Improved automated solid-phase microsequencing of peptides using DABITC.
Improved automated solid-phase microsequencing of peptides using DABITC.
复制标题
使用 DABITC 改进肽的自动化固相微测序。
DOI：
10.1016/0003-2697(81)90803-4
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发表时间：
1981
影响因子：
2.9
通讯作者：
Brigitte Wittmann
中科院分区：
文献类型：
--
作者：
J. Salnikow;A. Lehmann;Brigitte Wittmann
与EDC（1-乙基-3-二甲氨基丙基碳二亚胺）的连接研究表明，如果符合最佳条件，肽基范围为4到31个，可以以满意的收率与氨基丙基玻璃连接。然而，具有羧基末端赖氨酸的肽的附着收率明显较低。采用Edman试剂DABITC （4- n， n -二甲氨基偶氮苯4′-异硫氰酸酯）与苯基异硫氰酸酯结合使用，可以对固定化肽进行高灵敏度固相测序。采用了间歇式氮冲洗、逐步泵送DABITC试剂以及采用基于三氟乙酸出水即时水稀释的自动转化装置等技术改进措施。固相微测序是一种普遍适用于1- 10-nmol范围内肽的方法，具有多达30-35个可识别的降解步骤。
Attachment studies with EDC (1-ethyl-3-dimethylaminopropyl carbodiimide) have revealed that peptides, ranging from 4 to 31 residues, can be linked to aminopropyl glass with satisfactory yield, if optimal conditions are obeyed. The attachment yields are, however, significantly lower with peptides possessing carboxyl terminal lysine. High-sensitivity solid-phase sequencing of immobilized peptides can be performed if DABITC (4-N, N-dimethylaminoazobenzene 4′-isothiocyanate), an Edman reagent with a covalently linked chromophore, is used in conjunction with phenyl isothiocyanate. Technical improvements have been introduced by utilization of intermittent nitrogen flushes, pumping of the DABITC reagent in a stepwise manner, and incorporation of an automated conversion device based on instant aequous dilution of the trifluoroacetic acid effluent. Solid-phase microsequencing constitutes a method of general applicability for peptides in the 1- to 10-nmol range, with up to 30–35 identifiable degradation steps.