Takeda A, Cone R E
Biochem Biophys Res Commun. 1984 Aug 16;122(3):932-7. doi: 10.1016/0006-291x(84)91180-x.
The peptide mapping method described by Cleveland, et al. (1) was improved to a two-dimensional analysis applicable to minute amounts (less than 0.5 microgram) of proteins. Radioiodinated proteins for analysis were purified by electrophoretic elution of the proteins from polyacrylamide gels into buffer containing 0.1% sodium dodecyl sulfate. The proteins were digested enzymatically in the presence of 0.1% sodium dodecyl sulfate and an excess of nonlabeled bovine serum albumin (0.2 mg/ml) relative to labeled substrate in order to attain reproducibility by maintaining a consistent substrate concentration among different samples. The peptides of these limited proteolytic products were resolved by two-dimensional polyacrylamide gel electrophoresis (isoelectric focusing followed by SDS-gels). The resulting 2D-peptide maps of murine and bovine albumin and a murine lymphocyte membrane protein, Tp100, showed excellent resolution and reproducibility.
克利夫兰等人（1）描述的肽图谱分析方法被改进为一种适用于微量（小于0.5微克）蛋白质的二维分析方法。用于分析的放射性碘化蛋白质通过将蛋白质从聚丙烯酰胺凝胶电泳洗脱到含有0.1%十二烷基硫酸钠的缓冲液中进行纯化。蛋白质在0.1%十二烷基硫酸钠和相对于标记底物过量的未标记牛血清白蛋白（0.2毫克/毫升）存在下进行酶消化，以便通过在不同样品之间保持一致的底物浓度来实现可重复性。这些有限的蛋白水解产物的肽通过二维聚丙烯酰胺凝胶电泳（等电聚焦后接SDS凝胶）进行分离。所得的小鼠和牛白蛋白以及小鼠淋巴细胞膜蛋白Tp100的二维肽图谱显示出优异的分辨率和可重复性。
