Proximity-based labeling systems and applications thereof

Abstract

To provide, in one aspect, compositions and methods for providing a microenvironment mapping platform operable to selectively identify various features, including protein-protein interactions on cellular membranes.SOLUTION: In some embodiments, a composition comprises a catalyst and a protein labeling agent, where the catalyst activates the protein labeling agent to a reactive intermediate. The catalyst, in some embodiments, can have electronic structure for permitting energy transfer to the protein labeling agent to form the reactive intermediate. The reactive intermediate reacts or cross-links with a protein or other biomolecule within a diffusion radius of the reactive intermediate.SELECTED DRAWING: Figure 6A

Description

Technical Field

[0001] Related Application Data This application claims the benefit of priority under Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application No. 62 / 858,539, filed on Jun. 7, 2019, and U.S. Provisional Patent Application No. 62 / 982,576, filed on Feb. 2 7, 2020, and the entire contents of each of these applications are incorporated herein by reference.

[0002] The present invention relates to a proximity-based labeling technique, and more particularly to compositions and methods that enable high-resolution labeling of proteins in a cellular environment.

Background Art

[0003] Protein proximity labeling has emerged as a powerful approach for profiling protein interaction networks. The ability to label associated proteins or bystander proteins via proximity labeling can have important implications for further understanding the cellular environment and biological roles of proteins of interest. Current proximity labeling methods all involve the use of enzymes to generate reactive intermediates that label adjacent proteins through diffusion or physical contact on a small number of selected amino acid residues. Despite the revolutionary impact of this technology, for example, phenoxyl radical (t > 100 μs) via peroxidase activation or biotin-AMP (t > 60 s) via biotin ligase, etc., the inherent stability of these reactive intermediates can promote diffusion far from their site of generation. As a result, profiling within a tight microenvironment with these enzyme-generated reactive intermediates > 100 μs) or biotin-AMP (t 1 / 2 > 100 μs) via peroxidase activation or biotin-AMP (t 1 / 2 > 60 s) via biotin ligase, etc., the inherent stability of these reactive intermediates can promote diffusion far from their site of generation. As a result, profiling within a tight microenvironment with these enzyme-generated reactive intermediates is difficult. Circling poses a challenge. Additionally, the large enzyme size, dependence on specific amino acids for labeling, and the inability to temporally control these labeling systems present further challenges in profiling within a limited spatial region.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Considering these limitations, a new approach for proximity-based labeling is needed. There is.

Means for Solving the Problems

[0005] In one aspect, provided herein are compositions and methods for a microenvironment mapping platform that can be engineered to selectively identify various features, including protein-protein interactions, on the cell membrane. In some aspects, the composition includes a catalyst and a protein labeling agent, and the catalyst activates the protein labeling agent into a reactive intermediate. In some embodiments, the catalyst can have an electronic structure that enables energy transfer to the protein labeling agent to form a reactive intermediate. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. If the protein or other biomolecule is not within its diffusion radius, the reactive intermediate is quenched by the surrounding aqueous or aqueous-based environment. As further described herein, the diffusion radius of the reactive intermediate can be adjusted according to the considerations of specific microenvironment mapping and can be limited to the nanometer scale. In some embodiments, operable to selectively identify various features including protein-protein interactions on the cell membrane. Compositions and methods are described herein. In some aspects, the composition includes a catalyst and a protein labeling agent, and the catalyst activates the protein labeling agent into a reactive intermediate . In some embodiments, the catalyst can have an electronic structure that enables energy transfer to the protein labeling agent to form a reactive intermediate. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. If the protein or other biomolecule is not within its diffusion radius, the reactive intermediate is quenched by the surrounding aqueous or aqueous-based environment. As further described herein, the diffusion radius of the reactive intermediate can be adjusted according to the considerations of specific microenvironment mapping and can be limited to the nanometer scale. In some embodiments, the diffusion radius of the reactive intermediate can be adjusted according to the considerations of specific microenvironment mapping and can be limited to the nanometer scale. In some embodiments, it can be limited to the nanometer scale. In some embodiments, Thus, for example, the diffusion radius can be less than 10 nm or less than 5 nm. Further, in some embodiments, the reactive intermediate can have a half-life of less than 5 ns. In certain embodiments the protein labeling agent can be functionalized with a marker such as biotin or a luminescent marker, for example, to assist in an analysis. Any catalyst that can act to participate in energy transfer to the protein labeling agent to provide the reactive intermediate can be used. In some embodiments, a transition metal catalyst is used. Alternatively, a non-transition metal organic catalyst may be used. Energy transfer from the catalyst to the protein labeling agent can occur via various mechanisms further described herein, including Dexter energy transfer.

[0006] In another aspect, a complex (conjugate) for proximity-based labeling is described herein. The complex includes a catalyst coupled to a biomolecule binder. The catalyst can have an electronic structure for energy transfer to a protein labeling agent to generate a reactive intermediate as described above. In some embodiments, the biomolecule binder can be used to selectively place or target the catalyst to a specific environment for mapping. The biomolecule binder can, for example, place a catalyst in a desired cellular environment for proximity labeling and related assays. The biomolecule binder can, in some embodiments, include a protein, a polysaccharide, a nucleic acid, or a lipid. In some cases, the biomolecule binder can include a multivalent display system including a protein, a polysaccharide, a nucleic acid, or a lipid. Also, the biomolecule binder can be a small molecule ligand having a specific binding affinity for a target protein.

[0007] In another aspect, a system for proximity-based labeling is described herein. In one embodiment, the system comprises a complex comprising a catalyst coupled to a biomolecule binder and a protein labeling agent activated by the catalyst for binding to a protein. The conjugate and the protein labeling agent can have any composition and / or properties described in the above and following detailed

[0008] In a further aspect, a method for proximity-based labeling is described herein. The method for proximity-based labeling comprises providing a catalyst and activating a protein labeling agent with the catalyst to a reactive intermediate. The reactive intermediate couples or binds to a protein. In some embodiments, the catalyst is coupled to a biomolecule binder for selectively positioning or targeting the catalyst to a specific environment for protein mapping with the protein labeling agent. The catalyst, complex, and protein labeling agent can have the compositions and / or properties described in the above and following detailed

[0009] These and other embodiments are further described in the following detailed BRIEF DESCRIPTION OF THE DRAWINGS

[0010]

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[0013] The embodiments described herein can be more readily understood by reference to the following detailed description and examples, as well as their preceding and following descriptions. However, the elements, devices, and methods described herein are not limited to the specific embodiments presented in the detailed description and examples. It should be recognized that these embodiments are merely exemplary of the principles of the present invention. Many modifications and adaptations will readily become apparent to those skilled in the art without departing from the spirit and scope of the present invention.

[0014] [Definitions] As used herein, the term "alkyl," used alone or in combination, refers to a straight-chain or branched-chain saturated hydrocarbon group optionally substituted with one or more substituents. For example, alkyl is C1-C30 or C1 to C 18 may be.

[0015] The term "aryl", when used alone or in combination herein, refers to an aromatic monocyclic or polycyclic ring system optionally substituted with one or more ring substituents.

[0016] The term "heteroaryl", as used alone or in combination herein, refers to an aromatic monocyclic or polycyclic ring system in which one or more of the ring atoms are elements other than carbon, such as nitrogen, boron, oxygen and / or sulfur.

[0017] The term "heterocyclic", as used alone or in combination herein, refers to a monocyclic or polycyclic ring system in which one or more of the atoms of the ring system are elements other than carbon, such as boron, nitrogen, oxygen, and / or sulfur or phosphorus, where the ring system is optionally substituted with one or more ring substituents. Heterocyclic ring systems can include aromatic rings and / or non-aromatic rings.

[0018] The term "alkoxy", as used alone or in combination herein, refers to the moiety RO-, where R is alkyl, alkenyl, or aryl as defined above.

[0019] The term "halo", as used alone or in combination herein, refers to an element of Group VIIIA of the Periodic Table (halogen). Depending on the chemical environment, halo can be in the neutral or anionic state.

[0020] Terms not specifically defined herein are given their ordinary meaning in the art.

[0021] [I. Proximity-Based Labeling Compositions] To selectively identify various features, including protein-protein interactions on the cell membrane Compositions are described herein for providing a microenvironment mapping platform that can be operably manipulated as such In some embodiments, the composition includes a catalyst and a protein labeling agent wherein the catalyst activates the protein labeling agent into a reactive intermediate. In some embodiments the catalyst can have an electronic structure that enables energy transfer to the protein labeling agent to form a reactive intermediate In some embodiments, for example the catalyst undergoes Dexter energy transfer with the protein labeling agent. In some embodiments the energy transfer can proceed by single electron transfer

[0022] In some embodiments, the energy transfer to the protein labeling agent can be due to the excited state of the catalyst electronic structure The excited state of the catalyst can be, for example, a singlet excited state or a triplet excited state The excited state of the catalyst can be generated by one or more mechanisms including energy absorption by the catalyst In some embodiments, the catalyst is a photocatalyst and the excited state is induced by the absorption of one or more photons. In other embodiments, the catalyst can be placed in an excited state by interaction with one or more chemical species in the ambient environment Alternatively, the energy transfer to the protein labeling agent, including electron transfer can occur from the ground state of the catalyst electronic structure

[0023] The energy transfer to the protein labeling agent, including electron transfer, forms a reactive intermediate of the protein labeling agent The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate If the protein or other biomolecule is not within the diffusion radius​ In this case, the reactive intermediate is quenched by the surrounding aqueous or water-based environment. The diffusion radius of the reactive intermediate can be adjusted according to the considerations of specific microenvironment mapping (proximity-based labeling) and can be limited to the nanometer scale. In some embodiments, for example, the diffusion radius of the reactive intermediate can be less than 10 nm, less than 5 nm, less than 4 nm, less than 3 nm, or less than 2 nm before quenching in an aqueous environment. Thus, the reactive intermediate can react or crosslink with a protein or other biomolecule within the diffusion radius or, if no protein or biomolecule is present, is quenched by the aqueous environment. In this way, through the coordinated efforts of the catalyst and the protein labeling agent, the local environment can be mapped with high resolution. Further, in some embodiments, the reactive intermediate can exhibit a t less than 5 ns, less than 4 ns, or less than 2 ns prior to quenching. In further embodiments, the diffusion radius can be extended to 5 - 500 nm by 1 / 2 extending the half-life of the reactive intermediate.

[0024] Any combination of a catalyst - protein labeling agent that exhibits the aforementioned electronic structural properties for energy transfer and for the generation of reactive intermediates and the accompanying binding of proteins or biomolecules can be used for microenvironment mapping. In some embodiments, the catalyst is a transition metal complex. The transition metal complex can exhibit a long-lived triplet excited state (T1) that promotes energy transfer to the protein labeling agent in some embodiments. The T1 state can have, for example, a t of 0.2 - 2 μs. 1 / 2may have. The transition metal complexes described herein are photocatalytic and may be, and in some embodiments, absorb light in the visible region of the electromagnetic spectrum. Upon absorption of electromagnetic radiation , the transition metal complex is excited to the S1 state and subsequently undergoes quantitative intersystem crossing to the T1 state. The transition metal catalyst can then return to the ground state S0 via short-range Dexter energy transfer to the protein labeling agent. Energy transfer to the labeling agent activates the labeling agent for reaction with the protein or other biomolecule. In some embodiments, the T1 state of the transition metal complex can be greater than 60 kcal / mol. The metal center can be selected from, for example, transition metals of the platinum group . In some embodiments, the metal center can be iridium.

[0025] The transition metal complex can have any composition and structure consistent with the principles of excitation and energy transfer to the protein labeling agent described above. In some embodiments, the photo catalytic transition metal complex is hexacoordinate. The transition metal photocatalyst of the proximity-based labeling composition described herein is, in some embodiments, represented by the following formula (I):

Chemical formula

[0026] derivative thereof. The ligand of the transition metal complex can also be selected from the species provided in Table I below .

[0027] Table I - Ligands of Photocatalytic Transition Metal Complexes

Table 1

[0028] In some embodiments, the photocatalytic transition metal complex of formula (I) is [Ir(dF(CF3)ppy)2(dt bbpy)](PF6), [Ir(dF(CF3)ppy)2(bpy)](PF6), or a derivative thereof.

[0029] Furthermore, the ligands of the photocatalytic transition metal complexes described herein, including the complex of formula (I), are water To enhance the solubility of transition metal complexes in a nonpolar or aqueous-based environment, one or more polar or hydrophilic moieties may be modified. Suitable moieties include, but are not limited to, alkylene oxy groups such as carboxyl, hydroxyl, and / or polyethylene glycol.

[0030] As further described herein, the transition metal complex can incorporate reactive functional groups for coupling biological molecule binders. For example, in some embodiments, transition metal catalysts, including the transition metal photocatalyst of formula (I), can include one or more click chemistry moieties including, but not limited to, BCN (bicyclononine, bicyclo[6.1.0]nonine), DBCO (dibenzocyclooctyne), TCO (trans-cyclooctene), tetrazine, alkyne, and azide.

[0031] The catalyst operable for energy transfer to a protein labeling agent that generates a reactive intermediate may, in some embodiments, include a non-transition metal catalyst. Various organic catalysts for use in the proximity- based labeling compounds and methods described herein can, in some embodiments, include thioxanthone, phenothiazine, flavin, phenoxazine, benzophenothiazine, coumarin, acetophenone, or a benzophenone group. Optionally, the organic catalyst can include a triarylmethane group, rose bengal, porphyrin, chlorin, bacteriochlorin, methylene blue, acridine dye, xanthene dye, or arylmethane dye. The non-transition metal organic catalyst can have the electronic structure and / or other properties of the transition metal complex described herein, which is tan ​​​ Including singlet and / or triplet excited states for energy transfer to a protein labeling agent . Energy transfer from an organic catalyst to a protein labeling agent can occur via any of the mechanisms described herein, such as Dexter energy transfer or single electron transfer. In some embodiments, the organic catalyst is a photocatalyst, and the excited state for energy transfer to the protein labeling agent is generated by absorption of one or more photons by the organic catalyst. For example , the organic catalyst can absorb light in the visible region of the electromagnetic spectrum.

[0032] The protein labeling agent receives energy transfer from the catalyst to form a reactive intermediate. The reactive intermediate reacts or crosslinks with a protein or other biomolecule within the diffusion radius of the reactive intermediate. The diffusion radius of the reactive intermediate is as described above. The specific identity of the protein labeling agent can be selected according to several considerations, including the identity of the catalyst, the nature of the reactive intermediate formed, the lifetime and diffusion radius of the reactive intermediate.

[0033] For example, in embodiments where the catalyst is a transition metal photocatalyst, the protein labeling agent can be diazirine. Triplet energy transfer from the excited state photocatalyst can promote diazirine to its triplet (T1) state. The diazirine triplet undergoes N2 dissociation to release a free triplet carbene, which reaches a reactive singlet state on the picosecond time scale (t <1 ns), which crosslinks with nearby proteins or is quenched in an aqueous environment. Figure 1 shows several embodiments described herein. 1 / 2 <1 ns), which crosslinks with nearby proteins or is quenched in an aqueous environment. Figure 1 shows several embodiments described herein. Energy transfer between an iridium photocatalyst of formula (I) and a diazirine protein labeling agent according to the morphology is shown. In particular, the electromagnetic radiation used to generate the excited state of the transition metal catalyst does not activate the diazirine to a reactive carbene. In some embodiments, the extinction coefficient of the transition metal complex is three to five orders of magnitude greater than that of the diazirine. Any diazirine that is consistent with the technical principles discussed herein, including the reaction mechanism of FIG. 1, can be used. For example, diazirine sensitization can be extended to various p- and m-substituted aryltrifluoromethyl diazirines having useful payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. FIG. 2 shows various diazirines that can be engineered to form reactive intermediates via energy transfer from a transition metal photocatalyst for the labeling of proteins or other biomolecules according to some embodiments. As shown in FIG. 2, the diazirine can be functionalized with a marker such as biotin. In one embodiment, the marker is desthiobiotin. The marker can assist in the identification of proteins labeled by a protein labeling agent. The marker can be useful, for example, in assay results via Western blot and / or other analytical techniques. The marker can include alkynes, azides, FLAG tags, fluorophores, and chloroalkane functionality in addition to biotin and desthiobiotin. In some embodiments, the transition metal complex has an extinction coefficient that is three to five orders of magnitude greater than that of the diazirine. 。

[0034] Any diazirine that is consistent with the technical principles discussed herein, including the reaction mechanism of FIG. 1, can be used. For example, diazirine sensitization can be extended to various p- and m-substituted aryltrifluoromethyl diazirines having useful payloads for microscopy and proteomics applications, including free carboxylic acids, phenols, amines, alkynes, carbohydrates, and biotin groups. FIG. 2 shows various diazirines that can be engineered to form reactive intermediates via energy transfer from a transition metal photocatalyst for the labeling of proteins or other biomolecules according to some embodiments. As shown in FIG. 2, the diazirine can be functionalized with a marker such as biotin. In one embodiment, the marker is desthiobiotin. The marker can assist in the identification of proteins labeled by a protein labeling agent. The marker can be useful, for example, in assay results via Western blot and / or other analytical techniques. The marker can include alkynes, azides, FLAG tags, fluorophores, and chloroalkane functionality in addition to biotin and desthiobiotin.

[0035] ​​​​​​In some embodiments where the catalyst is a transition metal photocatalyst, the protein labeling agent is an azide. Triplet energy transfer from the excited state photocatalyst can promote nitrene formation from the azide. The reactive nitrene either crosslinks with neighboring proteins or is quenched in an aqueous environment. Any azide that can be engineered to undergo energy transfer by a transition metal photocatalyst for nitrene formation can be used. In some embodiments, the azide is an aryl azide.

[0036] In some embodiments where the catalyst is an organic catalyst, the protein labeling agent can include one or more moieties for receiving single electron transfer from the organic catalyst. The single electron transfer to the protein labeling agent can generate reactive radicals having a diffusion radius and / or lifetime as described herein for reacting with the protein in the local environment of the organic catalyst. For example the organic catalyst can be a flavin photocatalyst. The excited flavin photocatalyst can generate reactive phenoxyl radicals via single electron transfer with a phenolic moiety. FIG. 3 shows the reaction mechanism for flavin-based protein labeling according to some embodiments. As shown in FIG. 3, the phenolic protein labeling agent is functionalized with a tag or marker to assist in the identification of the protein labeled by that protein labeling agent.

[0037] [II. Conjugate] In another aspect, conjugates (complexes) for proximity-based labeling are described herein. The conjugate includes a catalyst bound to a biomolecule binder. The biomolecule binder The catalyst linked to the agent includes the transition metal catalysts and organic catalysts detailed in Section I above and may include any of the catalysts described herein. Additionally, the biomolecule binder may, in some embodiments, include proteins, polysaccharides, nucleic acids, or lipids. Optionally, the biomolecule binder may include a multivalent display system including proteins, polysaccharides, nucleic acids, or lipids. In certain embodiments, the biomolecule binder may be a small molecule ligand having a specific binding affinity for a target protein. The biomolecule binder can be used to position the catalyst in the desired extracellular environment for proximity labeling and related assays. Thus, the specific identity of the biomolecule binder is selected according to the chemical and / or steric requirements of the desired target site for the placement of the catalyst in the proximity-based labeling process. Any biomolecular target site can be selected, and the target site is not limited in the present disclosure. In certain embodiments, the target site can be a protein for studying protein-protein interactions, which includes interactions with cell membrane receptors. In some embodiments, for example, the biomolecule binder is an antibody, such as a secondary antibody that interacts with a primary antibody bound to a desired antigen. In other embodiments, the biomolecule binder is a ligand having specificity for a protein receptor on the cell membrane, such as a G protein-coupled receptor.

[0038] The biomolecule binder can be bound to the catalyst. In some embodiments, the catalyst includes a reactive handle or functional group for coupling the biomolecule binder. In some embodiments, for example, the catalyst includes BCN, DBCO, TCO, tetrazine, alkyne, and azide. ​may include one or more click chemistry moieties, including but not limited to these. Figure 4 shows various transitions of formula (I) having reactive functionality for coupling a biomolecule binder. Figure 4 shows a metal photocatalyst. As shown in Figure 4, various lengths can be used between the reactive functional group and the coordination ligand. of an alkylene oxide linker. The length of an alkylene oxide linker such as ethylene oxide can be selected according to several considerations, including the steric conditions of the target site. Figure 5 shows several transition metal photocatalysts of formula (I) that form conjugates with biomolecule binders according to several embodiments.

[0039] [III. System for proximity-based labeling] In another aspect, a system for proximity-based labeling is described herein. The system includes, for example, a conjugate including a catalyst bound to a biomolecule binder and a protein labeling agent activated by the catalyst for binding to a protein. The conjugate can include any of the catalysts and biomolecule binders described herein, including the embodiments detailed in Section II above. For example, the catalyst can have an electronic structure for activating the protein labeling agent to a reactive intermediate via energy transfer. Further more, the protein labeling agent can include any of the labeling agents described herein, including the protein labeling agents described in Section I above. The specific identities of the conjugate and the associated protein labeling agent can be selected according to several considerations, such as the chemical nature and / or steric requirements of the biological environment to be mapped in the proximity-based labeling system.

[0040] The systems for proximity-based labeling described herein can be used for various applications. In some embodiments, the system enables targeted identification that allows conjugate and related protein labeling agents to identify one or more molecules in a biological context by proteomics. Further, a system comprising a conjugate and a protein labeling agent facilitates interactome mapping. Targeting the conjugate and the protein labeling agent enables the detection and identification of one or more molecules and adjacent interacting factors in a biological context by proteomics. Identification of such molecules by the systems described herein can enable the enrichment and / or purification of such molecules and adjacent interacting factors. In addition, a system comprising a conjugate and a protein labeling agent further enables the detection and identification of one or more molecules in a biological context via microscopy.

[0041] [IV - Method of Proximity-Based Labeling] In another aspect, a method of proximity-based labeling is described herein. The proximity-based labeling method provides a conjugate comprising a catalyst linked to a biomolecule binder, and activates a protein labeling agent to a reactive intermediate by the catalyst, and binds the reactive intermediate to the protein. The conjugate can comprise any catalyst and biomolecule binder described herein, including the embodiments detailed in Section II above. It is possible to include any of the labeling agents described herein, including the chemical agent. Conjugate and the specific identity of the related protein labeling agent depend on the chemical nature and / or steric requirements of the biological environment to be mapped in the proximity-based labeling system and can be selected according to several considerations. In some embodiments of proximity-based labeling, the catalyst can be provided in the absence of the biomolecule binder.

[0042] The methods described herein can be utilized to map various biological environments, including local regions of the cell membrane and / or the local extracellular environment. A conjugate containing a catalyst and a biomolecule binder can be targeted to a specific local region of the cell membrane (e.g., a target receptor). Activation of the protein labeling agent can identify the protein(s) and / or other molecules in the target local region. Notably, the activated protein labeling agent can also identify or label molecules associated with another cell in contact with the targeted cell region. Thus, the systems and methods described herein can elucidate and map cell-cell interactions and the cell-cell environment. The methods described above enable interactome mapping and allow for the identification of one or more molecules and adjacent interacting factors in a biological context by proteomics. Identification of such molecules by the methods described herein can enable the enrichment and / or purification of such molecules and adjacent interacting factors.

[0043] Figure 6A shows proximity-based labeling as described herein, according to some embodiments. ​​​​​​​​​​​​​Shows the operation of the system and method. As shown in FIG. 6A, a conjugate containing a photocatalyst and an antibody targets a specific local region of the cell membrane. After the photocatalyst is irradiated to generate an excited state, energy transfer occurs to the diazirine protein labeling agent. The diazirine protein labeling agent contains tags for identification purposes in the analysis. The energy transfer activates the protein labeling agent into a reactive carbene. The reactive carbene binds to proteins and / or other biomolecules within a tight diffusion radius, as described herein. The photocatalyst activation or the diazirine labeling agent outside the energy transfer radius is not activated. Further, proteins and / or other biomolecules outside the diffusion radius of the reactive carbene are not labeled. In this way, high-resolution proximity-based labeling can be achieved. After the conjugate targets a specific local region of the cell membrane and the photocatalyst is irradiated to generate an excited state, energy transfer occurs to the diazirine protein labeling agent. The diazirine protein labeling agent contains tags for identification purposes in the analysis. The energy transfer activates the protein labeling agent into a reactive carbene. The reactive carbene binds to proteins and / or other biomolecules within a tight diffusion radius, as described herein. The photocatalyst activation or the diazirine labeling agent outside the energy transfer radius is not activated. Further, proteins and / or other biomolecules outside the diffusion radius of the reactive carbene are not labeled. In this way, high-resolution proximity-based labeling can be achieved. After the conjugate targets a specific local region of the cell membrane and the photocatalyst is irradiated to generate an excited state, energy transfer occurs to the diazirine protein labeling agent. The diazirine protein labeling agent contains tags for identification purposes in the analysis. The energy transfer activates the protein labeling agent into a reactive carbene. The reactive carbene binds to proteins and / or other biomolecules within a tight diffusion radius, as described herein. The photocatalyst activation or the diazirine labeling agent outside the energy transfer radius is not activated. Further, proteins and / or other biomolecules outside the diffusion radius of the reactive carbene are not labeled. In this way, high-resolution proximity-based labeling can be achieved.

[0044] FIG. 6B shows the operation of the proximity-based labeling system and method described herein for the identification and verification of membrane G protein-coupled receptors (GPCRs) according to some embodiments. As provided in FIG. 6B, the conjugate contains a photocatalyst coupled to a biomolecule binding agent having specificity for the GPCR. The conjugate binds to the local site of the GPCR. After irradiating the photocatalyst with blue light to provide an excited state, energy transfer occurs to the diazirine protein labeling agent. The diazirine protein labeling agent contains an affinity handle or tag for identification purposes in the analysis. This energy transfer activates the protein labeling agent into a reactive carbene. The reactive carbene binds to proteins and / or other biomolecules within a tight diffusion radius, as described herein. Multiple After irradiating the photocatalyst with blue light to provide an excited state, energy transfer occurs to the diazirine protein labeling agent. The diazirine protein labeling agent contains an affinity handle or tag for identification purposes in the analysis. This energy transfer activates the protein labeling agent into a reactive carbene. The reactive carbene binds to proteins and / or other biomolecules within a tight diffusion radius, as described herein. At the local GPCR site, multiple A labeling event can occur, leading to signal amplification.

[0045] These and other embodiments are further illustrated by the following non-limiting examples.

Example

[0046] Example 1 - Photocatalytic Diazirine Sensitization for Protein Labeling It has been demonstrated that the carbene generated via photocatalytic diazirine sensitization can label proteins. As shown in FIG. 7A, photocatalyst 3 was combined with diazirine 4 and BSA in DPBS to obtain a reaction mixture with a total solution volume of 100 μL and the desired component concentrations. Photocatalyst 3 is shown in FIG. 7B and Table I summarizes the sample conditions. These samples were then either placed in the dark or irradiated with UV (375 nm) light or visible (450 nm) light at 100% intensity for 10 minutes in a bioreactor. Then, 30 μL of the sample was taken out and combined with 10 μL of 4× reducing Laemmli sample buffer (5% β-mercaptoethanol), vortexed, and heated at 9 5 °C for 10 minutes. Then, 10 μL of each sample was analyzed by Western blot.

[0047] Table I. Sample Conditions

Table 2

[0048] Biotinylation of BSA was detected for Sample 2. When a solution of BSA and biotinylated diazirine probe was irradiated with 450 nm light (Sample 3), the degree of biotinylation was less than 0.5% and it was established that the background signal of diazirine was minimal at this wavelength. ​However, in the presence of water-soluble photocatalyst 3, catalyst-dependent biotinylation of BSA was observed. The photocatalytic labeling of BSA was further confirmed by mass spectrometry of the intact protein. Unlike traditional enzyme-based labeling methods, this approach uses Continuous delivery of visible light is required to maintain diazirine sensitization via photocatalyst 3. By utilizing this feature, we can determine how the light source can be turned on or off, as shown in FIG. 7A. It was demonstrated how fine temporal control over the labeling process could be obtained.

[0049] Example 2 - Antibody-photocatalyst conjugates and their use for proximity-based labeling A general entry point for spatially targeted photocatalytic proximity labeling on cell surfaces As an index, a secondary antibody-photocatalyst conjugate was prepared: goat anti-mouse (Gt / α-Ms). The antibody was first synthesized by reaction with azidobutyric acid N-hydroxysuccinimide ester. functionalized with an isopropyl group and then coupled to an alkyne-containing iridium catalyst via click chemistry. The antibody-photocatalyst ratio was 1:6. The iridium catalyst was iridium complex 3 shown in Figure 7B. do.

[0050] Next, to address protein targeting labeling on surfaces, we used α-human immunoglobulin Human Fc-tagged vascular endothelial growth factor receptor 2 (VEGF) bound to IgG agarose beads A model system was prepared containing the R2 protein and the epidermal growth factor receptor (EGFR) protein ( These beads were then incubated sequentially with 20αMs / α-VEGFR2 antibody and Ir-Gt / α-Ms. The iridium catalyst was then brought into close proximity with the VEGFR2 protein on the bead surface. - Irradiating these beads with 450 nm light in the presence of biotin probe selectively labeled VEGFR2 rather than EGFR. It was obtained that. When Ms / α-EGFR was used as the primary antibody (1°Ab), the selectivity of labeling was reversed. As shown in Figure 8B, in a similar experiment using peroxidase-based labeling, it was important to note that EGFR or VEGFR2 could not be distinguished. In the case of, the selectivity of labeling was reversed. As shown in Figure 8B, in a similar experiment using peroxidase-based labeling, it was important to note that EGFR or VEGFR2 could not be distinguished. In the case of, the selectivity of labeling was reversed. As shown in Figure 8B, in a similar experiment using peroxidase-based labeling, it was important to note that EGFR or VEGFR2 could not be distinguished. In the case of, the selectivity of labeling was reversed. As shown in Figure 8B, in a similar experiment using peroxidase-based labeling, it was important to note that EGFR or VEGFR2 could not be distinguished.

[0051] Example 3 - Microenvironment mapping on the cell membrane Antibody-targeted photocatalytic diazirine activation (i.e., μMap) was applied to the surface of live cells. For these experiments, the addition of antibodies to the cell surface was maintained at 4°C to limit antibody-mediated protein bridging. CD45, a very abundant tyrosine phosphatase on the surface of T cells involved in antigen receptor signaling, was initially selected as the target. Western blot analysis of CD45-targeted μMap on Jurkat cells showed light- and time-dependent protein biotinylation compared to an isotype-targeted control (Figure 9A). Antibody-targeted photocatalytic diazirine activation (i.e., μMap) was applied to the surface of live cells. For these experiments, the addition of antibodies to the cell surface was maintained at 4°C to limit antibody-mediated protein bridging. CD45, a very abundant tyrosine phosphatase on the surface of T cells involved in antigen receptor signaling, was initially selected as the target. Western blot analysis of CD45-targeted μMap on Jurkat cells showed light- and time-dependent protein biotinylation compared to an isotype-targeted control (Figure 9A). Antibody-targeted photocatalytic diazirine activation (i.e., μMap) was applied to the surface of live cells. For these experiments, the addition of antibodies to the cell surface was maintained at 4°C to limit antibody-mediated protein bridging. CD45, a very abundant tyrosine phosphatase on the surface of T cells involved in antigen receptor signaling, was initially selected as the target. Western blot analysis of CD45-targeted μMap on Jurkat cells showed light- and time-dependent protein biotinylation compared to an isotype-targeted control (Figure 9A). Antibody-targeted photocatalytic diazirine activation (i.e., μMap) was applied to the surface of live cells. For these experiments, the addition of antibodies to the cell surface was maintained at 4°C to limit antibody-mediated protein bridging. CD45, a very abundant tyrosine phosphatase on the surface of T cells involved in antigen receptor signaling, was initially selected as the target. Western blot analysis of CD45-targeted μMap on Jurkat cells showed light- and time-dependent protein biotinylation compared to an isotype-targeted control (Figure 9A). Antibody-targeted photocatalytic diazirine activation (i.e., μMap) was applied to the surface of live cells. For these experiments, the addition of antibodies to the cell surface was maintained at 4°C to limit antibody-mediated protein bridging. CD45, a very abundant tyrosine phosphatase on the surface of T cells involved in antigen receptor signaling, was initially selected as the target. Western blot analysis of CD45-targeted μMap on Jurkat cells showed light- and time-dependent protein biotinylation compared to an isotype-targeted control (Figure 9A). Antibody-targeted photocatalytic diazirine activation (i.e., μMap) was applied to the surface of live cells. For these experiments, the addition of antibodies to the cell surface was maintained at 4°C to limit antibody-mediated protein bridging. CD45, a very abundant tyrosine phosphatase on the surface of T cells involved in antigen receptor signaling, was initially selected as the target. Western blot analysis of CD45-targeted μMap on Jurkat cells showed light- and time-dependent protein biotinylation compared to an isotype-targeted control (Figure 9A). Next, quantitative proteome analysis based on tandem mass tag (TMT) of streptavidin-enriched proteins was used to identify CD45 and two known binding partners (CD45AP and CD2) as part of a broader subset of enriched cell membrane proteins (Figure 9B). Next, quantitative proteome analysis based on tandem mass tag (TMT) of streptavidin-enriched proteins was used to identify CD45 and two known binding partners (CD45AP and CD2) as part of a broader subset of enriched cell membrane proteins (Figure 9B). Next, quantitative proteome analysis based on tandem mass tag (TMT) of streptavidin-enriched proteins was used to identify CD45 and two known binding partners (CD45AP and CD2) as part of a broader subset of enriched cell membrane proteins (Figure 9B).

[0052] Having obtained a proof of concept for cell surface labeling, it was examined whether μMap could distinguish spatially separated microenvironments on the same cell membrane. For this purpose, CD29 and CD47 were selected as ideal targets that do not have a known spatial co-association on the cell surface. Indeed, on Jurkat cells Having obtained a proof of concept for cell surface labeling, it was examined whether μMap could distinguish spatially separated microenvironments on the same cell membrane. For this purpose, CD29 and CD47 were selected as ideal targets that do not have a known spatial co-association on the cell surface. Indeed, on Jurkat cells Having obtained a proof of concept for cell surface labeling, it was examined whether μMap could distinguish spatially separated microenvironments on the same cell membrane. For this purpose, CD29 and CD47 were selected as ideal targets that do not have a known spatial co-association on the cell surface. Indeed, on Jurkat cells μ-mapping of CD45, CD29, or CD47 was performed using known interactors (CD29:CD49D, CD45:CD45A Unique proteins containing both P:CD2) and previously unknown interactors This resulted in enrichment of the quality set (Figure 9B). Importantly, several proteins were targeted shared between pairs of proteins but shared by all three proteins This demonstrates the ability of μMap to distinguish between unrelated microenvironments. When using state-of-the-art peroxidase-based proximity labeling methods, cell surface CD45 and The associated proteins were not selectively separated from CD29 or CD47 (Fig. 9C).

[0053] Example 4 - Microenvironment mapping on cell membranes The selectivity of the compositions and methods described herein is due to the ability to target PD-L1 proximity proteins in B cells. It has also been used to investigate the protein interactome. 1 acts as an immune checkpoint ligand that can accelerate tumor progression through suppressing T cell activity. In this event, PD-L1-targeted μ-MAP The tumor necrosis factor receptor family member CD30 and the immunosuppressant receptor 5, CD300 A (Fig. 10A) was identified as a possible novel interactor based on significant enrichment. These results provide new insight into the microenvironment of checkpoint proteins. This paper highlights the potential of μ-mapping to provide

[0054] Further investigation of enriched subsets of proteins identified by PD-L1μ mapping To verify this, targeted labeling of 10 of these two highly enriched proteins was performed. Therefore, targeted μ-mapping of these proteins within the PD-L1 microenvironment should provide a similar condensed list to verify their spatial relatedness. Indeed, μ-mapping by induction of α-CD30, α- CD300A, and α-PD-L1 identified the same set of 12 surface receptors (Figure 10B).

[0055] Example 5 - Intra-synaptic / extra-synaptic μ-mapping within a two-cell system It is well recognized that the development of new cancer treatment strategies requires an understanding of the mechanisms underlying cell-to-cell communication, particularly in the context of T cell activation and differentiation. Furthermore, considering that the localization of PD-L1 is found within the T cell / antigen presenting cell (APC) immune synapse (i.e., at the interface between two interacting immune cells), it was hypothesized that PD-L1-directed μ-mapping using the complexes and methods described herein would result in not only biotinylation (cis-labeling) of the PD-L1-expressing APC surface but also biotinylation (trans-labeling) of the adjacent synapse T cell (Figure 11A). Additionally, as an important control experiment, it was hypothesized that μ-maps with minimized diffusion radii would prevent biotinylation of the cell membranes of distant trans when targeting proteins excluded from the synapse such as CD45RO.

[0056] In a two-cell system composed of PD-L1-expressing JY-B lymphocytes as antigen-presenting cells and Jurkat T lymphocytes that specifically express PD-1 and CD45RO isoforms, μ-mapping targeting PD-L1 and CD45 was evaluated. Immune cell-APC interactions involve multiple receptor types Considered to be driven by the binding of proteins (e.g., adhesiveness, co-stimulatory / co-inhibitory, and T cell receptor (TCR)-major histocompatibility complex (MHC)5), staphylococcal enterotoxin D (SED) was used to promote MHC class II and TCR engagement and thus promote B cell / T cell immune synapse formation and signaling (Figure 11A). After application of the μ-map protocol based on blue light irradiation, the degree of cis / trans labeling selectivity was assayed via flow cytometry analysis. As expected, the PD-L1-targeted μ-map resulted in both cis and trans cell labeling, whereas the CD45RO-targeted μ-map resulted in selective cis labeling on CD45RO-expressing Jurkat cells without any labeling of adjacent B cells (Figures 11B and 11C). In stark contrast to the μ-map, peroxidase-based proximity labeling of PD-L1 or CD45RO within this two-cell line resulted in complete labeling of both cell types within 30 seconds, which was clearly visualized by flow cytometry and confocal microscopy (Figures 11B, 11C and 11D). In contrast, the PD-L1-targeted μ-map showed high selectivity for trans labeling only in the cis and trans cell contact regions (Figure 11D). Importantly, application of the μ-map technology to PD-1 in the Jurkat-JY co-culture system resulted in a reverse trend of cis and trans cell labeling. Collectively, these findings clearly demonstrate that the ability of the μ-map to elucidate protein-protein interactions can be directly applied to highly selective labeling of dynamic interfaces within complex multi-cell lines.

[0057] ​​​​​​​​Example 6 - Antibody-Photocatalyst Conjugates and Proximity-Based Labeling Therewith Labeling Conjugates comprising a flavin photocatalyst combined with a phenolic protein labeling agent can be used in the proximity-based labeling compositions and methods described herein FIG. 12A shows bead-based protein labeling in which CD45-Fc or PDL1-Fc fusion proteins are bound on the same beads, and then bound to a primary antibody and a secondary antibody flavin conjugate (AFC). Next, CD45 or PDL1 is labeled in the presence of biotin phenol and visible light. FIG. 12B is a Western blot analysis of light-dependent CD45 biotinylation at the indicated time points FIG. 12C is a schematic diagram of CD45RO or CD45RA targeted cell labeling using AFC in a mixed T cell population. FIG. 12D is a flow cytometry analysis of the time course of photolabeling of biotinylation of CD45RA+ or CD45RO+ T cells using isotype (top), α-CD45RA+AFC (middle), and α-CD45RO+AFC (bottom) FIG. 12D is a flow cytometry analysis of the time course of photolabeling of biotinylation of CD45RA+ or CD45RO+ T cells using isotype (top), α-CD45RA+AFC (middle), and α-CD45RO+AFC (bottom) FIG. 12D is a flow cytometry analysis of the time course of photolabeling of biotinylation of CD45RA+ or CD45RO+ T cells using isotype (top), α-CD45RA+AFC (middle), and α-CD45RO+AFC (bottom)

[0058] FIG. 12D is a flow cytometry analysis of the time course of photolabeling of biotinylation of CD45RA+ or CD45RO+ T cells using isotype (top), α-CD45RA+AFC (middle), and α-CD45RO+AFC (bottom) FIG. 12D is a flow cytometry analysis of the time course of photolabeling of biotinylation of CD45RA+ or CD45RO+ T cells using isotype (top), α-CD45RA+AFC (middle), and α-CD45RO+AFC (bottom) FIG. 12D is a flow cytometry analysis of the time course of photolabeling of biotinylation of CD45RA+ or CD45RO+ T cells using isotype (top), α-CD45RA+AFC (middle), and α-CD45RO+AFC (bottom) FIG. 12D is a flow cytometry analysis of the time course of photolabeling of biotinylation of CD45RA+ or CD45RO+ T cells using isotype (top), α-CD45RA+AFC (middle), and α-CD45RO+AFC (bottom)

[0059] Example 7 - Microenvironment Mapping on the Cell Membrane FIG. 13A is a schematic diagram showing proximity photolabeling of CD45 on Jurkat cells using a secondary antibody flavin conjugate (AFC). FIG. 13B is a Western blot analysis of CD45 target labeling of Jurkat cells The biotinylation level increased as a function of visible light irradiation time in CD45-directed labeling, but not in the isotype control FIG. 13C shows CD45-directed labeling FIG. 13C shows CD45-directed labeling Confocal imaging of the cells, showing that biotinylation (magenta staining) is limited to the cell surface and depends on the light exposure time. The nucleus is labeled with Hoechst staining, and the scale bar indicates 5 mm. Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments). Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments). Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments). Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments). Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments). Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments). Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments). Figure 13D provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of CD45 vs. isotype-targeted biotinylation in Jurkat cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are indicated by blue dots, known CD45 interactors within this enriched group are indicated by orange dots, and CD45 is indicated by red dots (n = 3 experiments).

[0060] Example 8 - Microenvironment Mapping on the Cell Membrane Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 Figure 14A is a schematic diagram showing proximity photolabeling of PDL1 on JY-PDL1 cells using a secondary antibody flavin conjugate (AFC). Figure 14B provides a volcano plot of significance vs. enrichment fold for targeted biotinylation of PDL1 vs. isotype-targeted biotinylation on PDL1-expressing Raji cells according to 2-minute blue light irradiation, harvesting, capture on streptavidin beads, and quantitative mass spectrometry-based proteomics analysis. Significantly enriched proteins (with an FDR-corrected p-value of less than 0.05 and an enrichment fold over the isotype-targeted control of more than 2.5 (1.32 log2-fold change)) are shown as orange or blue dots, and PDL1 is shown as a red dot (n = 3 experiments). Figure 14C shows PDL1 on JY and Raji cells expressing PDL1 A Venn diagram of significantly enriched proteins identified from target labeling. Shown in the center are the proteins identified in both cell types. Figure 14D is a list of significantly enriched proteins identified from PDL1 targeted in JY and Raji cells having known PDL1-related functions. Figure 14E is a STRING protein-protein interaction network and GO term analysis of significantly enriched proteins for the PDL1 targeting experiment. The shading indicates attributes among a broad set of gene ontology (biological process) terms. Nodes with multiple shading tones indicate attributes in multiple terms. Thick edges indicate experimental evidence of interactions from StringDB, and thin edges indicate interactions from other sources.

[0061] Example 9 - In-synapse / extra-synapse μ-mapping within a two-cell system Figure 15A is a schematic diagram showing a two-cell line consisting of recombinant Jurkat and Raji cells. Antibody target labeling with a photocatalyst (PC) or peroxidase (HRP) against PDL1 is illustrated. Figure 15B is a flow cytometry analysis where biotinylation is detected in both Raji and Jurkat cells using antibody flavin conjugate (AFC) for PDL1 targeting, but not detected with isotype targeting or in the absence of visible light irradiation. No intercellular labeling was observed between PDL1-labeled Raji cells and suspended A375 cells. Figure 15C is a confocal microscopy image of a Raji-Jurkat two-cell line with PDL1 targeting on Raji cells, showing labeling at both the cell contact points on Raji and Jurkat cells when AFC is used. On the other hand, in HRP, excessive labeling was observed in both cell types (indicated by white arrows). Cells were imaged for biotinylation, CD3 surface expression, and nuclei. Figure 15D is a schematic diagram showing a two-cell line consisting of recombinant Jurkat at and Raji cells. Photocatalyst (PC) against CD45 or antibody-target labeling by peroxidase (HRP) is illustrated. Figure 15E is flow cytometry analysis, and the target labeling of CD45RO (known to be excluded from synapses) on Jurkat cells resulted in low-level inter-Raji cell labeling using AFC, while using HRP resulted in almost quantitative labeling.

[0062] Example 10 - Selective Protein Labeling Using Small Molecule-Based Conjugates To first confirm the ability of small molecule iridium conjugates to directly label specific proteins, a simple two-protein biochemical assay was designed. A carbonic anhydrase (CA) as the target protein and bovine serum albumin (BSA) as the competing protein were selected at equimolar ratios. According to this hypothesis, irradiation of this mixture in the presence of biotin-tagged diazirine and sulfo honamide-iridium conjugate would result in selective labeling of CA rather than BSA. Analysis of the labeling ratio by immunoblot using streptavidin would provide an indicator of both reaction efficiency and selectivity. Recognizing the possible influence of the iridium catalyst on ligand binding, the catalyst-ligand conjugate was prepared with a PEG3 (triethylene glycol) linker to spatially separate the two components. Happily, after irradiation with 450 nm light for 10 minutes in the presence of biotin-peg3-diazirine, 3 ​​​​​ Labeling biased towards the target protein CA was observed at 1.5:1. Figure 16 shows the configuration and results of experiments involving sulfonamide-iridium conjugates and biotin-tagged diazirine.

[0063] In contrast, when labeling was performed in the presence of the non-conjugated photocatalyst, BSA was selectively biotinylated over CA at a ratio of 5:1. Overall, this represents a three-fold increase in labeling selectivity when using the Ir-ligand conjugate, providing confidence in this approach for target ID. Importantly, this selectivity was completely abolished when the targeting experiment was carried out with an excess of non-conjugated sulfonamide ligand, confirming that the observed selectivity was the result of ligand-protein binding events. Furthermore, significant enrichment of CA was also found when labeling was performed in HEK 293T cell lysates, verifying that this methodology is also compatible with one of the most complex biological settings.

[0064] Example 11 - Selective Protein Labeling Using Small Molecule-Based Conjugates The generality of small molecule-based catalytic conjugates as a platform for ligand-directed target identification of proteins was further investigated. Using the copper-catalyzed azide-alkyne click reaction (CuAAC) between the iridium alkyne of Figure 4 and a series of ligand-azide conjugates, small molecule-iridium conjugates were readily prepared in an operationally simple manner. The ability of the ligand-iridium conjugate to label its corresponding protein target was determined by their selectivity as compared to BSA, as shown in Figure 17. Bruto Dasatinib, a commercially available therapeutic agent for myeloid leukemia, which exhibits nanomolar binding to n-tyrosine kinase (BTK, 5 nM), was first examined. The corresponding dasatinib-I r conjugate resulted in a 12-fold increase in BTK-biased labeling compared to the BSA lane by Western blotting. The bromodomain inhibitor JQ-114 (50 nM binding to BRD4) was similarly effective in directing the label, providing a 6-fold increase in BRD4 labeling relative to BSA. Next, lenalidomide, a less potent binder and a therapeutic agent for multiple myeloma, was investigated. Lenalidomide acts as a molecular glue with the cereblon (CRBN) protein. This ligand exhibits a lower binding affinity (178 nM), but was found to be equally effective in inducing the photocatalytic targeting ID approach described herein, resulting in a 3-fold change relative to the off-compete control. Collectively, these data suggest that the signal amplification brought about by the catalytic nature of this labeling platform can overcome the long-standing challenges of PAL with weaker affinity ligands. Furthermore, it was found that the selectivity of labeling can be increased with increasing irradiation time, which further supports our catalytic labeling hypothesis. In addition to small molecules, it was also sought to establish whether other targeting modalities are compatible with the catalytic labeling technology described herein. ATSP7041, an α-helix stapled cyclic peptide targeting the E3 ligase MDM2, can be readily conjugated to the photocatalyst via a modified azidolyzine

[0065] ​​​​​​​​​​It was achieved. As provided in Figure 17, a significant enrichment (3-fold) of the target protein was observed compared to the free photocatalyst control. The synthesis of the corresponding inert cyclic peptide having D-phenylalanine did not result in labeling that favored MDM2 over BSA, which further confirms that the interaction is substrate-specific and not based on background affinity.

[0066] Example 12 - Investigation of Small Molecule Protein-Protein Interaction (PPI: Protein-Protein Interactio n) PPI is essential for cell function and constitutes a challenging class for small molecule drug discovery. This challenge stems in part from the transient nature of these interactions, which makes it difficult to detect them biochemically. However, many prominent small molecule ligands are known to bind to protein complexes or to proteins that function through dynamic complexes. Therefore, a method that can effectively discriminate the components of a protein complex will be an important tool for PPI research. First, we set out to study AT7519, a small molecule that binds to cyclin-dependent kinase 2 (CDK2). CDK2 is well known to form a PPI with cyclin A protein and contribute to the regulation of the cell cycle. As shown in Figure 18, the exposure of the Ir conjugate AT7519 to recombinant cyclin A, CDK2, and (as a control) BSA showed significant enrichment of both CDK2 (5-fold) and cyclin A (2-fold) compared to the BSA control, exemplifying the ability of this methodology to capture a transient interaction that would otherwise be difficult to examine. A (2-fold) compared to the BSA control, exemplifying the ability of this methodology to capture a transient interaction that would otherwise be difficult to examine. interaction that would otherwise be difficult to examine. interaction that would otherwise be difficult to examine.

[0067] Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Example 13 - Investigation of Protein-Protein Interaction (PPI) Western blotting and MS analysis were used to interrogate whether the tight labeling of the μ-map platform using the conjugates and labeling agents described herein could be utilized to identify the ligand-binding site. To best examine this, we selected the 3-protein complex calcineurin A / FKBP12 / calcineurin B that binds to the macrolide tacrolimus. During the synthesis of a series of tacrolimus-Ir conjugates with different linker lengths, short linkers were found to result in labeling in the immediate vicinity of the binding site of the complex, enabling the footprinting of the molecular recognition site. However, as the length of the tether increased, the labeling radius increased to include other members of the protein complex, and the primary site of labeling changed. MS analysis of the PEG3 linker supported this data and showed labeled residues directly adjacent to the binding site (Figure 20 left).

[0068] Example 13 - Investigation of Protein-Protein Interaction (PPI) Western blotting and MS 2 analysis were used to interrogate whether the tight labeling of the μ-map platform using the conjugates and labeling agents described herein could be utilized to identify the ligand-binding site. To best examine this, we selected the 3-protein complex calcineurin A / FKBP12 / calcineurin B that binds to the macrolide tacrolimus. During the synthesis of a series of tacrolimus-Ir conjugates with different linker lengths, short linkers were found to result in labeling in the immediate vicinity of the binding site of the complex, enabling the footprinting of the molecular recognition site. However, as the length of the tether increased, the labeling radius increased to include other members of the protein complex, and the primary site of labeling changed. MS analysis of the PEG3 linker supported this data and showed labeled residues directly adjacent to the binding site (Figure 20 left). Next, a 4-protein complex in which adjacent proteins do not directly interact with the small molecule ligand was examined, demonstrating that labeling of protein interactors through space can be achieved. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. 2 Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst. Next, we initiated a search for the rapamycin / mTOR axis in which rapamycin recruits FKBP12 to the mTOR complex, resulting in the suppression of the immune response. Notably, as shown in Figure 19, upon irradiation, both proteins were enriched compared to the control (FKBP12: 2-fold, mTOR: 12-fold), despite the interference with the binding efficiency caused by the pendant Ir catalyst.

[0069] Next, a 4-protein complex in which adjacent proteins do not directly interact with the small molecule ligand was examined, demonstrating that labeling of protein interactors through space can be achieved. Next, a 4-protein complex in which adjacent proteins do not directly interact with the small molecule ligand was examined, demonstrating that labeling of protein interactors through space can be achieved. Therefore, the E3 ligase complex CRBN / DDB1 / Cul4a / RBX1 was selected. Consistent with the previous example described in this specification, when a short tether was used, only the primary target CRBN was labeled, but as the tether length increased, adjacent proteins in the E3 ligase complex could be captured by this technique. Proteomic analysis of the labeling reaction using a PEG3 linker showed, in addition to the molecular footprint of the small molecule binding site on CRBN, a labeling site on adjacent Cul4a, presumably arising from secondary protein-protein interactions (right in Figure 20). As an exemplary membrane target, adenosine receptor A2a (ADORA2A). This GPCR has become an important target for immunotherapy, but importantly, it has never been identified by chemical proteomics of living cells. Using A2a, a reported ligand for ADORA2A, an Ir conjugate (A2a-Ir) was synthesized, and a tethered diazirine conjugate (A2a-Dz) was obtained as described by Yao (Figure 21). Next, to confirm that the potency against the proposed target is retained upon conjugation, the binding affinity of the small molecule conjugate was measured. The Yao diazirine retained binding similar to that of the parent compound (0.8 nM compared to 4.8 nM for the parent A2a), but surprisingly, the Ir conjugate showed a >100-fold lower binding affinity (643 nM). Nevertheless, in ADORA2A-expressing HEK293T cells, the photocatalytic labeling method described herein, followed by streptavidin immunoprecipitation and western blot analysis

[0070] Example 14 - Identification of Cell Surface Receptors ​​​​​​​​​​​​​​The analysis showed that the labeling between stoichiometric Yao-type diazirine-A2a and A2a-iridium probes Immunostaining revealed clear differences in the enrichment of ADORA after labeling with Yao-type diazirine-A2a. 2A did not show a signal corresponding to that of the photocatalytic labeling platform described herein. Significant enrichment was observed when using the TMT-based chemical protocol for these reactions. Biogenomic analysis confirmed the initial results and demonstrated that the photocatalytic labeling method described herein is a novel method for the detection of ADORA2A showed an enrichment of 3 log2 fold change for GPCRs, providing indisputable target identification. In contrast, when stoichiometric diazirine was used, ADORA2A was consistent with previous data. Importantly, the binding sites for the target protein were not enriched statistically (Figure 22). This labeling platform is more likely to be used for the chemiluminescence assay than for the chemiluminescence assay, as the significant loss in affinity of the ligand-iridium conjugate is more likely to be due to the lack of The catalytic amplification of the signal provided by the platform is far superior.

[0071] Example 15 - Identification of cell surface receptors The human GPR40 receptor (hGPR40) can also be labeled using the compositions and methods described herein. The hGPR40-Ir conjugate was subjected to a tandem reaction with biotinylated diazirine (Diaz-PEG3-Bt). The hGPR40 ligand was also used as a competitor. 40 cells were treated with hGPR40-Ir conjugate or free Ir-alkyne photocatalyst for 30 min and washed The cells were then treated with (Diaz-PEG3-Bt) and irradiated at 450 nm for 10 min. The cell lysates were processed and As shown in 24A and 24B, Western blot and Analyzed by total protein staining.

[0072] Various embodiments of the present invention have been described to achieve various objects of the present invention. It should be recognized that these embodiments are merely illustrative of the principles of the present invention. Numerous changes and adaptations will be readily apparent to those skilled in the art without departing from the spirit and scope of the present invention. ​

Claims

1. A composition for labeling based on proximity, comprising a catalyst and a protein labeling agent, wherein the catalyst has an electronic structure that activates the protein labeling agent to a reactive intermediate via energy transfer. Composition.

2. The composition according to claim 1, wherein the reactive intermediate crosslinks with the protein.

3. The composition according to claim 1, wherein the reactive intermediate is inserted into the C-H bond of the protein.

4. The composition according to claim 1, wherein the reactive intermediate is a carbene, nitrene or phenoxyl radical. Composition.

5. The composition according to claim 1, wherein the reactive intermediate has a diffusion radius before quenching in an aqueous or aqueous-based environment of less than 4 nm. Composition.

6. The reactive intermediate has a half-life (t 1 / 2 ) of less than 5 nanoseconds (ns), as recited in claim 1 Composition.

7. The composition according to claim 1, wherein the energy transfer is Dexter energy transfer.

8. The composition according to claim 1, wherein the energy transfer is single electron transfer.

9. The composition according to claim 1, wherein the energy transfer is from the triplet excited state of the catalyst. Composition.

10. The composition according to claim 9, wherein the catalyst is a photocatalyst.

11. The composition according to claim 10, wherein the photocatalyst absorbs light in the visible region of the electromagnetic spectrum. Composition.

12. The composition according to claim 1, wherein the catalyst is a transition metal photocatalyst having absorption in the visible region of the electromagnetic spectrum. Composition.

13. The composition according to claim 12, wherein the energy transfer is from the triplet excited state of the photocatalyst, and the triplet excited state has an energy of more than 60 kcal / mol. Composition.

14. The transition metal photocatalyst is represented by the following formula: 【Chemical 1】 wherein M is a transition metal. A, D, E, G, Y and Z are independently selected from C and N. R 1 to R 6 each represents one to four optional ring substituents, and each of the one to four optional ring substituents Each is independently alkyl, heteroalkyl, haloalkyl, halo, hydroxy, alkoxy. Si, amine, amide, ether, -C(O)O - , -C(O)OR 7 , and -R 8 selected from the group consisting of OH and R 7 is selected from the group consisting of hydrogen and alkyl, and R 8 is alkyl, X - is a counter ion, Composition according to claim 12.

15. The composition according to claim 12, wherein the transition metal photocatalyst is soluble in an aqueous or aqueous-based environment. Composition.

16. The composition according to any one of claims 12 to 15, wherein the protein labeling agent is diazirine or azide. Composition.

17. The composition according to claim 1, wherein the catalyst is an organic photocatalyst.

18. The organic photocatalyst is thioxanthone, phenothiazine, flavin, phenazine. The composition according to claim 17, which is selected from the group consisting of coumarin, acetophenone, and benzophenone groups.

19. The composition according to claim 18, wherein the energy transfer is single electron transfer.

20. The protein labeling agent is functionalized with a marker, and the marker is biotin, desthiobiotin, alkyne, azide, FLAG tag, fluorophore, and chloroalkane functionality The composition according to claim 1, which is selected from the group consisting of .

21. A complex comprising a transition metal photocatalyst bound to a biomolecule binder.

22. The complex according to claim 21, wherein the biomolecule binder comprises a protein, polysaccharide, nucleic acid, or lipid.

23. The complex according to claim 21, wherein the biomolecule binder comprises a ligand specific for a cell surface receptor.

24. The complex according to claim 21, wherein the transition metal photocatalyst has a triplet excited state exceeding 60 kcal / mol.

25. The transition metal complex is represented by the following formula: 【Chemical 2】 In the formula, M is a transition metal. A, D, E, G, Y, and Z are independently selected from C and N. R 1 ~R 6 each represents one to four optional ring substituents, and each of the one to four optional ring substituents Each is independently alkyl, heteroalkyl, haloalkyl, halo, hydroxy, alkoxy Si, amine, amide, ether, -C(O)O - , -C(O)OR 7 , and -R 8 selected from the group consisting of OH and R 7 is selected from the group consisting of hydrogen and alkyl, and R 8 is alkyl, X - is a counter ion, The complex according to any one of claims 21 to 24.

26. The complex according to any one of claims 21 to 24, further comprising a hydrophilic linker between the transition metal photocatalyst and the biomolecule binder.

27. The complex according to claim 26, wherein the linker comprises an alkylene oxide.

28. The complex according to claim 21, wherein the transition metal photocatalyst absorbs light in the visible region of the electromagnetic spectrum.

29. The complex according to claim 21, wherein the biomolecule binder is covalently bonded to the transition metal photocatalyst.

30. A system for labeling based on proximity, comprising A complex comprising a catalyst bound to a biomolecule binder and a protein labeling agent, The catalyst has an electronic structure that activates the protein labeling agent to a reactive intermediate via energy transfer. System.

31. The system according to claim 30, wherein the reactive intermediate crosslinks with the protein.

32. The biomolecule binder comprises a ligand specific for a cell surface receptor, and the reactive intermediate The system according to claim 30, which crosslinks with the surface cell receptor.

33. The system according to claim 30, wherein the reactive intermediate is inserted into a C-H bond of a protein 。

34. The system according to claim 33, wherein the reactive intermediate is a carbene.

35. The diffusion radius of the reactive intermediate before quenching in an aqueous or water-based environment is less than 4 nm, the system according to claim 30.

36. The system according to claim 30, wherein the energy transfer is Dexter energy transfer 。

37. The system according to claim 30, wherein the energy transfer is single electron transfer.

38. The system according to claim 30, wherein the energy transfer is from a triplet excited state of the catalyst described.

39. The catalyst according to claim 38, which absorbs light in the visible region of the electromagnetic spectrum system.

40. The catalyst is a transition metal photocatalyst having absorption in the visible region of the electromagnetic spectrum, according to claim 30 of the described system.

41. The energy transfer is from a triplet excited state of the photocatalyst, and the triplet state has an energy exceeding 60 kcal / mol, the system according to claim 40.

42. The transition metal photocatalyst is represented by the following formula, [Chemical Formula 3] wherein M is a transition metal,[[]] A, D, E, G, Y and Z are independently selected from C and N, R 1 to R 6 each represents one to four optional ring substituents, and each of the one to four optional ring substituents each is independently alkyl, heteroalkyl, haloalkyl, halo, hydroxy, alkoxy Si, amine, amide, ether, -C(O)O - , -C(O)OR 7 , and -R 8 selected from the group consisting of OH wherein, R 7 is selected from the group consisting of hydrogen and alkyl, and R 8 is alkyl, X - is a counter ion, The system according to claim 40 or 41.

43. The system according to claim 30, wherein the biomolecule binder comprises a protein, a polysaccharide, a nucleic acid, or a lipid described.

44. The system according to claim 40 or 41, wherein the protein labeling agent is diazirine or azide described.

45. A method of labeling based on proximity, comprising providing a complex comprising a catalyst bound to a biomolecule binder, positioning the complex at a target region of the cell membrane by the biomolecule binder, activating a protein labeling agent to a reactive intermediate by the catalyst, and binding the reactive intermediate to one or more proteins within the target region including the method.

46. The method according to claim 45, wherein the biomolecule binder comprises a ligand specific for a cell surface receptor described.

47. The method according to claim 45, wherein the reactive intermediate is inserted into a C-H bond of the one or more proteins described.

48. The reactive intermediate has a diffusion radius before quenching in an aqueous or water-based environment of less than 4 nm, the method according to claim 45.

49. The catalyst has an electronic structure that activates the protein labeling agent to the reactive intermediate via energy transfer , the method according to claim 45.

50. The energy transfer is Dexter energy transfer, the method according to claim 49.

51. The energy transfer is single electron transfer, the method according to claim 49.

52. The energy transfer is from the triplet excited state of the catalyst, as described in claim 49 .

53. The excited triplet state has an energy exceeding 60 kcal / mol, as described in claim 52 .

54. The catalyst is a transition metal photocatalyst, the method according to any one of claims 49 to 53.

55. The transition metal photocatalyst is of the following formula, 【Chemical Formula 4】 wherein M is a transition metal, A, D, E, G, Y and Z are independently selected from C and N, R 1 to R 6 each represents one to four optional ring substituents, and each of said one to four optional ring substituents each is independently alkyl, heteroalkyl, haloalkyl, halo, hydroxy, alkoxy Si, amine, amide, ether, -C(O)O - , -C(O)OR 7 , and -R 8 selected from the group consisting of OH wherein, R 7 is selected from the group consisting of hydrogen and alkyl, and R 8 is alkyl, X - is a counter ion, , the method according to claim 54.

56. The protein labeling agent is diazirine or azide, the method according to claim 54 。

57. further comprising concentrating the one or more proteins bound to the reactive intermediate , the method according to claim 45.

58. The biomolecule binder includes protein, polysaccharide, nucleic acid, or lipid, as described in claim 45 .

59. The one or more proteins are bound to the cell membrane, the method according to claim 45.

60. The one or more proteins are bound to adjacent cells in contact with the cell membrane, as described in claim 45.

61. further comprising identifying the one or more proteins bound to the reactive intermediate for interactome mapping , the method according to claim 45.

62. further comprising purifying the one or more proteins bound to the reactive intermediate , the method according to claim 45.

63. The purifying includes affinity chromatography, the method according to claim 62.

64. One or more proteins in the target region that are not labeled with the protein labeling agent The method according to claim 62, further comprising purifying. **Claim 65** The method according to claim 45, wherein the one or more proteins bound by the reactive intermediate are identifiable within the target region by microscopy. target region by microscopy.

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