Jie Shi, Yuting Xiong, Junyan Li, Baolei Gao, Guangyan Qing,* Qianying Sheng,* and Minbo Lan*

Anal. Chem., 2025, 10.1039/D4SC07379G

https://doi.org/10.1021/acs.analchem.4c06041

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 non-modified sites, and their ultra-low 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 and non-methylated peptides through the adsorption of the SC4A-K(Me3) complex. By utilizing the interaction between calix[4]arene cavities and trimethylated lysine residues, methylated peptides could be specifically separated from peptide samples. This method significantly improves signal-to-noise ratio (S/N), even in samples containing a 10-fold excess of bovine serum albumin (BSA) trypsin digests. Additionally, we successfully enrich 12 methylated peptides from histones. 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.