Fig. 3: Target engagement of ABP, quantification of targets and live cell and in vivo imaging.

Schematic representation of a measurement of target (UCHL1) engagement by gel fluorescence and western blotting using ABPs. Generally, ABP labelling is directed towards a particular target protein or protein class. ABPs may contain latent handles such as an alkyne, which may be bioorthogonally ligated with a reporter such as a fluorophore or a biotin affinity tag. If a fluorophore is used, labelled proteins may be separated by SDS-PAGE and directly visualised using fluorescence scanning, while biotinylated target proteins are generally enriched on a neutravidin-immobilised resin and visualised on western blot using target-specific antibodies; b quantification of on- and off-targets using ABPs and competitive mass spectrometry (MS)-based proteomics. Cells are treated with selective inhibitors/drugs, followed by intracellular labelling with ABPs, lysis, bioorthogonal ligation to biotin and enrichment of biotinylated proteins on Neutravidin-agarose resin. On-resin digestion generates peptides from probe-labelled proteins, which are subsequently analysed using liquid chromatography-mass spectrometry. Differential labelling in inhibitor-treated and untreated samples are quantified, enabling the identification of novel drug targets as well as sites of protein modification for further drug development; c Imaging target protein activity in live cell or in in vivo (e.g. zebrafish embryo) cancer models using fluorescently labelled ABPs and fluorescence microscopy.