Tertiary Amine Coupling by Oxidation for Selective Labeling of Dimethyl Lysine Post-Translational Modifications

Lysine dimethylation (Kme₂) is a crucial post-translational modification (PTM) that regulates biological processes and is implicated in diseases. There is significant interest in globally identifying these methylation marks. Unfortunately, this remains challenging due to the lack of robust technologies for selectively labeling Kme₂. To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme₂ to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme₂ were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme₂ peptides and proteins in complex cell lysate mixtures with varying payloads, including affinity tags and fluorophores. We further showed the application of TACO chemistry for the identification of Kme₂ sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme₂ sites, in addition to previously known 5 Kme₂ sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme₂, facilitating the global identification of low-abundance Kme₂-PTMs and their sites within complex cell lysate mixtures.