Separation of Methylated Histone Peptides via Host-Assisted Capillary Electrophoresis

Abstract

Lysine methylation in protein is one important epigenetic mechanism that regulates diverse biological processes but is challenging to study due to the large variability in methylation levels and sites. Here, we show that supramolecular hosts such as calixarenes and cucurbiturils can be applied in the background electrolyte (BGE) of capillary electrophoresis (CE) for highly effective separation of post-translationally methylated histone peptides. The molecular recognition event causes a shift in the electrophoretic mobility of the peptide, allowing affinity measurement for binding between the synthetic receptor and various methylated lysine species. Successful separation of the H3 peptides carrying different methylation levels at the K9 position can be achieved using CX4 and CX6 as the BGE additives in CE, enabling monitoring of the activity of the histone lysine demethylase JMJD2E. This reveals the power of combining high resolution CE with synthetic hosts for study of protein methylation, and the method should be capable of analyzing complex biological samples for better understanding of the functions of histone methylation.

A new, deep quinoxaline-based cavitand receptor for the complexation of benzene

“…In our group, we have been exploiting two main types of receptors, in which the bridging groups at the upper rim are either phosphonate RPO 3 moieties or quinoxaline ring systems. Both families have been extensively used in sensing in solution (Lee et al, 2018;Liu et al 2018) and in the gas phase (Melegari et al, 2013;Tudisco et al, 2016). Indeed, the demand for fast and reliable detection of biological and chemical hazards is rising continuously and optimal sensors for environmental, security and biomedical applications must be sufficiently responsive to allow detection of the target analyte at low concentrations, and selective enough to respond primarily to a single chemical species in the presence of interferents.…”

Synthetic Receptors for the Recognition and Discrimination of Post‐Translationally Methylated Lysines

“…Zhong and co‐workers employed sulfonatocalixarenes 1 and 5 , as well as cucurbituril 39 , in the separation of methylated histone proteins by host‐mediated capillary electrophoresis . The molecular recognition event changes the electrophoretic mobility of differently methylated peptides.…”

Cucurbit[7]uril‐Dimethyllysine Recognition in a Model Protein

“…The larger Q8 can accommodate the side chains of two aromatic residues . While this binding mode has featured prominently in numerous Qn ‐protein systems, Qn can interact also with lysine, in particular, methylated lysine side chains . Mono‐, di‐ and tri‐methylation of the lysine ammonium (N ζ ) are common post translational modifications that occur most notably in histones, with vast ramifications for gene expression .…”

“…Q7 ‐KMe n interactions have been characterized for the amino acids revealing dramatic increases in affinity with the degree of methylation . Recent experiments with a KMe 3 ‐containing histone peptide indicate a ≈10‐fold drop in K d relative to the amino acid, pointing to a favorable contribution by the N α group . To date, there are no literature reports of Qn ‐KMe n binding with a protein.…”