Stoichiometry of histidine kinases by mass spectrometry

Lauren J Tomlinson, Alice K M Clubbs Coldron , Patrick A Eyers, Claire E Eyers (2020) Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry. Methods Mol Biol 2077:83-91. doi: 10.1007/978-1-4939-9884-5_6.

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.

Keywords: Histidine phosphorylation; Intact mass spectrometry; NME1; NME2.