A Method for Selective Isolation of the Amino-Terminal Peptide from α-Amino-Blocked Proteins
A Method for Selective Isolation of the Amino-Terminal Peptide from α-Amino-Blocked Proteins
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从 α-氨基封闭蛋白中选择性分离氨基末端肽的方法
DOI：
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发表时间：
1994
期刊：
影响因子：
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通讯作者：
K. Titani
中科院分区：
文献类型：
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作者：
T. Akiyama;T. Sasagawa;Motoshi Suzuki;K. Titani
本文介绍了一种从α-氨基（Nα）封闭蛋白质中选择性分离氨基N端肽的方法。该方法包括四个步骤。首先，蛋白质中赖氨酸残基的N-氨基被琥珀酰化。第二，通过酶或化学裂解消化衍生的蛋白质。第三，使消化物与溴化氰活化的琼脂糖凝胶反应。只有N-末端封闭的肽不能反应，而其他肽与琼脂糖凝胶共价结合。第四，通过反相色谱法纯化未偶联的肽。包括封闭基团的肽的氨基酸序列可以通过质谱法测定。这些程序成功地测试了三个已知的Nα-阻断蛋白，包括牛脑S100蛋白，马细胞色素c，卵清蛋白。
Abstract A method for selective isolation of the amino N-terminal peptide from an α-amino (Nα)-blocked protein is presented. The method consists of four steps. First, ϵ-amino groups of lysine residues in the protein are succinylated. Second, the derivatized protein is digested by either enzymatic or chemical cleavage. Third, the digest is subjected to reaction with cyanogen bromide-activated Sepharose. Only the N-terminal-blocked peptide fails to react, while the other peptides are covalently bound to the Sepharose. Fourth, uncoupled peptides are purified by reversed-phase chromatography. The amino acid sequence of the peptide including the blocking group can be determined by mass spectrometry. These procedures were successfully tested with three known Nα-blocked proteins including bovine brain S100 protein, horse cytochrome c, and ovalbumin.