Identification of histidine phosphorylations in proteins using mass spectrometry and affinity-based techniques.
Histidine phosphorylation plays a key role in prokaryotic signaling and accounts for approximately 6% of the protein phosphorylation events in eukaryotics. Phosphohistidines generally act as intermediates in the transfer of phosphate groups from donor to acceptor molecules. Examples include the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) and the histidine kinases found in two-component signal transduction pathways. The latter are utilized by bacteria and plants to sense and adapt to changing environmental conditions. Despite the importance of histidine phosphorylation in two-component signaling systems, relatively few proteins have so far been identified as containing phosphorylated histidine residues. This is largely due to the instability of phosphohistidines, which, unlike the phosphoesters formed by serine, threonine, and tyrosine, are labile and susceptible to acid hydrolysis. Nevertheless, it is possible to preserve and identify phosphorylated histidine residues in target proteins using appropriate sample preparation, affinity purification, and mass spectrometric techniques. This chapter provides a brief overview of such techniques, describes their use in confirming histidine phosphorylation of a known PTS protein (HPr), and suggests how this approach might be adapted for large-scale identification of histidine-phosphorylated proteins in two-component systems.
Ross
Andrew R S
AR
Plant biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada.
D001426:Bacterial Proteins / Q000737:chemistry; D001671:Biochemistry / Q000379:methods*; D006639:Histidine / Q000031:analogs & derivatives / Q000737:chemistry*; D000071677:Histidine Kinase; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D001426:Bacterial Proteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D006639:Histidine; D011494:Protein Kinases; D000071677:Histidine Kinase; C005436:phosphohistidine