A pan-specific antibody for direct detection of protein histidine phosphorylation

• Published in: Nature chemical biology Vol. 9; no. 7; pp. 416 - 421
• Main Authors: Kee, Jung-Min, Oslund, Rob C, Perlman, David H, Muir, Tom W
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.07.2013; Nature Publishing Group
• Subjects: 631/92/1643; 631/92/458; 631/92/612/100/2285; 631/92/96; Antibodies - chemistry; Biochemical Engineering; Biochemistry; Bioorganic Chemistry; Carbon sources; Cell Biology; Chemistry; Chemistry/Food Science; Dose-Response Relationship, Drug; E coli; Enzyme-Linked Immunosorbent Assay; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Histidine - analogs & derivatives; Histidine - chemistry; Hydrogen-Ion Concentration; Immunoglobulins; Ions; Ketoglutaric Acids - metabolism; Mass Spectrometry; Nitrogen; Phosphorylation; Phosphotransferases (Paired Acceptors) - metabolism; Proteins; Proteins - chemistry; Pyruvate Synthase - chemistry; Recombinant Proteins - chemistry; Signal transduction
• ISSN: 1552-4450; 1552-4469
• DOI: 10.1038/nchembio.1259

Studies of histidine phosphorylation have been limited owing to a lack of appropriate tools. The synthesis of a stable phosphohistidine mimic now leads to a pan antibody, enabling detection and further functional investigations of this little-known post-translational modification. 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.