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).

Multifunctional Photo-Cross-Linking Probes: From Target Protein Searching to Imaging Applications

“…Its longer lifetime leads to diffusion effects. The half-life and labeling radius of the reactive intermediates should be further investigated and recently demonstrated in the MacMillan groups μMap development . This work presented a systematic investigation of the labeling radius between carbene-based and phenoxy radical-based methods.…”

“…The half-life and labeling radius of the reactive intermediates should be further investigated and recently demonstrated in the MacMillan groups μMap development. 51 This work presented a systematic investigation of the labeling radius between carbene-based and phenoxy radical-based methods. In addition, they also examined the diffusion effect by controlling probes' PEG linker lengths.…”

Finding a vocation for validation: taking proteomics beyond association and location

“…Proteins also undergo important subcellular changes in localization that can have severe implications for their interactomes, functions 28,44 and drug mechanisms. 45 Several methods have arisen to probe protein localization, including APEX(2), 46,47 Bio-ID, 48 Turbo-ID, 49 μMap, 50 and so on (Fig. 3B).…”

“…Indeed, dependent on the diffusion distance of the generated electrophile, and the timing of the experiment, subtly different aspects of local proteomes can be uncovered. 50 As these methods can tag a relatively spatially-defined region, 51,52 (which is defined by the half-life of the reactive species liberated), they are also increasingly used to profile protein associations or complex associations. Moreover, these methods also have the ability to catch transient interactions, subcellular-specific, or tissue-specific interactions, among others.…”

High resolution photocatalytic mapping of SARS-CoV-2 Spike protein-host cell membrane interactions

“…Due to fast quenching and the short half-life of the carbene intermediate (T 1/2 < 1 ns) the activated probe is unable to diffuse farther than 4 nm (Geri et al ., 2020). This drives a shorter labeling radius and greater spatiotemporal control compared to enzyme methods with higher resolution protein microenvironment snapshots (Oakley et al, 2022). Furthermore, the bioconjugation of a small molecular weight photocatalyst (< 1000 Da) to the targeting modality of interest results in a smaller perturbation compared to attachment of a larger enzyme.…”