Chemical proteomic profiling of lysine crotonylation using minimalist bioorthogonal probes in mammalian cells

Protein lysine crotonylation has been found to be closely related to the occurrence and development of various diseases. Currently, site identification of crotonylation is mainly dependent on antibody enrichment; however, due to the cost, heterogeneity, and specificity of antibodies, it is desired to develop an alternative chemical tool to detect crotonylation. Herein, we report an alkynyl-functionalized bioorthogonal chemical probe, Cr-alkyne, for the detection and identification of protein lysine crotonylation in mammalian cells. Our in-gel fluorescence and chemical proteomic analyses demonstrated that Cr-alkyne can be metabolically incorporated into lysine of histones and directly label known crotonylated proteins. We further applied Cr-alkyne to the proteome-wide profiling of crotonylation and revealed a large number of previously unreported modification sites, some of which could be validated by co-elution with synthetic peptides. Moreover, by integrating Cr-alkyne with quantitative chemical proteomics, we also explored the crotonylation sites regulated by HDACs, unveiling new HDAC regulated sites. Our study thus provides an enabling chemical tool for characterizing protein crotonylation and greatly expands our understanding of substrate proteins and functions of this important modification.