A tyrosine kinase assay using reverse-phase high-performance liquid chromatography.
A tyrosine kinase assay using reverse-phase high-performance liquid chromatography.
复制标题
使用反相高效液相色谱法进行酪氨酸激酶测定。
DOI：
10.1006/abio.1997.2077
复制
发表时间：
1997
影响因子：
2.9
通讯作者：
Kohanski,RA
中科院分区：
文献类型：
--
作者：
Cann,AD;Wolf,I;Kohanski,RA
反相 HPLC 可作为一种非常精确且准确的蛋白激酶肽磷酸化常规检测方法，与其他方法相比具有优势。特别地，可以使用具有天然氨基酸序列的肽而不需要放射性同位素。然而，所采用的反应条件通常会给磷酸肽和脱辅基肽的回收和定量带来困难。遇到了两个普遍问题：首先，肽保留时间的变化和注射前沿宽度的增加可能干扰定量，这两者都是由重复样品注射造成的。这些主要是由于用于减少反应过程中肽损失的载体牛血清白蛋白的存在以及用于研究酶催化反应动力学的高浓度 ATP 造成的。这些问题通过定期清洗反相柱得到解决，从而可以使用多种肽和 ATP 浓度。其次，测定中使用的肽的稳定性受到二硫苏糖醇与锰的组合的影响。前者是激酶纯化的常用试剂，后者通常是激酶反应中使用的金属辅因子。最小化二硫苏糖醇的浓度或使用镁解决了这些困难。因此，在使用反相 HPLC 监测蛋白酪氨酸激酶测定中的肽磷酸化时，考虑这些因素非常重要。
Reverse-phase HPLC can be used as a very precise and accurate routine assay for peptide phosphorylation by protein kinases that has advantages over other methods. In particular, peptides with native amino acid sequences can be used without the need for radioisotopes. However, reaction conditions that are employed can often present difficulties in recovery and quantitation of phospho- and apo-peptides. Two general problems were encountered: First, variation in the retention times of peptides and an increasing width of the injection front which can interfere with quantitation both resulted from repeated sample injections. These were caused mostly by the presence of carrier bovine serum albumin used to reduce loss of peptides during the reaction and by high concentrations of ATP used to study the kinetics of enzyme catalyzed reactions. These problems were solved by regular washing of the reverse-phase column, thus allowing a broad range of peptide and ATP concentrations to be used. Second, the stability of peptides used in the assay was affected by dithiothreitol in combination with manganese. The former is a common reagent of kinase purifications and the latter is often the metal cofactor used in kinase reactions. Minimizing the concentration of dithiothreitol or using magnesium resolved these difficulties. Consideration of these factors is therefore important when using reverse-phase HPLC to monitor peptide phosphorylation in protein tyrosine kinase assays.