Monitoring the crosstalk between methylation and phosphorylation on histone peptides with host-assisted capillary electrophoresis

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.

Capillary Zone Electrophoresis-Tandem Mass Spectrometry As an Alternative to Liquid Chromatography-Tandem Mass Spectrometry for Top-down Proteomics of Histones

“…We need to note that the CZE conditions (e.g., BGE composition) can modulate the μ ef of histones substantially as demonstrated by the works from the Zhong group. 60,61 We expect that systematic optimizations of the CZE condition could amplify the effect of the PTMs (e.g., N-terminal acetylation) on histone proteoforms' μ ef , thus leading to better separations of modified and unmodified histone proteoforms.…”

Applications of Synthetic Receptors in Bioanalysis and Drug Transport

“…The method was also be exploited to analyze the activities of methylation enzymes. 48 A remarkably stable host−guest pair is formed between cucurbit [7]uril (CB [7]) and adamantylammonium (AdA), with a K a > 10 12 M −1 , 49−51 comparable to that of the streptavidin−biotin or antibody−antigen complexes. CB [7] was recently employed to purify protein therapeutics (e.g., monoclonal antibody or cytokine proteins) site-specifically modified by AdA.…”

“…Since they bind differentially to methylated lysines with different methylation states or at various sites on the peptide, they can resolve histone peptides carrying different lysine methylations effectively. The method was also be exploited to analyze the activities of methylation enzymes …”

Recent (2018–2020) development in capillary electrophoresis

“…Peptides with different methylation levels at the same lysine position were separated using CX4 and CX6 in the BGE, which was further used to monitor the activity of the histone lysine demethylase JMJD2E. Later, this method was employed to analyze dual modifications, i.e., methylation and phosphorylation, on histone peptides using a linear polyacrylamide-coated capillary [35]. With the capability of separating the peptides carrying different modifications, the crosstalk between serine phosphorylation and lysine methylation was revealed.…”