Fragmentation of protein using trypsin.
Fragmentation of protein using trypsin.
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DOI：
10.1101/pdb.prot4550
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发表时间：
2006-10-01
期刊：
影响因子：
--
通讯作者：
Simpson, Richard J
中科院分区：
文献类型：
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作者：
Simpson, Richard J
在这个方案中，高度特异的胰蛋白酶被用来完全水解蛋白质。蛋白分解是用高水平的胰酶进行的，以确保完全的蛋白分解。(胰酶是一种强健的酶；因此，蛋白质分解也可以在变性条件下进行，如2M盐酸胍、0.1%十二烷基硫酸钠和10%乙腈，以确保完全消化。)胰酶能裂解精氨酸的羧基或赖氨酸的羧基与相邻氨基酸的氨基之间的肽键。当赖氨酸和精氨酸残基在序列中与酸性氨基酸或胱氨酸相邻时，切割速度较慢。当赖氨酸或精氨酸后面跟着脯氨酸时，不会发生切割。通过琥珀酸化作用，胰酶的裂解位点可以限制在精氨酸肽键上。或胰酶消化前的柠檬酰化反应。
INTRODUCTIONIn this protocol, the highly specific protease trypsin is used to hydrolyze a protein completely. Proteolysis is carried out with high levels of trypsin to ensure total proteolysis. (Trypsin is a robust enzyme; hence, proteolysis can also be carried out under denaturing conditions such as 2 M guanidine-HCl, 0.1% SDS, and >10% acetonitrile to ensure complete digestion.) Trypsin cleaves the peptide bond between the carboxyl group of arginine or the carboxyl group of lysine and the amino group of the adjacent amino acid. The rate of cleavage occurs more slowly when the lysine and arginine residues are adjacent to acidic amino acids in the sequence or cystine. Cleavage does not occur when lysine or arginine is followed by proline. The sites of trypsin cleavage can be limited to arginine peptide bonds by succinylation. or citraconylation prior to trypsin digestion.