Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization.
Biotinylated peptides/proteins. I. Determination of stoichiometry of derivatization.
复制标题
生物素化肽/蛋白质。
DOI：
10.1016/0003-2697(91)90384-6
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发表时间：
1991
影响因子：
2.9
通讯作者：
Kurosky,A
中科院分区：
文献类型：
--
作者：
Smith,JS;Miller,BT;Knock,SL;Kurosky,A
描述了一种在与含有延伸间隔臂6-氨基己酸(NHS-ϵAhx-生物素)的生物素N-羟基琥珀酰亚胺酯反应后测定肽和蛋白质生物素化化学计量的方法。该分析方法基于 6-氨基己酸的苯硫基氨基甲酰衍生物的定量，能够测量低皮摩尔量的生物素衍生物。使用自动在线水解仪-衍生仪进行分析，然后使用高效液相色谱法进行分析。已知肽的成分分析与质谱分析结果非常一致。还描述了生物素化蛋白质探针的生产程序，该探针可以重复标记以含有特定量的生物素。 6-氨基己酸间隔臂提供的分析优势和空间自由度有力地证明了 NHS-ϵAhx-生物素试剂是肽和蛋白质生物素化的首选试剂。
A method is described for the determination of the stoichiometry of biotinylation of peptides and proteins after reaction with an N-hydroxysuccinimide ester of biotin containing the extended spacer arm 6-aminohexanoic acid (NHS-ϵAhx-biotin). The method of analysis, based on the quantification of phenylthiocarbamyl derivatives of 6-aminohexanoic acid, is able to measure low picomolar amounts of biotinyl derivative. Analyses were performed using an automated on-line hydrolyzer-derivatizer followed by high-performance liquid chromatography. Compositional analyses determined for known peptides were in excellent agreement with analyses obtained by mass spectrometry. Procedures are also described for the production of biotinylated protein probes that can be labeled reproducibly to contain specific amounts of biotin. The analytical advantage and steric freedom provided by the 6-amino-hexanoic acid spacer arm argue strongly for the NHS-ϵAhx-biotin reagent to be a reagent of choice for the biotinylation of peptides and proteins.