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

Lysine dimethylation (Kme) 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 Kme. To address this, we present a chemical method named tertiary amine coupling by oxidation (TACO). This method selectively modifies Kme to aldehydes using Selectfluor and a base. The resulting aldehydes from Kme were then functionalized using reductive amination, thiolamine, and oxime chemistry. We successfully demonstrated the versatility of TACO in selectively labeling Kme 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 Kme sites at a single-molecule level by fluorosequencing. We discovered novel 30 Kme sites, in addition to previously known 5 Kme sites, by proteomics analysis of TACO-modified nuclear extracts. Our work establishes a unique strategy for covalently modifying Kme, facilitating the global identification of low-abundance Kme-PTMs and their sites within complex cell lysate mixtures.