Supramolecular Affinity Chromatography for Methylation-Targeted Proteomics

Abstract

Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation-a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

Selective Binding of a Lower Lysine Methylation State: An N,N-Dimethyllysine Selective Host Molecule and Its Use in Methyl Proteomics

“…The calixarene-modified gel was then packed into capillaries. [35] (c) The resulting capillary were used to enrich proteolyzed sample from cell extract of S. cerevisiae (SI7). (d) Distribution of methylated peptides seen in the samples before (input) and after enrichment (unretained and retained) is summarised in the Venn diagram.…”

“…[38][39][40] We have previously used chlorosulfonyl aromatics to modify the upper rim of amino-substituted calixarenes via sulfonamide bonds. [23,35,41,42] While carrying out additional chemistry of that type, we accidentally identified a new lower-rim-modifying reaction. We report here a new lower-rim sulfonate ester modification of calix [4]arenes, and its key role in creating a Kme2-selective host molecule.…”

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

“…Due to its high affinities for trimethylated peptides, calixarene 14 has been employed in supramolecular affinity chromatography for methylation‐targeted proteomics. Linking 14 to agarose beads allowed the resolution of histone peptides (H3K4me x , H3K27me x and H3K27me x ) on the basis of their methylation …”

Proteins and Proteoforms: New Separation Challenges

“…Selectivity of the p-sulfonocalix(4)arene for methyllysine residues in peptides is reported to be superior to that of a cation-exchange chromatography resin. Attaching this supramolecular affinity selector to agarose 120 (Figure 4C) allowed the enrichment of peptides bearing methylated lysine residues from a trypsin digested nuclear extract prior to a bottom-up LC–MS/MS analysis. The advantages of this method are the enhanced selectivity and enrichment of methylated lysine carrying species.…”