Host-Guest Protein Assembly for Affinity Purification of Methyllysine Proteomes

Anal Chem. 2020 Jul 7;92(13):9322-9329. doi: 10.1021/acs.analchem.0c01643. Epub 2020 Jun 22.

Abstract

Protein-protein interactions drive self-assembly of biomacromolecules and thus enable important physiological functions at a cellular level. Supramolecular chemists have developed artificial host-guest interactions that are similar with, yet distinct from and orthogonal to, the natural protein-protein interactions. For instance, cucurbit[n]urils are synthetic receptors that can specifically recognize proteins with N-terminal aromatic residues with high affinities, yet this interaction can be reversed by the competition of small molecules such as amantadine. Herein, we develop a site-specific, oriented protein-display method by combining the host-guest interaction based on cucurbit[7]uril and a covalent protein-peptide reaction. A methyllysine-binding protein HP1β chromodomain (CD) is immobilized via host-guest interactions and used as the "bait" to capture methyllysine proteomes from cancer cells. The captured "fish"-methyllysine-containing proteins-can be released via competitive displacement by amantadine in a nondenaturing and traceless manner. This affinity purification method found 73 novel methyllysine sites from 101 identified sites among 66 methylated proteins from 255 HP1β CD-binding proteins in cancer cells via subsequent mass spectrometric analysis. This work thereby presents a new strategy of artificial host-guest protein assembly in affinity purification of methyllysine proteins in coupling to mass spectrometry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amantadine / chemistry
  • Bridged-Ring Compounds / chemistry*
  • Chromatography, Affinity*
  • Chromobox Protein Homolog 5
  • Chromosomal Proteins, Non-Histone / chemistry
  • Chromosomal Proteins, Non-Histone / metabolism
  • HeLa Cells
  • Humans
  • Imidazoles / chemistry*
  • Lysine / chemistry
  • Lysine / metabolism*
  • Methylation
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Peptides / chemistry*
  • Peptides / isolation & purification
  • Peptides / metabolism

Substances

  • Bridged-Ring Compounds
  • CBX1 protein, human
  • Chromosomal Proteins, Non-Histone
  • Imidazoles
  • Peptides
  • cucurbit(7)uril
  • Chromobox Protein Homolog 5
  • Amantadine
  • Lysine