Molecular Spatiomics by Proximity Labeling

Abstract

Conspectus Proximity labeling can be defined as an enzymatic “in-cell” chemical reaction that catalyzes the proximity-dependent modification of biomolecules in live cells. Since the modified proteins can be isolated and identified via mass spectrometry, this method has been successfully utilized for the characterization of local proteomes such as the sub-mitochondrial proteome and the proteome at membrane contact sites, or spatiotemporal interactome information in live cells, which are not “accessible” via conventional methods. Currently, proximity labeling techniques can be applied not only for local proteome mapping but also for profiling local RNA and DNA, in addition to showing great potential for elucidating spatial cell–cell interaction networks in live animal models. We believe that proximity labeling has emerged as an essential tool in “spatiomics,” that is, for the extraction of spatially distributed biological information in a cell or organism. Proximity labeling is a multidisciplinary chemical technique. For a decade, we and other groups have engineered it for multiple applications based on the modulation of enzyme chemistry, chemical probe design, and mass analysis techniques that enable superior mapping results. The technique has been adopted in biology and chemistry. This “in-cell” reaction has been widely adopted by biologists who modified it into an in vivo reaction in animal models. In our laboratory, we conducted in vivo proximity labeling reactions in mouse models and could successfully obtain the liver-specific secretome and muscle-specific mitochondrial matrix proteome. We expect that proximity reaction can further contribute to revealing tissue-specific localized molecular information in live animal models. Simultaneously, chemists have also adopted the concept and employed chemical “photocatalysts” as artificial enzymes to develop new proximity labeling reactions. Under light activation, photocatalysts can convert the precursor molecules to the reactive species via electron transfer or energy transfer and the reactive molecules can react with proximal biomolecules within a definite lifetime in an aqueous solution. To identify the modified biomolecules by proximity labeling, the modified biomolecules should be enriched after lysis and sequenced using sequencing tools. In this analysis step, the direct detection of modified residue(s) on the modified proteins or nucleic acids can be the proof of their labeling event by proximal enzymes or catalysts in the cell. In this Account, we introduce the basic concept of proximity labeling and the multidirectional advances in the development of this method. We believe that this Account may facilitate further utilization and modification of the method in both biological and chemical research communities, thereby revealing unknown spatially distributed molecular or cellular information or spatiome.

Cell-Selective Multifunctional Surface Covalent Reconfiguration Using Aptamer-Enabled Proximity Catalytic Labeling

“…2 On the other hand, it theoretically allows the simultaneous introduction of many different functional molecules into a given cell membrane, with little restriction on the number of molecular species. At present, the state-of-the-art covalent labeling strategies with spatial specificity from outside of cells mainly include two categories: (1) genetically fusing peroxidases (APEX, 23 APEX2, 24 or HRP 25 ) or biotin ligases (BioID 26 and TurboID 27 ) to a target region, which can catalyze the covalent labeling to proximal proteins 28,29 and (2) using affinity recognition to drive covalent labeling to occur at specific locations. 2,30−42 In a groundbreaking approach by MacMillan et al, Ir complex-conjugated antibodies were utilized for spatially localizing carbene generation to map the microenvironment of immune cells.…”

“…At present, the state-of-the-art covalent labeling strategies with spatial specificity from outside of cells mainly include two categories: (1) genetically fusing peroxidases (APEX, APEX2, or HRP) or biotin ligases (BioID and TurboID) to a target region, which can catalyze the covalent labeling to proximal proteins , and (2) using affinity recognition to drive covalent labeling to occur at specific locations. ,− …”

“…], thus allowing labeling to be restricted to target cells . Furthermore, by linking tyramine to different functional moieties, phenol-containing molecules can be simply synthesized in a modular manner …”

“…37 Furthermore, by linking tyramine to different functional moieties, phenol-containing molecules can be simply synthesized in a modular manner. 29 Herein, we report an aptamer-enabled proximity catalytic labeling (APCL) platform for cell-selective multifunctional surface reconfiguration in mixed cell populations (Scheme 1). We carefully investigate the labeling process and attribute the cell selectivity to the increased local concentration of phenoxy radicals on the surface of target cells by Apt, as well as the short lifetime of phenoxy radicals.…”

Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling

“…Proximity labeling using small-molecule catalysts has recently been investigated [ 22 , 23 , 24 ]. We also developed photocatalytic proximity-labeling reactions that proceed in proximity to photocatalysts.…”