Campbell David G, Morrice Nicholas A
MRC Protein Phosphorylation Unit, University of Dundee, Dundee, Scotland.
J Biomol Tech. 2002 Sep;13(3):119-30.
The analysis of protein phosphorylation sites is one of the major challenges in the post-genomic age. To understand the role of reversible phosphorylation in cell signaling, the precise location of phosphorylation sites must be determined in a phosphoprotein as well as the effect that these post-translational modifications have on the function of the protein. The use of solid phase Edman degradation of (32)P-labeled phosphoproteins and peptides was described over 10 years ago as a method for the identification of phosphorylation sites. Since that time a number of laboratories have used this technique as the standard method for phosphorylation site analysis. In this report, we will describe how we routinely use this technology to perform hundreds of successful phosphorylation site analyses per annum. By combining mass spectrometry to identify the phosphopeptide and solid phase Edman degradation to localize the site of phosphorylation, subpmole quantities of phosphopeptides can be successfully characterized.
蛋白质磷酸化位点分析是后基因组时代的主要挑战之一。为了解可逆磷酸化在细胞信号传导中的作用，必须确定磷蛋白中磷酸化位点的精确位置以及这些翻译后修饰对蛋白质功能的影响。10多年前就描述了使用固相Edman降解法分析（32）P标记的磷蛋白和肽，作为鉴定磷酸化位点的一种方法。从那时起，许多实验室都将该技术用作磷酸化位点分析的标准方法。在本报告中，我们将描述我们如何每年常规使用该技术进行数百次成功的磷酸化位点分析。通过结合质谱法鉴定磷酸肽和固相Edman降解法定位磷酸化位点，可以成功鉴定亚皮摩尔量的磷酸肽。
