Two-Step Activity-Based Protein Profiling with the Proteasome System as Model of Study.
Activity-based protein profiling (ABPP) is a method to highlight enzymatic activities in a biological sample, which uses chemical probes that react covalently with the catalytic nucleophile of the enzyme. To circumvent disadvantages associated with the presence of reporter tags on chemical probes, the probe is equipped with a ligation handle to which the reporter can be reacted at the desired time and place in the ABPP workflow. This chapter demonstrates the power of a triple bioorthogonal ligation strategy which addresses the three activities of the proteasome: the β5-subunit selective norbornene-tagged probe is reacted with fluorescent tetrazine, the β1-selective azide-functionalized probe was addressed with a biotinylated phosphine, followed by an alkyne-substituted pan-reactive probe to label the remaining β2 activity to which an azide-coupled fluorophore was ligated. The result of the triple ligation was similar to each reaction performed separately demonstrating the value of the triple ligation strategy for a single experiment.
['Soriano|Guillem Paniagua|GP|', 'Overkleeft|Herman S|HS|', 'Florea|Bogdan I|BI|']
D001386:Azides D057930:Click Chemistry D057809:HEK293 Cells D006801:Humans D008956:Models, Chemical D015335:Molecular Probes D046988:Proteasome Endopeptidase Complex D011506:Proteins
Proteasome Activity-based protein profiling (ABPP) Bioorthogonal chemistry Copper(I)-catalyzed azide-alkyne cycloaddition (click reaction) Inverse-electron-demand Diels-Alder reaction Staudinger-Bertozzi ligation