Utilizing 4-Sulfonylcalix[4]arene as a Selective Mobile Phase Additive for the Capture of Methylated Peptides

Protein methylation has attracted increasing attention due to its significant regulatory roles in various biological processes. However, the diversity of methylation forms, subtle differences between methylated and nonmodified sites, and their ultralow abundances pose substantial challenges for capturing and isolating methylated peptides from biological samples. Herein, we develop a chromatographic method that utilizes 4-sulfonylcalix[4]arene (SC4A) as a mobile phase additive and Click-Maltose as the stationary phase to separate methylated/nonmethylated peptides through the adsorption of the SC4A-K(Me3) complex. By utilization of the interaction between calix[4]arene cavities and trimethylated lysine residues, methylated peptides could be specifically separated from peptide samples. This method significantly improves the signal-to-noise ratio (S/N), even in samples containing a 10-fold excess of bovine serum albumin (BSA) trypsin digests. Additionally, we successfully enriched 12 methylated peptides from histone digests. This study paves the way for the selective enrichment of lysine methylated peptides in post-translational modification proteomics (PTMs), enhancing both the capture efficiency and selectivity of methylated peptides and providing robust technical support for subsequent proteomics research.