McCroskey M C, Colca J R, Pearson J D
Biopolymer Chemistry, Upjohn Co., Kalamazoo, MI 49001.
J Chromatogr. 1988 Jun 17;442:307-15. doi: 10.1016/s0021-9673(00)94478-9.
We have developed a simple method for phosphoamino acid analysis of 32P-labeled phosphoproteins using anion-exchange high-performance liquid chromatography (HPLC). Phosphoproteins undergo partial acid hydrolysis and the resulting hydrolysate is injected directly onto a column. The sample is then isocratically eluted from the column by 35 mM phosphoric acid at pH 3.0 with collected fractions analyzed by Cerenkov counting. Phosphoamino acid identification is accomplished by the comparison of the retention times of 32P-labeled peaks to retention times of phosphoamino acid standards which had been monitored at 206 nm. This method has greater sensitivity and is more reliable than cellulose thin-layer electrophoresis and the results obtained by high-efficiency Cerenkov counting can be evaluated immediately, instead of waiting days or weeks for autoradiographic development of cellulose plates. This HPLC protocol is an improvement over other published HPLC protocols in that there is no need for pre- or post-column derivatization and the free [32P]phosphate elutes long after the phosphoamino acids. Thus sensitivity is increased as there is no interference from the free phosphate. Selection of an HPLC anion-exchange column is critical for this separation. Only two of the four columns that we tested performed well. We present data from several phosphoproteins including calcium-calmodulin dependent protein kinase, the beta-subunit of the insulin receptor, and phosphorylated calmodulin to demonstrate the utility of this procedure.
我们开发了一种使用阴离子交换高效液相色谱（HPLC）对32P标记的磷蛋白进行磷酸氨基酸分析的简单方法。磷蛋白进行部分酸水解，所得水解产物直接注入柱中。然后用pH 3.0的35 mM磷酸等度洗脱柱上的样品，收集的馏分通过切伦科夫计数进行分析。通过将32P标记峰的保留时间与在206 nm处监测的磷酸氨基酸标准品的保留时间进行比较来完成磷酸氨基酸鉴定。该方法比纤维素薄层层析具有更高的灵敏度和可靠性，并且通过高效切伦科夫计数获得的结果可以立即评估，而无需等待纤维素板放射自显影片显影数天或数周。此HPLC方案是对其他已发表的HPLC方案的改进，因为无需柱前或柱后衍生化，且游离[32P]磷酸盐在磷酸氨基酸之后很久才洗脱。因此，由于没有游离磷酸盐的干扰，灵敏度得以提高。选择HPLC阴离子交换柱对于这种分离至关重要。我们测试的四根柱子中只有两根表现良好。我们展示了来自几种磷蛋白的数据，包括钙调蛋白依赖性蛋白激酶、胰岛素受体的β亚基和磷酸化钙调蛋白，以证明该方法的实用性。
