Fig. 3: Diverse SMART targeting enables AND-gated proximity labelling.
a, SMART targeting modalities encompass antibody fragments (AbFs), antibody mimetics (AbMs) and synthetic ligands (peptides and small molecules). b–d, SMART-SpyCatcher was tested with an anti-CEACAM6 single-domain antibody, a CXCR4-targeting cyclic peptide BKT140, an ADORA2A-targeting small molecule SCH58261 and the DARPins anti-HER2, anti-EGFR and anti-EpCAM. OE19 (HER2hi, EGFRlow and EpCAMhi; b) or derivatives stably expressing CXCR4 (OE19CXCR4DOX; c) or ADORA2A (OE19A2ADOX; d) were treated with SMART-SpyCatcher and fluorescently labelled SpyTag003 (100 nM each), followed by flow cytometry. e, Schematic of AND-gated protein proximity labelling by the delivery of a SpyTag003 catalyst. f, K562HER2+/EGFR+, OE19, A431 and OE19CXCR4DOX were treated with SMART-SpyCatcher (100 nM), SpyTag003–APEX2 (300 nM) and then biotin-phenol (250 μM) and H2O2 (100 mM, 2 min) before western blot analysis. AND gates predicted to enable labelling are shown in red; asterisks indicate endogenously biotinylated proteins. g, Densitometry analysis was done using FIJI (National Institutes of Health). Signals were normalized to tubulin (loading control). a.u., arbitrary units. h, Schematic of cell-selective photocatalytic proximity labelling using SMART-SpyCatcher and a SpyTag003-Ir conjugate. i, Mixed K562 cells (phenotypes indicated) were treated with SMART-SpyCatcher (100 nM) and SpyTag003-Ir (200 nM). After washing, cells were incubated with biotin-diazirine (100 μM), irradiated with 450-nm light (5 min), stained with Streptavidin-Alexa Fluor 546 and analysed by flow cytometry; histograms are normalized to the mode. j, Quantification of the data in i; mean ± s.e.m. (n = 3 independent biological replicates). Statistical analysis: applied unpaired two-sided t-test (b–d); applied one-way analysis of variance (ANOVA) followed by Dunnett’s test (j) (Supplementary Tables 7 and 8).
