Methods for studying human sirtuins with activity-based chemical probes

Abstract Sirtuins are unique posttranslational modification enzymes that utilize NAD+ as the co-substrate to remove acyl groups from lysine residues. The deacylation events result in profound biological consequences, from transcription silencing to metabolism regulation. This article focuses on a newly developed technology using activity-based chemical probes to report sirtuin functional state in various settings. These chemical probes, thioacyllysine peptides carrying photo-cross-linker as well as bioorthogonal functionality, target the active site of sirtuins to form stalled reaction intermediate. Subsequently, the probe forms covalent adduct with the protein through photocrosslinking. Ultimately, the active sirtuin can be visualized via “click” chemistry-mediated conjugation to a fluorescent tag. Here, we describe the labeling protocols on recombinant protein, whole cell lysate, and in situ labeling.