswaran D, Smith M A, Perry G, Anderson V E
Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935, USA.
Bioconjug Chem. 1999 Jan-Feb;10(1):112-8. doi: 10.1021/bc980098v.
A universal procedure to isolate cross-linked peptides has been demonstrated. The procedure relies on initially converting the epsilon-amino groups of lysine to dimethyl lysine by reductive methylation with sodium cyanoborohydride and formaldehyde. The lysine-modified protein is proteolytically cleaved and the resulting alpha-amino groups derivatized with methoxypoly(ethylene glycol)5000 succinyl hydroxysuccinimide. Any unintentional derivatization of tyrosine side chains can be reversed by incubation under mildly alkaline conditions. The cross-linked polypeptides contain two poly(ethylene glycol)5000 chains while non-cross-linked peptides contain a single poly(ethylene glycol)5000 chain. The two populations of peptides can be separated by gel filtration chromatography. This procedure has been shown capable of isolating cross-linked peptides using glutathione, lysozyme, chemically cross-linked hemoglobin, and neurofibrillary tangles isolated from the brain of a case of Alzheimer's disease.
已证明了一种分离交联肽的通用方法。该方法首先依靠用氰基硼氢化钠和甲醛进行还原甲基化反应，将赖氨酸的ε-氨基转化为二甲基赖氨酸。对赖氨酸修饰的蛋白质进行蛋白水解切割，然后用甲氧基聚（乙二醇）5000琥珀酰羟基琥珀酰亚胺对产生的α-氨基进行衍生化。酪氨酸侧链的任何意外衍生化都可以通过在温和碱性条件下孵育来逆转。交联多肽含有两条聚（乙二醇）5000链，而非交联肽含有一条聚（乙二醇）5000链。这两种肽可以通过凝胶过滤色谱法分离。已表明该方法能够使用谷胱甘肽、溶菌酶、化学交联的血红蛋白以及从一例阿尔茨海默病患者大脑中分离出的神经原纤维缠结来分离交联肽。
