Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry

Protein histidine phosphorylation has largely remained unexplored due to the challenges of analyzing relatively unstable phosphohistidine-containing proteins. We describe a procedure for determining the stoichiometry of histidine phosphorylation on the human histidine kinases NME1 and NME2 by intact mass spectrometry under conditions that retain this acid-labile protein modification. By characterizing these two model histidine protein kinases in the absence and presence of a suitable phosphate donor, the stoichiometry of histidine phosphorylation can be determined. The described method can be readily adapted for the analysis of other proteins containing phosphohistidine.