Radius measurement via super-resolution microscopy enables the development of a variable radii proximity labeling platform

Abstract

The elucidation of protein interaction networks is critical to understanding fundamental biology as well as developing new therapeutics. Proximity labeling platforms (PLPs) are state-of-the-art technologies that enable the discovery and delineation of biomolecular networks through the identification of protein-protein interactions. These platforms work via catalytic generation of reactive probes at a biological region of interest; these probes then diffuse through solution and covalently "tag" proximal biomolecules. The physical distance that the probes diffuse determines the effective labeling radius of the PLP and is a critical parameter that influences the scale and resolution of interactome mapping. As such, by expanding the degrees of labeling resolution offered by PLPs, it is possible to better capture the various size scales of interactomes. At present, however, there is little quantitative understanding of the labeling radii of different PLPs. Here, we report the development of a superresolution microscopy-based assay for the direct quantification of PLP labeling radii. Using this assay, we provide direct extracellular measurements of the labeling radii of state-of-the-art antibody-targeted PLPs, including the peroxidase-based phenoxy radical platform (269 ± 41 nm) and the high-resolution iridium-catalyzed µMap technology (54 ± 12 nm). Last, we apply these insights to the development of a molecular diffusion-based approach to tuning PLP resolution and introduce a new aryl-azide-based µMap platform with an intermediate labeling radius (80 ± 28 nm).

Keywords: STED microscopy; photoredox catalysis; proximity labeling.

Fig. 1.

(A) General proximity labeling regime. (B) State-of-the-art PLPs cover a range of spatial resolution. This work focuses on the development of a strategy for the direct measurement and comparison of state-of-the-art PLP labeling radii and the development of new PLPs with novel degrees of spatial selectivity.

Fig. 2.

(A) Proximity labeling of the EGFR interactome using dual antibody targeting. (B) STED image of radial clusters observed on the extracellular membrane of A549 cells upon µMap labeling (250 µM diazirine (1), 2 min irradiation). These radial clusters colocalize with EGFR (scale bar, 2 µm). (C) representative clusters highlighting colocalization of streptavidin and EGFR (scale bar, 200 nm). (D) STED image of peroxidase-based labeling (250 µM Bt-Tyr (2), 1 mM H₂O₂, 1 min, scale bar, 1 µm). (E) Measurement of peroxidase-based (Left, 5 mM [2], 1 mM H₂O₂, 15 min) and µMap (Right, 5 mM [1], 450 nm irradiation for 15 min) labeling on the surface of BSA-coated coverslips. (F) Biotin-antibody conjugates to probe tether-length limited hypothesis. (G) STED image of radial clusters observed with antibody-biotin conjugates on the coverslip surface (scale bar, 200 nm). (H) TMT-based proteomics of µMap labeling of EGFR on A549 cells (250 µM diazirine [1], 2 min irradiation). (I) TMT-based proteomics of peroxidase-based (250 µM Bt-Tyr [2], 1 mM H₂O₂, 1 min) labeling of EGFR on A549 cells.

Fig. 3.

(A) The development of a PLP that offers intermediate spatial resolution remains an unmet challenge. (B) Triplet nitrenes undergo quenching process at diminished rates compared to carbenes. (C) Measurement for the labeling radius of phenyl azide (3) using the BSA-coated coverslip model (scale bar, 200 nm). (D) Measurement for the labeling radius of F₄PhN₃ (4) using the BSA-coated coverslip model (scale bar, 200 nm). (E) Targeted labeling of the EGFR interactome on the surface of A549 cells using PhN₃ probe (3) (scale bar, 0.5 µm). (F) Targeted labeling of the EGFR interactome on the surface of A549 cells using PhN₃ probe (4) (scale bar, 0.5 µm).

Fig. 4.

(A) Truncating labeling radius by modulating the diffusion coefficient. (B) 2D DOSY NMR of a mixture of PEG3-PhN₃ (3) and PEG24-PhN₃ (5). Analysis of cross-peaks in the DOSY spectra reveals that the PEG24 analog has a ∼1.6-fold reduced diffusion coefficient relative to PEG3. (C) STED image of BSA labeling on the surface of a coverslip with extended linker azide probe (5) reveals a labeling radius of 80 ± 28 nm. (D) Targeted cell-surface EGFR interactome labeling using PEG24 PhN₃ (5) reveals smaller clusters in the diffusion reduced labeling (scale bar, 0.5 µm). (E) TMT-based proteomics for PEG3 PhN₃-based µMap. (F) TMT-based proteomics for PEG24 PhN₃-based µMap. (G) Range of labeling radii measured across PLPs analyzed in this manuscript. These mean FWHM values (averages of 100 measurements) were calculated from clusters observed on BSA-coated coverslips.