Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Loomis WF, Shaulsky G, Wang N (1997) Histidine kinases in signal transduction pathways of eukaryotes. J Cell Sci 110:1141–1145
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298(5600):1912
Cohen P (2002) The origins of protein phosphorylation. Nat Cell Biol 4(5):127–130
Adam K, Hunter T (2018) Histidine kinases and the missing phosphoproteome from prokaryotes to eukaryotes. Pathol Focus 98:233–247
Kee JM, Muir TW (2012) Chasing phosphohistidine, an elusive sibling in the phosphoamino acid family. ACS Chem Biol 7(1):44–51
Klumpp S, Krieglstein J (2009) Reversible phosphorylation of histidine residues in proteins from vertebrates. Sci Signal 2(61):1–4
Fuhs SR et al (2015) Monoclonal 1- and 3-phosphohistidine antibodies: new tools to study histidine phosphorylation. Cell 162(1):198–210
Srivastava S et al (2016) Histidine phosphorylation relieves copper inhibition in the mammalian potassium channel KCa3.1. eLife 5:pii: e16093
Puttick J, Baker E, Delbaere L (2008) Histidine phosphorylation in biological systems. Biochim Biophys Acta 1784(1):100–105
Attwood PV (2013) P/N bond protein phosphatases. Biochim Biophys Acta 1834:470–478
Attwood PV, Muimo R (2018) The actions of NME1/NDPK-A and NME2/NDPK-B as protein kinases. Lab Investig 98:283–290
Attwood PV, Wieland T (2015) Nucleoside diphosphate kinase as protein histidine kinase. Naunyn Schmiedeberg's Arch Pharmacol 388(2):153–160
Wu Z, Tiambeng TN, CaI W, Chen B, Lin Z, Zachery R, Gregorich ZR, Ge Y (2018) Impact of phosphorylation on the mass spectrometry quantification of intact phosphoproteins. Anal Chem 90(8):4935–4939
Haydon CE, Eyers PA, Aveline-Wolf LD, Resing KA, Maller JL (2003) Identification of novel phosphorylation sites on Xenopus laevis Aurora A and analysis of phosphopeptide enrichment by immobilized metal-affinity chromatography. Mol Cell Proteomics 2(10):1055–1067
Schweppe RE, Haydon CE, Lewis TS, Resing KA, Ahn NG (2003) The characterization of protein post-translational modifications by mass spectrometry. Acc Chem Res 36(6):453–456
Johnson H, Eyers CE, Eyers PA, Beynon RJ, Gaskell SJ (2009) Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry. J Am Soc Mass Spectrom 20(12):2211–2220
Byrne DP, Vonderach M, Ferries S, Brownridge PJ, Eyers CE, Eyers PA (2016) cAMP-dependent protein kinase (PKA) complexes probed by complementary differential scanning fluorimetry and ion mobility-mass spectrometry. Biochem J 473(19):3159–3175
Ferries S, Perkins S, Brownridge PJ, Campbell A, Eyers PA, Jones AR, Eyers CE (2017) Evaluation of parameters for confident phosphorylation site localization using an orbitrap fusion tribrid mass spectrometer. J Proteome Res 16(9):3448–3459
Attwood PV, Besant PG, Piggott MJ (2011) Focus on phosphoaspartate and phosphoglutamate. Amino Acids 40(4):1035–1051
Besant PG, Attwood PV, Piggott MJ (2009) Focus on phosphoarginine and phospholysine. Curr Protein Pept Sci 10(6):536–550
Hardman G, Perkins S, Ruan Z, Kannan N, Brownridge P, Byrne DP, Eyers PA, Jones AR, Eyers CE (2017) Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics. bioRxiv 202820. https://doi.org/10.1101/202820
Hardman G, Perkins S, Brownridge PJ, Clarke CJ, Byrne DP, Campbell AE, Anton Kalyuzhnyy A, Myall A, Eyers PA, Jones AR, Eyers CE (2019) Strong anion exchange‐mediated phosphoproteomics reveals extensive human non‐canonical phosphorylation. The EMBO Journal. https://doi.org/10.15252/embj.2018100847
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Tomlinson, L.J., Clubbs Coldron, A.K.M., Eyers, P.A., Eyers, C.E. (2020). Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry. In: Eyers, C. (eds) Histidine Phosphorylation. Methods in Molecular Biology, vol 2077. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9884-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9884-5_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9883-8
Online ISBN: 978-1-4939-9884-5
eBook Packages: Springer Protocols