Barbieri Christopher M, Stock Ann M
Center for Advanced Biotechnology and Medicine, Department of Biochemistry and Howard Hughes Medical Institute, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Anal Biochem. 2008 May 1;376(1):73-82. doi: 10.1016/j.ab.2008.02.004. Epub 2008 Feb 13.
Recent development of the phosphate chelator, Phos-tag, together with Phos-tag pendant reagents, has provided new methods for detection of phosphorylated serine, threonine, tyrosine, and histidine residues in phosphoproteins. We have investigated the use of Phos-tag for detection and quantification of phospho-aspartate in response regulator proteins that function within two-component signaling systems. Alternative methods are especially important, because the labile nature of the acylphosphate bond in response regulator proteins has restricted the application of many traditional methods of phosphoprotein analysis. We demonstrate that Phos-tag gel stain can be used to detect phospho-Asp in response regulators and that Phos-tag acrylamide gel electrophoresis can be used to separate phosphorylated and unphosphorylated forms of response regulator proteins. The latter method, coupled to Western blot analysis, enables detection of specific phosphorylated proteins in complex mixtures such as cell lysates. Standards of phosphorylated proteins can be used to correct for hydrolysis of the labile phospho-Asp bond that invariably occurs during analysis. We have employed Phos-tag methods to characterize the phosphorylation state of the Escherichia coli response regulator PhoB both in vitro, using purified protein, and in vivo, by analyzing lysates of cells grown under different conditions of induction of the PhoR/PhoB phosphate assimilation pathway.
磷酸盐螯合剂Phos-tag及其相关试剂的最新进展，为检测磷蛋白中磷酸化的丝氨酸、苏氨酸、酪氨酸和组氨酸残基提供了新方法。我们研究了使用Phos-tag检测和定量在双组分信号系统中起作用的响应调节蛋白中的磷酸天冬氨酸。替代方法尤为重要，因为响应调节蛋白中酰基磷酸键的不稳定性质限制了许多传统磷蛋白分析方法的应用。我们证明，Phos-tag凝胶染色可用于检测响应调节蛋白中的磷酸天冬氨酸，并且Phos-tag丙烯酰胺凝胶电泳可用于分离响应调节蛋白的磷酸化和未磷酸化形式。后一种方法与蛋白质免疫印迹分析相结合，能够检测复杂混合物（如细胞裂解物）中特定的磷酸化蛋白。磷酸化蛋白标准品可用于校正分析过程中不可避免发生的不稳定磷酸天冬氨酸键的水解。我们已采用Phos-tag方法，通过使用纯化蛋白在体外以及通过分析在不同诱导条件下生长的细胞裂解物在体内来表征大肠杆菌响应调节蛋白PhoB的磷酸化状态，该诱导条件涉及PhoR/PhoB磷酸盐同化途径。
