Jue R A, Doolittle R F
Biochemistry. 1985 Jan 1;24(1):162-70. doi: 10.1021/bi00322a023.
We have developed a new method for obtaining information about protein sequences that uses an approach analogous to that used to determine DNA sequences. In essence, three steps are involved. First, a detectable label is attached exclusively to the amino terminus of a polypeptide. Next, the labeled chain is subjected to partial specific cleavage in a way that produces roughly equimolar amounts of fragments of different sizes. Cleavages for methionine, tryptophan, arginine, aspartyl-proline bonds, and asparaginyl-glycine bonds have been employed. Lastly, the labeled fragments are separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of target amino acids along the polypeptide chain can be deduced from the specific pattern of labeled bands by reading the "ladder" in the same way that DNA sequencing gels are read. Although the method can be conducted with a radioactive label, we have chosen to use a fluorescent label. We have applied the method successfully to the three subunit chains of two different fibrinogens.
我们开发了一种获取蛋白质序列信息的新方法，该方法采用了一种类似于用于确定DNA序列的方法。实质上，涉及三个步骤。首先，将可检测的标记专门连接到多肽的氨基末端。接下来，以产生大致等摩尔量的不同大小片段的方式对标记的链进行部分特异性切割。已采用针对甲硫氨酸、色氨酸、精氨酸、天冬氨酰 - 脯氨酸键和天冬氨酰胺 - 甘氨酸键的切割。最后，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳按大小分离标记的片段。通过以读取DNA测序凝胶的相同方式读取“梯状条带”，可以从标记条带的特定模式推断目标氨基酸沿多肽链的分布。虽然该方法可以用放射性标记进行，但我们选择使用荧光标记。我们已成功将该方法应用于两种不同纤维蛋白原的三条亚基链。
