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Separation of Modified Biomolecules With Synthetic Receptors in Capillary Electrophoresis


Post-translational modifications (PTMs) on histone proteins affect many essential biological processes. With the abundance of PTMs on histone proteins, these modifications can collectively work together to alter chromatin structure and impact gene expression in this complex system. Detection and understanding of these PTMs, as well as the enzymes that mediate them, can elucidate their biological outcomes and connection to pathogenesis. Mass spectrometry (MS) is a powerful tool that is most often used for identification and characterization of modification patterns, and it has greatly expanded the library of histone modifications. However, MS does require coupling to separation techniques, such as reversed-phase liquid chromatography and hydrophilic interaction chromatography, in order to reduce sample complexity and improve analysis. While chromatographic techniques have been widely used, multiple columns and long separation times are required for peptides that are more challenging to separate. For instance, methylation does not alter the charge of lysine residues; in addition, it introduces only subtle changes in size and hydrophobicity. Recently, there has been an increasing interest in synthetic receptors that have affinity for lysine methylation. Such receptors include water-soluble calixarenes, cucurbiturils, and deep cavitands. Capillary electrophoresis (CE) is an extremely suitable separation method that can be combined with these synthetic receptors, which can be easily included in the separation buffer. In this work, we first developed a method to induce mobility shifts for methylated small guests and peptides. After combining the high resolving power of CE and the selective recognition of host molecules, separation of modified and unmodified peptides was achieved. This host-assisted CE method was able to discriminate between different lysine methylation levels; essentially, this enabled the monitoring of various PTM enzyme reactions, including demethylation, methylation, and phosphorylation. This technique has also demonstrated its capability for analysis of a crosstalk event in which a pre-existing histone modification can impact the activity of an enzyme for another PTM. Furthermore, there is potential of host-assisted CE to separate proteins with PTMs as well.

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