Department of Chemistry, Princeton University, Princeton, New Jersey, USA.
Nat Chem Biol. 2013 Jul;9(7):416-21. doi: 10.1038/nchembio.1259. Epub 2013 May 26.
Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems because of the lack of adequate research tools. We report the development of the first pan-phosphohistidine (pHis) antibody using a stable pHis mimetic as the hapten. This antibody was successfully used in ELISA, western blotting, dot blot assays and immunoprecipitation and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with MS analysis. We also observed that the amount of protein pHis in Escherichia coli lysates depends on carbon source and nitrogen availability in the growth medium. In particular, we found that the amount of pHis on phosphoenolpyruvate synthase (PpsA) is sensitive to nitrogen availability in vivo and that α-ketoglutarate inhibits phosphotransfer from phosphorylated PpsA to pyruvate. We expect this antibody to open opportunities for investigating other pHis proteins and their functions.
尽管蛋白质组氨酸磷酸化在中心代谢和细菌细胞信号转导中具有重要意义，但由于缺乏足够的研究工具，其在生物系统中的广泛程度和功能作用仍然难以捉摸。我们报告了第一个泛磷酸组氨酸（pHis）抗体的开发，该抗体使用稳定的 pHis 模拟物作为半抗原。该抗体在 ELISA、western blot、斑点印迹分析和免疫沉淀中成功应用，并与 MS 分析结合，可用于检测和鉴定来自天然细胞裂解物的组氨酸磷酸化蛋白。我们还观察到大肠杆菌裂解物中的蛋白质 pHis 含量取决于生长培养基中的碳源和氮源可用性。特别是，我们发现磷酸烯醇丙酮酸合酶（PpsA）上的 pHis 含量对体内氮源可用性敏感，并且α-酮戊二酸抑制从磷酸化的 PpsA 到丙酮酸的磷酸转移。我们希望该抗体为研究其他 pHis 蛋白及其功能提供机会。
