磷酸化底物肽的硫代磷酸化及荧光标记

以蛋白激酶Ａ的一磷酸化底物肽ＬＲＲＡＳＬＧ为模型肽，研究了硫代磷酸化及荧光标记反应条件。荧光标记试剂５－｛「（（２－碘代乙酰）氨）乙基」氨基｝萘－１－磺酸（１，５－ＩＡＥＤＡＮＳ）适宜浓度为１．６ｍｍｏｌ／Ｌ，标记反应缓冲液的适宜ｐＨ为７至８。实验了标记肽分别在Ｎ－端序列分析、电喷雾质谱和０．１％三氟乙酸存在下的稳定性。比较了标记肽和标记肽的紫外吸收光谱的差异特征。初步显示高效液相色谱蛋白酶解肽谱

Thiophosphorylation and Fluorescent Labeling of Substrate Peptide of Protein Kinase

Study on the conditions of thiophosphorylation reaction and fluorescent labeling reaction of the substrate of protein kinase A was carried out by using Kemptide LRRASLG as a model. The suitable concentration of the fluorescent regent 5 {[((2 iodoacetyl)amino)ethyl]amino} naphthelene 1 sulfonic acid (1,5 IAEDANS) and the suitable pH of labeling reaction buffer were 1.6 mmol/L and 8, respectively. Stability of the labeled peptide under the conditions of automatic N terminal protein sequencing, electrospray mass spectrometry and in the presence of 0.1% trifluoroacetic acid was investigated, respectively. The specific differences in UV spectra between the labeled and unlabeled peptides were observed. Therefore, the possibility to detect the thiophosphorylated and fluorophore labeled peptide during high performance liquid chromatography peptide mapping was primarily shown.