Triple Bioorthogonal Ligation Strategy for Simultaneous Labeling of Multiple Enzymatic Activities

Three at the same time: A ligation strategy combining tetrazine–norbornene cycloaddition, Staudinger–Bertozzi ligation, and copper(I)-catalyzed click reaction was used to label the three catalytic activities of the proteasome simultaneously in a single experiment. The orthogonality of the three ligation reactions enables selective monitoring of multiple targets at the same time in complex biological samples.