Monitoring the crosstalk between methylation and phosphorylation on histone peptides with host-assisted capillary electr
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Monitoring the crosstalk between methylation and phosphorylation on histone peptides with host-assisted capillary electrophoresis Jiwon Lee 1 & Junyi Chen 2 & Priyanka Sarkar 1 & Min Xue 1,2 & Richard J. Hooley 1,3 & Wenwan Zhong 1,2 Received: 17 December 2019 / Revised: 27 January 2020 / Accepted: 3 February 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Post-translational modifications (PTMs) greatly increase protein diversity and regulate their functions by changing the structures, properties, and molecular interactions of proteins. In peptide regions with high density of PTMs, PTMs can influence modification on residues in proximity or even at distal positions, adding another layer of regulation. Methods that can monitor the activities of PTM enzymes on peptides carrying multiple modifications are valuable tools for better understanding of PTM crosstalk. Herein, we developed a host-assisted capillary electrophoresis (CE) method to separate histone peptides with methylation and phosphorylation and applied it to monitor the crosstalk between serine phosphorylation and lysine methylation when they were added by Aurora B kinase and G9a lysine methyltransferase, respectively. A synthetic receptor molecule, 4hexasulfonatocalix[6]arene (CX6), was included in the CE buffer to improve the resolution of the corresponding substrates and products. A linear polyacrylamide-coated capillary was employed to effectively reduce wall adsorption of the cationic histone peptides. The peptide substrates were labeled with fluorescein to enhance their detectability during CE separation. Our method successfully revealed that the activity of G9a methyltransferase was completely inhibited by the adjacent phosphorylation, while 25% reduction in the activity of Aurora B kinase was observed with the presence of dimethylation on the nearby residue. The PTM crosstalk was examined not only using a pure peptide substrate, but also in a competitive reaction environment, in which the modified and unmodified peptides were mixed and the enzyme actions on both peptides were monitored simultaneously. Our work demonstrates that host-assisted CE is an effective method for study of PTM crosstalk, which could offer the advantages of fast separation, high resolution, and low sample consumption.
Keywords Capillary electrophoresis . Enzyme assay . Post-translational modification . PTM crosstalk . Synthetic receptor
Introduction Published in the topical collection featuring Female Role Models in Analytical Chemistry. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02486-y) contains supplementary material, which is available to authorized users. * Wenwan Zhong [email protected] 1
Department of Chemistry, University of California-Riverside, 900 University Ave., Riverside, CA 92521, USA
2
Department of Environmental Toxicology Program, University of California-Riverside, 00 University Ave., Riverside, CA 92521, USA
3
Department of Biochemistry and
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