Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry

  • Lauren J. Tomlinson
  • Alice K. M. Clubbs Coldron
  • Patrick A. Eyers
  • Claire E. EyersEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2077)


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.

Key words

NME1 NME2 Histidine phosphorylation Intact mass spectrometry 


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Lauren J. Tomlinson
    • 1
  • Alice K. M. Clubbs Coldron
    • 2
  • Patrick A. Eyers
    • 2
  • Claire E. Eyers
    • 1
    Email author
  1. 1.Department of Biochemistry, Centre for Proteome Research, Institute of Integrative BiologyUniversity of LiverpoolLiverpoolUK
  2. 2.Department of Biochemistry, Institute of Integrative BiologyUniversity of LiverpoolLiverpoolUK

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