Tryptic peptide mapping of picomolar quantities of protein labeled with the Bolton--Hunter reagent.
Tryptic peptide mapping of picomolar quantities of protein labeled with the Bolton--Hunter reagent.
复制标题
用 Bolton--Hunter 试剂标记的皮摩尔量蛋白质的胰蛋白酶肽图谱。
DOI：
10.1016/0003-2697(81)90391-2
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发表时间：
1981
影响因子：
2.9
通讯作者：
Stevens,JG
中科院分区：
文献类型：
--
作者：
Feitelson,MA;Wettstein,FO;Stevens,JG
用Bolton-Hunter试剂(125I-3-[4-羟基苯基]丙酸N-羟基丁二酰亚胺酯)对5-25pmoL的纯化蛋白进行了胰酶肽图谱的表征。在放射性酰化之后，与未标记的酯反应和还原甲基化，以确保赖氨酰残基对胰蛋白酶的抗性。还原的和烷基化的蛋白质用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析，从单独的凝胶切片中胰酶消化，然后在薄层上进行二维定位。该方法可以绘制比活性为1到2×104cpm/ng的蛋白质的肽图，比直接的碘化程序产生更多的斑点(通常也更多的结构信息)，并且可以用于表征不能被生物合成标记的蛋白质。
Characterization of 5 to 25 pmol of purified proteins by tryptic peptide mapping has been accomplished using the Bolton-Hunter reagent (125I-3-[4-hydroxyphenyl]propionic acid N-hydroxysuccinimide ester). Radioacylation is followed by reaction with unlabeled ester and reductive methylation to ensure resistance of lysyl residues to trypsinization. Reduced and alkylated proteins are analzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trypsinized from individual gel slices, and mapped two-dimensionally on thin layers. The method permits peptide mapping of proteins with specific activities of 1 to 2 × 104cpm/ng, results in more spots (and often more structural information) than direct iodination procedures, and can be used for characterization of proteins that could not be biosynthetically labeled.