Post-translational prenylation of proteins is an essential process in eukaryotic cells, and plays a key role in signal transduction and vesicular trafficking.[1] In the case of protein geranylgeranylation, geranylgeranyl pyrophosphate (GGpp) acts as the prenyl donor, and the process is catalyzed either by geranylgeranyl transferase-1 (GGT-1), or by Rab geranylgeranyl transferase (RGGT), the latter requiring a Rab escort protein (REP-1 or REP-2). GGT-1 targets Rho family proteins, whilst RGGT acts on the Rab small GTPases; both sets of proteins are prenylated at C-terminal prenylation motifs.[2] Mis-prenylation of Rabs leads to several severe diseases,[3] and a deeper understanding of this process is essential in order to design effective therapies.

However, efficient detection and identification of prenylated proteins remains challenging, making use of quantities of 3H-GGpp with detection times typically extending from days to weeks.[4] Biotinylated or fluorophore-containing prenyl analogues offer enhanced detection,[5, 6] but their flexibility is limited by the substrate specificity of the prenyl transferase and they are associated with a significant reduction in affinity for native RGGT.[6] We and others have recently demonstrated that azide or alkyne tagging combined with bioorthogonal ligation chemistry can be used to detect post-translational protein lipidation (acylation or prenylation) in cell-free and live-cell systems.[4, 7–10] These highly biocompatible and biomimetic tags are minimally disruptive and enable subsequent labeling with a wide range of potential reporters. We describe here a versatile azidetag/bioorthogonal ligation system (Figure 1) that enables the rapid detection and affinity purification of proteins geranylgeranylated by either RGGT or GGT-1 by using multiple labels, applicable both to recombinant proteins and proteins from cell lines or mammalian tissues.