Photoactive Antibody Conjugates

Abstract

A kit comprising a photoreactive probe and a main subject probe, and a method of using the kit. The photoreactive probe (e.g., a ruthenium complex conjugated to an antibody) can be activated in a selected region of interest by optical radiation, and the activated photoreactive probe allows the main subject probe (e.g., a phenol connected to a desthiobiotin tag) to form a covalent bond with a molecule in a sample in the selected region of interest. The kit and method can be useful for analyzing biomolecules in biological samples (e.g., for identification of biomarkers in subcellular localization or via mass spectrometry).

Description

[Technical field]

[0001] (Priority Claim) This application claims priority to U.S. Provisional Patent Application No. 63 / 329,219, entitled "PHOTOACTIVE ANTIBODY CONJUGATE," filed April 8, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Incorporated by reference) All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0003] (Field) Described herein are methods and kits for identifying, tagging, and analyzing biomolecules. In particular, photoreactive kits useful for photoactivating and tagging a subset of biomolecules are described. In particular, the methods and kits may be useful for analyzing biological samples, such as identifying proximal biomolecules in cell or tissue samples. [Background technology]

[0004] (background) Cells are composed of many different types of biological molecules (biomolecules). Biomolecules in cells interact with nearby biomolecules in the intracellular environment to form complexes, organelles, or other aggregates and achieve a variety of important cellular functions. Uncovering the intracellular environment in which biomolecules interact with each other and how they function as a whole is extremely challenging. Biomolecules are small and exist with tens of millions of other molecules in the cellular environment. Interactions between neighboring biomolecules are often weak, and many of the techniques used to study biomolecules disrupt those interactions. Although techniques such as yeast two-hybridization assays and more recently proximity labeling have helped advance understanding of the cellular environment, these techniques have various limitations, such as nonspecific binding resulting in false positives and missed interactions, slow reaction times, and perturbation of the natural cellular environment. What is needed are better tools for determining naturally occurring biomolecular interactions to address these and other problems. Summary of the Invention

[0005] (Summary of disclosure) Described herein are systems, kits, and methods for identifying, tagging, and analyzing biomolecules. In particular, photoreactive kits useful for photoactivation and tagging a subset of biomolecules are described. In particular, the methods and kits may be useful for analyzing biological samples, such as identifying proximal biomolecules in cell or tissue samples. These kits may be particularly useful for selective tagging and proximity labeling of biomolecules via selective light illumination by a microscope system.

[0006] Described herein is a compound of formula (I): [ka] wherein moiety C is a single chemical bond or a linker; moiety B comprises and is attached to moiety C, wherein the 1-50 photoreactive moieties each have the formula (II): [ka]

[0007] (In formula (II): L 1 , L 2 , L 3 , and L 4 are each independently a ligand; and X 1 and X 2 are each independently a ligand having a reactive site, where the reactive site is configured for binding to the C moiety, derived from a ruthenium-based compound represented by: G moiety comprises a bait molecule bound to the C moiety, where the bait molecule is an antibody and is configured to conjugate with a first molecule in a sample. These and other embodiments can include a primary subject probe comprising a detectable tag moiety bound to a photoexcitable subject moiety, where the photoreactive probe is photoactivated at either a wavelength in the range of 700 nm to 1100 nm using a two-photon light source or a wavelength in the range of 200 nm to 1100 nm using a single light source, and where the photoexcited primary subject probe is configured to form a covalent bond with a target molecule in the sample when the photoreactive probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe.

[0008] In these and other embodiments, the photoreactive kit comprises X 1 and X 2 may each independently be selected from the group consisting of 3-ethynylpyridine, 3-(bromomethyl)pyridine, maleimide, 4'-methyl-4-carboxybipyridine-N-succinimidyl ester, nicotinaldehyde, 1-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethanone, 4-pentynenitrile, and 4-aminobutyne.

[0009] In these and other embodiments, the photoreactive kit comprises L 1and L 2 are linked to form a first bidentate ligand, and L 3 and L 4 are linked to form a second bidentate ligand, wherein the first bidentate ligand and the second bidentate ligand are independently selected from 2,2'-bipyridyl (bpy), 4,4'-dicyano-5,5'-dimethyl-2,2'-bipyridine (CN-Me-bpy), 4,4'-dimethyl-2,2'-bipyridine (dmb), 4,4'-di-tert-butyl-2,2'-bipyridine (dbpy), 4,4',5,5'-tetramethyl-2,2'-bipyridine (tmb), 2-phenylpyridine (ppy), ), 6-bromo-2,2'-bipyridine, 6,6'-dibromo-2,2'-bipyridine, 5-bromo-2,2'-bipyridine, 6-amino-2,2'-bipyridine, 6,6'-diamino-2,2'-bipyridine, 2,2'-bipyridine-6-carbonitrile, 2,2'-bipyridine-6,6'-bis(carbonitrile), 2,2'-bipyridine-6-carboxylic acid, 2,2'-bipyridine-6,6'-dicarboxylic acid, and biquinoline.

[0010] In these and other embodiments, the photoreactive kit comprises a ruthenium-based compound of formula (II): [ka] TIFF2025511874000005.tif149170 or a derivative thereof.

[0011] In these and other embodiments, the photoreactive kit comprises a photoreactive moiety comprising: [ka] or a moiety that is a derivative thereof.

[0012] In these and other embodiments, the linker is [ka] The moiety may include a moiety in which

[0013] In these and other embodiments, the photoreactive kit is provided, wherein the linker is an amino acid, (PEG) n , an oligonucleotide, or a peptide (wherein n is an integer of 1 to 20).

[0014] In these and other embodiments, the photoreactive kit may be configured such that when the photoreactive probe is photoactivated using a two-photon light source at a wavelength in the range of 700 nm to 1100 nm, and the primary subject probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe, the photoexcited primary subject probe forms a covalent bond with a target molecule in the sample. In these and other embodiments, the photoreactive kit may be further configured such that when the photoreactive probe is photoactivated using a single light source at a wavelength in the range of 300 nm to 800 nm, and the primary subject probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe, the photoexcited primary subject probe forms a covalent bond with a target molecule in the sample.

[0015] In these and other embodiments, the photoreactive kits may be such that the detectable tag moiety is at least one of a biotin derivative, a digoxigenin tag, a CLIP tag, a HaloTag, a SNAP tag, an oligonucleotide, a peptide tag, and a click chemistry tag, and the click chemistry tag includes an alkyne-based or azide-based moiety.

[0016] In these and other embodiments, the photoreactive kit comprises a photoexcitable subject moiety comprising: [ka] and at least one of their derivatives.

[0017] In these and other embodiments, the photoreactive kit can be one in which the main subject probe is desthiobiotin-phenol or biotin-phenol.

[0018] In these and other embodiments, the photoreactive kit comprises a photoexcitable subject moiety comprising: [ka] It may be that:

[0019] In these and other embodiments, the photoreactive kit comprises a kit in which the detectable tag moiety is at least one of a biotin derivative, a click chemistry tag, a CLIP tag, a digoxigenin tag, a HaloTag, an oligonucleotide, a peptide tag, a SNAP tag, and the photoreactive site is [ka] It may include a moiety in which:

[0020] In these and other embodiments, the photoreactive kit may further comprise a connector, the connector being capable of conjugating with the detectable tag portion of the main subject probe. In these and other embodiments, the photoreactive kit may be one in which the connector is a fluorescent connector. In these and other embodiments, the photoreactive kit may further comprise a tag-enzyme complex, the connector being capable of conjugating with the connector, and further comprising an enzyme of the tag-enzyme complex comprising a peroxidase. In these and other embodiments, the photoreactive kit may further comprise a connector, the connector being capable of conjugating with the detectable tag portion of the main subject probe; a tag-enzyme complex, the tag of the tag-enzyme complex being capable of conjugating with the connector, and further comprising an enzyme of the tag-enzyme complex comprising a peroxidase; and an additional subject probe configured to form an additional subject probe covalent bond with the target molecule by the catalytic activity of the peroxidase of the tag-enzyme complex. In these and other embodiments, the photoreactive kit can be one in which the additional subject probe is the same as or different from the primary subject probe and includes an additional subject probe tag portion and an additional subject probe subject portion, In these and other embodiments, the photoreactive kit can be one in which the connector is a fluorescent connector.

[0021] In these and other embodiments, the photoreactive kit may have a concentration of the photoreactive probe in the range of 0.1 ug / mL to 100 ug / mL and a concentration of the main subject probe in the range of 1 uM to 20 mM.

[0022] Also described herein is a compound of formula (I): [ka] wherein the C moiety is a single chemical bond or linker; the B moiety comprises at least one photoreactive moiety attached to the C moiety; and the G moiety comprises a bait molecule attached to the C moiety. In these and other embodiments, the photoreactive kit may include a primary subject probe comprising a detectable tag moiety bound to a photoexcitable subject moiety, wherein the bait molecule of the photoreactive probe is configured to conjugate with a first molecule in a sample, and the photoreactive probe is configured to form a covalent bond with a target molecule in the sample when the photoreactive probe is photoactivated and the primary subject probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe.

[0023] In these and other embodiments, the photoreactive kit can be one in which the bait molecule comprises at least one of an antibody, avidin, neutravidin, streptavidin, other biotin-binding proteins, CLIP tags, HaloTag, SNAP tags, other self-labeling protein tags, DNA or RNA fluorescent in situ hybridization (FISH) probes, other RNA molecules, other nucleic acid molecules, protein A, protein G, protein L, protein A / G, protein A / G / L, other immunoglobulin-binding peptides, drugs, or other small molecules.

[0024] In these and other embodiments, the photoreactive kit may be one in which the photoreactive moiety is at least one of riboflavin, lumiflavin, another flavin derivative, fluorescein or a derivative thereof, methylene blue or a derivative thereof, miniSOG photosensitizer protein, Killer Red photosensitizer protein, another photosensitizer protein, pterin or a derivative thereof, a ruthenium-based photocatalyst, and rose bengal or a derivative thereof.

[0025] In these and other embodiments, the photoreactive kit is one in which the bait molecule is an antibody and a number of the photoreactive sites are attached to the antibody via the C moiety, where the number can range from 1 to 50.

[0026] In these and other embodiments, the photoreactive kit may be one in which the photoreactive moiety is configured to enable the main subject probe to form the covalent bond with the molecule of the sample.

[0027] In these and other embodiments, the photoreactive kit comprises a photoreactive moiety having the formula (II): [ka] (In formula (II): L 1 , L 2 , L 3 , and L 4 are each independently a ligand; and X 1 and X 2 are each independently a ligand, 1 and X 2 at least one of the linking regions has a linking region, wherein the at least one linking region is attached to the C portion of the photoreactive probe. The ruthenium-based compound may be derived from a ruthenium-based compound represented by:

[0028] In these and other embodiments, the photoreactive kit comprises X 1 and X 2 may each independently be selected from the group consisting of 3-ethynylpyridine, 3-(bromomethyl)pyridine, maleimide, 4'-methyl-4-carboxybipyridine-N-succinimidyl ester, nicotinaldehyde, 1-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethanone, 4-pentynenitrile, and 4-aminobutyne.

[0029] In these and other embodiments, the photoreactive kit comprises L 1 and L 2 are linked to form a first bidentate ligand, and L 3 and L 4 are linked to form a second bidentate ligand, wherein the first bidentate ligand and the second bidentate ligand are independently selected from 2,2'-bipyridyl (bpy), 4,4'-dicyano-5,5'-dimethyl-2,2'-bipyridine (CN-Me-bpy), 4,4'-dimethyl-2,2'-bipyridine (dmb), 4,4'-di-tert-butyl-2,2'-bipyridine (dbpy), 4,4',5,5'-tetramethyl-2,2'-bipyridine (tmb), 2-phenylpyridine (ppy), ), 6-bromo-2,2'-bipyridine, 6,6'-dibromo-2,2'-bipyridine, 5-bromo-2,2'-bipyridine, 6-amino-2,2'-bipyridine, 6,6'-diamino-2,2'-bipyridine, 2,2'-bipyridine-6-carbonitrile, 2,2'-bipyridine-6,6'-bis(carbonitrile), 2,2'-bipyridine-6-carboxylic acid, 2,2'-bipyridine-6,6'-dicarboxylic acid, and biquinoline.

[0030] In these and other embodiments, the photoreactive kit comprises a ruthenium-based compound of formula (II): [ka] TIFF2025511874000014.tif148170 and any of their derivatives.

[0031] In these and other embodiments, the photoreactive kit comprises a photoreactive moiety comprising: [ka] or a moiety that is a derivative thereof.

[0032] In these and other embodiments, the photoreactive kit comprises a linker comprising: [ka] It may include a moiety in which:

[0033] In these and other embodiments, the photoreactive kit is provided, wherein the linker is an amino acid, (PEG) n , an oligonucleotide, or a peptide (wherein n is an integer of 1 to 20).

[0034] In these and other embodiments, the photoreactive kit may be one in which the photoreactive moiety is activatable using a light source at a wavelength in the range of 200 nm to 1100 nm to enable the main subject probe to form the covalent bond with the target molecule in the sample.

[0035] In these and other embodiments, the photoreactive kit can be one in which the photoreactive moiety is activatable with a two-photon light source at a wavelength in the range of 700 nm to 1100 nm to enable the primary subject probe to form the covalent bond with the target molecule in the sample. In these and other embodiments, the photoreactive kit can be one in which the photoreactive moiety is activatable with a light source at a wavelength in the range of 300 nm to 800 nm to enable the primary subject probe to form the covalent bond with the target molecule in the sample.

[0036] In these and other embodiments, the photoreactive kit may be one in which the photoreactive moiety is activatable using a two-photon light source at wavelengths ranging from 700 nm to 1100 nm to enable the main subject probe to form the covalent bond with the target molecule in the sample.

[0037] In these and other embodiments, the photoreactive kits may be such that the detectable tag moiety is at least one of a biotin derivative, a CLIP tag, a digoxigenin tag, a HaloTag, an oligonucleotide, a peptide tag, a SNAP tag, and a click chemistry tag, and the click chemistry tag includes an alkyne-based or azide-based moiety.

[0038] In these and other embodiments, the photoreactive kit is provided, wherein the subject site is: [ka] and one or more of their derivatives.

[0039] In these and other embodiments, the photoreactive kit can be one in which the main subject probe is desthiobiotin-phenol or biotin-phenol.

[0040] In these and other embodiments, the photoreactive kit comprises a photoexcitable subject moiety comprising: [ka] It may be that:

[0041] In these and other embodiments, the photoreactive kit comprises a kit in which the detectable tag moiety is at least one of a biotin derivative, a click chemistry tag, a CLIP tag, a digoxigenin tag, a HaloTag, an oligonucleotide, a peptide tag, a SNAP tag, and the photoreactive site is [ka] It may include the site.

[0042] In these and other embodiments, the photoreactive kit may further comprise a connector capable of conjugating to the detectable tag portion of the primary subject probe. In these and other embodiments, the photoreactive kit may be one in which the connector is a fluorescent connector. In these and other embodiments, the photoreactive kit may further comprise a tag-enzyme complex capable of conjugating to the connector, and further wherein the enzyme of the tag-enzyme complex comprises peroxidase.

[0043] In these and other embodiments, the photoreactive kit may further comprise: a connector capable of conjugating to the detectable tag portion of the primary subject probe; a tag-enzyme complex, the tag of which is capable of conjugating to the connector and further wherein the enzyme of the tag-enzyme complex comprises a peroxidase; and an additional subject probe configured to form an additional subject probe covalent bond with the target molecule through the catalytic activity of the peroxidase of the tag-enzyme complex.

[0044] In these and other embodiments, the photoreactive kit can be one in which the additional subject probe is different from the primary subject probe and includes an additional subject probe tag portion and an additional subject probe subject portion, In these and other embodiments, the photoreactive kit can be one in which the connector is a fluorescent connector.

[0045] In these and other embodiments, the photoreactive kit may have a concentration of the photoreactive probe in the range of 0.1 ug / mL to 100 ug / mL and a concentration of the main subject probe in the range of 1 uM to 20 mM.

[0046] Also described herein is a method for photoreactive labeling.These and other methods can include one or more of the following steps: delivering a plurality of photoreactive probes of a photoreactive kit to a sample; non-covalently conjugating the bait molecules of the photoreactive probes of the first portion to a plurality of first molecules in the sample; delivering a plurality of main subject probes of a photoreactive kit described herein to the sample; selectively irradiating a selected region of interest of the sample with optical radiation, thereby activating the photoreactive sites of the plurality of photoreactive probes to generate a plurality of photoactivated photoreactive probes in the selected region; photoexciting the photoexcitable subject sites of the main subject probes with the plurality of photoactivated photoreactive probes to generate a plurality of photoexcited main subject probes with photoexcited subject sites; and forming covalent bonds between the plurality of photoexcited main subject probes and a plurality of target molecules in the selected region of interest in the sample, thereby binding the plurality of main subject probes to the plurality of target molecules.

[0047] In these and other methods, where non-covalently conjugating the bait molecules of the photoreactive probes of a first portion to a plurality of first molecules in the sample leaves the photoreactive probes of a second portion unconjugated, the method may further include the step of: removing the unconjugated photoreactive probes from the sample.

[0048] These and other methods can further include one or more of the steps of: delivering a plurality of connectors to the sample, and conjugating the plurality of connectors to the detectable tag portions of the plurality of main subject probes.

[0049] In these and other methods where the plurality of connectors comprises fluorescent connectors, the method may further include one or more of the following steps: detecting the locations of the plurality of fluorescent connectors in the sample, and thereby identifying the locations of the plurality of main subject probes and the plurality of target molecules covalently bound thereto.

[0050] In these and other methods, the photoexcitation step comprises:

[0051] The method may further include a step of photoexciting the primary subject probe to generate a plurality of photoexcited primary subject probes, each having a free radical, and the step of forming a covalent bond includes forming a covalent bond between each of the plurality of photoexcited primary subject probes and an amino acid in each of the plurality of target molecules within the selected region of interest in the sample.

[0052] In these and other methods, the step of forming the covalent bond can include forming a covalent bond between each of the plurality of photoexcited primary subject probes and an amino acid in each of the plurality of target molecules within the selected region of interest in the sample.

[0053] In these and other methods, the amino acid can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0054] These and other methods may further include one or more of the following steps: delivering to the sample the connector of a photoreactive kit described herein, where the connector is capable of conjugating to the detectable tag portion of the primary subject probe; delivering to the sample the tag-peroxidase complex of a photoreactive kit described herein, where the tag of the tag-enzyme complex is capable of conjugating to the connector and further, where the enzyme of the tag-enzyme complex comprises a peroxidase; delivering to the sample an additional subject probe of a photoreactive kit described herein, where the additional subject probe is configured to form an additional subject probe covalent bond with the target molecule by catalytic activity of the peroxidase of the tag-enzyme complex; and conjugating the tag-peroxidase complex to the connector, where the tag-peroxidase catalyzes the additional subject probe to form a covalent bond between the additional subject probe and the sample.

[0055] In these and other methods, the tag-peroxidase complex activates the additional subject probe to cause the subject site of the additional subject probe to have a free radical and form a covalent bond between the subject site of the additional subject probe and a tyrosine of the sample.

[0056] Also described herein is an analytical method comprising one or more of the steps of: delivering a plurality of photoreactive probes of the photoreactive kit described herein to the sample; non-covalently conjugating the bait molecules of the first portion of the photoreactive probes to a plurality of first molecules in the sample; delivering a plurality of main subject probes of the photoreactive kit described herein to the sample; and illuminating the sample with an imaging light source of an image-guided system; imaging the illuminated sample with a camera; acquiring at least one image of the intracellular morphology of the sample in a first field of view with the camera; processing the at least one image and determining a region of interest in the sample based on the processed image; obtaining coordinate information of the region of interest; and selectively irradiating the region of interest with optical radiation according to the coordinate information to activate the photoreactive site, wherein the activated photoreactive site enables the main subject probe to form a covalent bond with the sample in the region of interest.

[0057] These and other methods may include illuminating the area with between 10us / pixel and 200us / pixel, between 25us / pixel and 400us / pixel, between 50us / pixel and 300us / pixel, between 75us / pixel and 200us / pixel, or between 400us / pixel and 5000us / pixel.

[0058] In these and other methods, the selectively irradiating step includes irradiating with a power intensity between 1 μW and 300 mW.In these and other methods, the selectively irradiating step includes irradiating an area defined by a point spread function.

[0059] These and other methods can further include the step of conjugating a connector to the primary subject probe and detectably proximity labeling a proximal neighbor to the target molecule with a detectable labeling activity.

[0060] In these and other methods, the detectably proximity-labeling step can include proximity-labeling an area less than 5 um, less than 2 um, less than 1 um, less than 500 nm, less than 300 nm, less than 200 nm, less than 100 nm, less than 50 nm, or less than 20 nm in diameter.

[0061] In these and other methods, the connector may include a catalytic label.

[0062] In these and other methods, the sample can comprise at least 1, at least 100, at least 1000, or at least 10,000 live or fixed cells. In these and other methods, the sample can comprise fixed cells, fixed tissues, cell extracts, or tissue extracts.

[0063] These and other methods may include performing mass spectrometry or sequencing on the selectively irradiated samples.

[0064] In these and other methods, the activated photoactive site can activate the main subject probe to have a free radical and form the covalent bond with an amino acid of the sample within the selected region of interest.

[0065] Also described herein is a mass spectrometry-implemented method for processing samples to predict biomarkers. Described herein are methods for separating samples into photolabeled and unlabeled sample groups; delivering photoreactive probes and primary subject probes as described herein to the photolabeled and unlabeled sample groups; selectively illuminating selected regions of interest of the photolabeled samples and keeping the unlabeled sample groups in the dark, where the illuminating step allows the primary subject probe to form a covalent bond with the sample; and selectively isolating the photolabeled and unlabeled sample groups by affinity precipitation between the primary subject probe and a plurality of affinity beads. extracting proteins bound to a plurality of probes from a sample group; performing mass spectrometry on the extracted proteins; calculating relative quantitative values ​​of individual proteins in the identified protein list between the photolabeled sample group and the unlabeled sample group according to intensity values ​​of peptide fragments of the individual proteins; determining a threshold for the relative quantitative values ​​between the photolabeled sample group and the unlabeled sample group; and once the threshold is determined, predicting at least one biomarker corresponding to the relative quantitative value of the individual protein that exceeds the threshold.

[0066] These and other methods can further include the step of non-covalently conjugating the bait molecule to a target molecule in the sample.

[0067] In these and other methods, the step of selectively irradiating the selected region of interest can further include the step of activating the photoreactive sites in the selected region such that the activated photoreactive sites enable the primary subject probe to form the covalent bond with the sample in the selected region of interest.

[0068] These and other methods can further include delivering the bait molecule of the photoreactive probe to the sample.

[0069] These and other methods can further include removing unconjugated photoreactive probes from the sample to allow the photoreactive probes to direct the selective irradiation to the selected region of interest.

[0070] These and other methods may further include the step of delivering the connector of a photoreactive kit described herein to the sample and allowing the connector to conjugate to the main subject probe due to the affinity between the connector and the main subject probe.

[0071] In these and other methods, the connector is a fluorescent connector for identifying the location of the sample that is covalently attached to the main subject probe.

[0072] In these and other methods, the activated photoreactive site can activate the main subject probe to have a free radical and form the covalent bond with an amino acid of the sample within the selected region of interest.

[0073] In these and other methods, the amino acid can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0074] These and other methods may further include delivering the tag-peroxidase of a photoreactive kit described herein to the sample and conjugating the tag-peroxidase to the connector such that the tag-peroxidase can catalyze the additional subject probe to form a covalent bond between the additional subject probe and the sample.

[0075] In these and other methods, the tag-peroxidase can activate the additional subject probe to cause the subject moiety of the additional subject probe to have a free radical and form the covalent bond between the subject moiety of the additional subject probe and a tyrosine of the sample.

[0076] Also described herein is a method implemented in mass spectrometry to process photolabeled samples to identify a list of biomarkers.Described herein is a method comprising one or more of the following steps: obtaining a sample; delivering a photoreactive kit described herein to the sample; selectively irradiating the selected region of interest of the biological sample so that the main subject probe can label the proteins of the sample in the selected region of interest; extracting a plurality of the probe-labeled proteins from the sample by affinity precipitation between the main subject probe and a plurality of affinity beads; performing mass spectrometry on the extracted proteins; and identifying the extracted proteins of the sample.

[0077] These and other methods may further comprise the step of calculating intensity values ​​of peptide fragments of each protein in the identified protein list of the sample.

[0078] These and other methods can further include ranking the list of identified proteins according to the intensity value of each protein.

[0079] These and other methods can further include the step of non-covalently conjugating the bait molecule to a target molecule in the sample.

[0080] In these and other methods, the step of selectively irradiating the selected region of interest can further include the step of activating the photoreactive sites in the selected region such that the activated photoreactive sites enable the primary subject probe to form the covalent bond with the sample in the selected region of interest.

[0081] These and other methods can further include delivering the bait molecule of the photoreactive probe to the sample.

[0082] These and other methods can further include removing unconjugated photoreactive probes from the sample to allow the photoreactive probes to direct the selective irradiation to the selected region of interest.

[0083] These and other methods may further include the step of delivering the connector of a photoreactive kit described herein to the sample and allowing the connector to conjugate to the main subject probe due to the affinity between the connector and the main subject probe.

[0084] In these and other methods, the connector can be a fluorescent connector configured to identify the location of the sample covalently attached to the main subject probe.

[0085] In these and other methods, the activated photoreactive site can activate the primary subject probe to have a free radical and form the covalent bond with an amino acid of the sample within the selected region of interest. In these and other methods, the amino acid can be selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0086] These and other methods may further include delivering the tag-peroxidase of a photoreactive kit described herein to the sample and conjugating the tag-peroxidase to the connector such that the tag-peroxidase can catalyze the additional subject probe to form a covalent bond between the additional subject probe and the sample.

[0087] In these and other methods, the tag-peroxidase can activate the additional subject probe to cause the subject site of the additional subject probe to have a free radical and form a covalent bond between the subject site of the additional subject probe and a tyrosine of the sample. [Brief description of the drawings]

[0088] BRIEF DESCRIPTION OF THE DRAWINGS A better understanding of the features and advantages of the methods and apparatus described herein will be obtained by reference to the following detailed description and accompanying drawings that set forth exemplary embodiments.

[0089] [Figure 1] FIG. 1 shows a schematic diagram of a system useful for optically selective spatial tagging and proximity labeling of cells on a substrate.

[0090] [Diagram 2] FIG. 2A shows a schematic diagram of a photoreactive probe having a tag moiety and a subject moiety.

[0091] FIG. 2B shows a schematic diagram of a subject probe having a tag portion and a subject portion.

[0092] FIG. 2C shows an example of a subject probe having a desthiobiotin tag moiety and a phenol subject moiety.

[0093] FIG. 2D shows a schematic of a subject probe having a tag moiety and a reactive phenolic site.

[0094] FIG. 2E shows the interaction of a photoreactive probe and a subject probe resulting in the labeling of an adjacent protein with the subject probe shown in FIG. 2D.

[0095] Figures 2F-2G show schematic steps in biomolecular proximity tagging at a target region using the probes shown in Figures 2A and 2B. Figure 2F shows schematic binding of a photoreactive probe to a first molecule.

[0096] FIG. 2G illustrates a schematic diagram of activation of a photoreactive probe by light and binding of the photoreactive probe to an adjacent biomolecule after binding of the photoreactive probe to a first molecule as shown in FIG. 2F.

[0097] FIG. 2H illustrates a schematic of a proximity labeling system that can be used to label biomolecules using the probes shown in FIGS. 2A and 2B.

[0098] Figure 2I shows a schematic comparing different methods of labeling biomolecules in small regions of interest (ROIs). The top right panel shows direct photochemical labeling, and the bottom panel shows photoassisted enzymatic labeling using the photoreactive and subject probes described herein to label proteins of interest.

[0099] [Diagram 3] Figures 3A-3N show examples of ruthenium-based photoreactive moieties that can be used in the photoreactive probes described herein. Figure 3A shows examples of ruthenium-based photoreactive moieties that can be used in the photoreactive probes and methods described herein.

[0100] FIG. 3B shows another example of a ruthenium-based photoreactive moiety.

[0101] FIG. 3C shows another example of a ruthenium-based photoreactive moiety.

[0102] FIG. 3D shows another example of a ruthenium-based photoreactive moiety.

[0103] FIG. 3E shows another example of a ruthenium-based photoreactive moiety.

[0104] FIG. 3F shows another example of a ruthenium-based photoreactive moiety.

[0105] FIG. 3G shows another example of a ruthenium-based photoreactive moiety.

[0106] FIG. 3H shows another example of a ruthenium-based photoreactive moiety.

[0107] FIG. 3I shows another example of a ruthenium-based photoreactive moiety.

[0108] FIG. 3J shows another example of a ruthenium-based photoreactive moiety.

[0109] FIG. 3K shows another example of a ruthenium-based photoreactive moiety.

[0110] FIG. 3L shows another example of a ruthenium-based photoreactive moiety.

[0111] FIG. 3M shows another example of a ruthenium-based photoreactive moiety.

[0112] FIG. 3N shows another example of a ruthenium-based photoreactive moiety.

[0113] FIG. 3O shows examples of Rose Bengal moieties and their derivatives that can be used in the photoreactive probes and methods described herein.

[0114] 3P-3Q show examples of fluorescein derivatives that can be used in the photoreactive probes and methods described herein.

[0115] FIG. 3R shows examples of methylene blue derivatives that can be used in the photoreactive probes and methods described herein.

[0116] 3S-3T show examples of lumiflavin derivatives that can be used in the photoreactive probes and methods described herein.

[0117] 3U-3V show examples of riboflavin and flavin derivatives that can be used in the photoreactive probes and methods described herein.

[0118] 3W and 3X show examples of pterin derivatives that can be used in the photoreactive probes and methods described herein.

[0119] FIG. 3Y shows another example of a ruthenium-based photoreactive moiety.

[0120] FIG. 3Z shows another example of a ruthenium-based photoreactive moiety.

[0121] FIG. 3AA shows another example of a ruthenium-based photoreactive moiety.

[0122] FIG. 3AB shows another example of a ruthenium-based photoreactive moiety.

[0123] FIG. 3AC shows another example of a ruthenium-based photoreactive moiety.

[0124] FIG. 3AD shows another example of a ruthenium-based photoreactive moiety.

[0125] FIG. 3AE shows another example of a ruthenium-based photoreactive moiety.

[0126] FIG. 3AF shows another example of a ruthenium-based photoreactive moiety.

[0127] FIG. 3AG shows another example of a ruthenium-based photoreactive moiety.

[0128] FIG. 3AH shows another example of a ruthenium-based photoreactive moiety.

[0129] FIG. 3AI shows another example of a ruthenium-based photoreactive site.

[0130] [Figure 4] 4A-4L show examples of linker moieties that can be used in the photoreactive probes described herein.

[0131] [Diagram 5] Figures 5A-5E show examples of tags that can be used in the subject probes described herein.Figures 5A-5E show click chemistry molecules.

[0132] 5F-5H show biotin derivatives that can be used in the probes and methods described herein.

[0133] FIG. 5I shows a digoxigenin molecule that can be used in the probes and methods described herein.

[0134] FIG. 5J shows examples of peptide tags that can be used in the probes and methods described herein.

[0135] FIG. 5K shows examples of SNAP tags that can be used in the probes and methods described herein.

[0136] [Figure 6] 6A-6L show examples of subject sites that can be used in the subject probes described herein.

[0137] [Figure 7] FIG. 7 shows a schematic of the conjugation of Ru(bpy) 3 2+ onto an antibody by NHS ester-based amide coupling to form a photoreactive probe.

[0138] [Figure 8] FIG. 8 shows experimental results of antibody-based ruthenium (Ab-Ru) targeted photolabeling (confocal imaging of nucleolus marker (magenta; first panel in the results on the XY and Z axes), antibody-based ruthenium (red; second panel from the left and second panel from the top on the XY axes), and photo(2P) labeling signal: desthiobiotin (green; third panel from the left and third panel from the top on the Z axis; scale bar: 20 μm).

[0139] [Figure 9] Figure 9 shows that a small region of interest (nucleolus) is precisely photolabeled using Ab-Ru photoreactive probe and biotin-phenol subject probe and the methods disclosed herein. Figure 9 shows all proteins identified in the nucleolus labeling experiment (Figure 8), where the proteins are ranked by their logarithmic fold change ratio (labeled cells / unlabeled cells).

[0140] [Figure 10] Figure 10 shows previously unidentified proteins in stress granules (SGs) identified using the probes and methods disclosed herein. The proteomic composition demonstrates the accuracy and power of discovering novel proteins using these methods.

[0141] [Figure 11] Figures 11A-11D show robust photolabeling using photoreactive moieties. Figures 11A-11C show the chemical structures of ruthenium, riboflavin, and rose bengal moieties. Figure 11D shows fluorescent labeling of cells using the photoreactive moieties shown in Figures 11A-11C.

[0142] [Figure 12] FIG. 12 shows successful conjugation of an antibody with a photoreactive moiety.

[0143] [Figure 13] 13A-13B show the results of successful antibody-photoreactive moiety conjugation.

[0144] [Figure 14] Figures 14A-C show the results of specific photolabeling of induced cells using conjugation of an antibody with a ruthenium-based photoreactive moiety. Figure 14A shows illumination at 470 nm. Figure 14B shows the control (no light). Figure 14C shows the Z-axis (side view) of the cell shown in Figure 14A.

[0145] [Figure 15] Figure 15 shows selective photolabeling of mouse brain tissue samples using antibodies and a ruthenium-based photoreactive site probe. Control (no light) shows no labeling.

[0146] [Figure 16] FIG. 16 shows the results of an experiment demonstrating the photolabeling of mouse brain tissue samples using antibodies and a ruthenium-based photoreactive site probe and neutravidin stain.

[0147] [Figure 17] Figure 17 shows the results of an experiment demonstrating the detection of nucleolar regions using a secondary antibody bait and a ruthenium-based photoreactive site probe. The photolabeled signal was labeled using two-photon technology.

[0148] [Figure 18] Figures 18A-C show experimental results showing detection of photolabeled subcellular compartments using a probe with a ruthenium-based photoreactive site and an Alexa fluor 568 secondary antibody bait that binds to a primary antibody. Figure 18A shows nucleolus detection using a rabbit anti-nucleolin antibody. Figure 18B shows nuclear pore complex detection using a mouse anti-NPC antibody. Figure 18C shows stress granule detection using a mouse anti-Ras GTPase-activating protein-binding protein 1 antibody.

[0149] [Figure 19] FIG. 19 shows the results of an experiment demonstrating specific two-photon labeling on fixed mouse brain tissue.

[0150] [Figure 20] FIG. 20 shows the results of an experiment demonstrating stress granules photolabeled with horseradish peroxidase-activated desthiobiotin, which is covalently bound to tyrosine residues on and proximal to the enzyme site.

[0151] [Figure 21] FIG. 21 shows the proximity labeling of a mouse liver tissue sample photolabeled with horseradish peroxidase-activated desthiobiotin, which is covalently attached to tyrosine residues on and proximal to the enzyme site.

[0152] [Figure 22] FIG. 22 shows an example of a workflow using a photoactivatable kit in combination with a microscopic photolabeling system and subsequent mass spectrometry, as described herein.

[0153] [Diagram 23] FIG. 23 shows experimental results showing confocal micrographs illustrating stress granules that were photolabeled (PL) with high precision and accuracy by antibody-ruthenium photoactive probes using a two-photon labeling system at wavelength=780 nm.

[0154] [Figure 24]Figures 24A-C show two-photon labeling with antibody-ruthenium photoactive probes. Figure 24A shows a Venn diagram of three biological replicates of two-photon labeling of Ab-Ru photoactive probes. Figure 24B shows a volcano plot of relative protein levels (PL / CTL ratio) in photolabeled samples versus control samples on a LOG2 scale. Over-represented (enriched) proteins are shown in the upper right portion bounded by arrows a and b. Figure 24C shows that a 74% true positive rate (annotated as stress granules (sg)) is identified in the top 50 proteins ranked by PL / CTL ratio.

[0155] [Diagram 25] Figures 25A-B show results demonstrating validation of potential stress granule proteins by immunofluorescence detection. Figure 25A shows that 37 proteins out of the top 50 proteins ranked by PL / CTL ratio were annotated as stress granule proteins. Figure 25B shows a proteome composition analysis confirming the accuracy of detecting stress granule proteins using the methods herein and the ability to use these methods to discover novel stress granule biomarkers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0156] (Detailed Description) Described herein are systems, kits, and methods useful for identifying, tagging, obtaining, and analyzing biomolecules and their adjacent biomolecules. The kits and methods may be particularly useful for analyzing biomolecular interactions in biological samples, such as analyzing proteins, nucleic acids, carbohydrates, or lipids in cell or tissue samples. The kits and methods may be advantageously used, for example, to identify and / or isolate previously unknown biomarkers (e.g., proximal or adjacent molecules) using protein sequencing and / or mass spectrometry. The kits and methods utilize photoreactive materials that can label biomolecules and their adjacent biomolecules. The photoreactive kits described herein may be particularly useful for specifically labeling a subset of biomolecules within the intracellular region of a cell using an image-guided microscope with precise illumination control, such as the system described in U.S. Patent Publication No. 2018 / 0367717, to enable automated labeling of cellular biomolecules of interest. The kits can be used for in situ tagging of biomolecules such as proteins inside cells or tissues, which can be followed by proximity labeling, for example, using Tyramide Signal Amplification (TSA). The biomolecules can be further analyzed by analytical techniques such as mass spectrometry and sequencing. These kits can be particularly useful for performing omics research, such as genomics, proteomics, and transcriptomics, and for discovering biomarkers suitable for diagnosis and treatment.

[0157] (Abbreviations and definitions:)

[0158] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analog" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. The amino acids described herein may be conservatively substituted so long as the conservatively substituted peptide still permits the desired function, such as recognition by a protease. Examples of conservative substitutions include Thr, Gly, or Asn for Ser, and His, Lys, Glu, Gln for Arg. Conservative substitutions are described, for example, in Molecular Cloning: A Laboratory Manual, 4th Edition, edited by Green and Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2014, and corrections and updates thereto.

[0159] The term "antibody" refers to immunoglobulins and related molecules, including monoclonal antibodies, polyclonal antibodies, monomers, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain only antibodies, three-chain antibodies, single chain Fvs, nanobodies. Antibodies can be polyclonal or monoclonal or recombinant antibodies. Antibodies can be murine, human, donkey, goat, humanized, chimeric, or derived from other species. As used herein, when an antibody or other entity "specifically recognizes" or "specifically binds" to an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules and binds to the antigen or epitope with substantially higher affinity than it does to other entities that do not display the antigen or epitope. A primary antibody binds to a specific antigen. A secondary (e.g., tertiary) antibody specifically binds to another antibody, typically of a class or subclass, usually via an Fc domain on the other antibody.

[0160] The term "antigen-binding fragment" refers to a fragment of an antibody that binds to an antigen or epitope.

[0161] The term "bait molecule" refers to a molecule that specifically interacts with a molecule of interest, sometimes called a target (or prey). Examples of bait molecules include antibodies, CLIP tags, drugs, nucleic acids, fluorescent in situ hybridization (FISH) probes, protein A, protein G, protein L, protein A / G, protein A / G / L, other small molecules, and SNAP tags.

[0162] The term "binding" refers to a physical interaction of a first moiety with a second moiety, where the first and second moieties are in physical contact with one another.

[0163] The term "biotin derivative" refers to biotin moieties including biotin and modifications of biotin such as biotin with open rings or substitutions. Typically, biotin derivatives are readily detectable using a biotin-binding entity or protein such as avidin, NeutrAvidin, or streptavidin.

[0164] The term "biotin-binding protein" refers to a protein that specifically binds biotin with high affinity. Examples of biotin detection reagents are avidin, NeutrAvidin, or streptavidin, structural analogs of which can each bind four biotins per biotin-binding protein molecule.

[0165] The term "catalyzed reporter deposition" (CARD) refers to the enzyme-catalyzed deposition of a detectable molecule on or near a target biomolecule (e.g., carbohydrate, lipid, nucleic acid, or protein). In some embodiments, the enzyme in the enzyme-catalyzed deposition is horseradish peroxidase (HRP) and the detectable molecule is tyramide or digoxigenin (DIG).

[0166] The term "click chemistry" refers to a chemical approach to easily link molecular building blocks. Click chemistry reactions are typically efficient, high-yielding, reliable, produce few or no by-products, and are compatible with aqueous environments or without added solvents. Examples of click chemistry are cycloadditions such as the copper(I)-catalyzed [3+2]-Husgen 1,3-dipolar cycloaddition of alkynes and azides to form 1,2,3-triazoles or Diels-Alder reactions. Click chemistry also includes copper-free reactions such as variants using substituted cyclooctynes ​​(see, for example, JM Baskin et al., Proc. Natl. Acad. Sci. USA 2007 Oct. 23, 104 (43), 16793-16797). Further examples of click chemistry are nucleophilic substitution; addition to CC multiple bonds (e.g., Michael addition, epoxidation, dihydroxylation, aziridination); and non-aldol-like chemistry (e.g., N-hydroxysuccinimide active ester coupling). Click chemistry reactions can be, but are not required to be, bioorthogonal reactions.

[0167] The term "conjugation" refers to the process by which two or more molecules specifically interact. In some embodiments, a tag and a label are conjugated. In some embodiments, a bait and a biomolecule are conjugated.

[0168] The term "conjugable" refers to a molecule that can specifically assemble with another molecule to which it can be conjugated. In some embodiments, the bait is conjugable to a biomolecule of interest. In some embodiments, the connector is conjugable to a main subject probe.

[0169] The term "detectable label" refers to a compound or composition that is directly or indirectly conjugated or configured to be conjugated to a molecule. The label itself may be detectable and may be a directly detectable label (e.g., a fluorescent label such as a fluorescent chemical adduct, a radioisotope label, etc.), or the label may be indirectly detectable (e.g., in the case of an enzymatically detectable label, the enzyme may catalyze a chemical change of a substrate compound or composition, and the product of the reaction may be detectable, etc.). Examples of detectable labels include, for example, biotin labels, fluorescent labels, horseradish peroxidase, immunologically detectable labels (e.g., hemagglutinin (HA) tags, poly-histidine tags), other luminescent labels, and radioactive labels. An example of an indirect label is biotin, which may be detected using streptavidin detection methods.

[0170] The term "immunoglobulin-binding peptide" refers to a peptide that can specifically bind with high affinity to a region of an immunoglobulin molecule (antibody) other than the complementarity determining region (CDR) / antigen-binding fragment (Fab) region. An immunoglobulin-binding peptide is a non-antibody (e.g., non-secondary antibody) that binds to another antibody. An immunoglobulin-binding peptide typically binds to the Fc (fragment, crystallizable) region of an immunoglobulin (antibody). An immunoglobulin-binding peptide is typically an immunoglobulin-binding protein, mimic, and variant thereof, including recombinant variants of immunoglobulin-binding bacterial proteins. Examples of non-antibody immunoglobulin-binding proteins include Protein A, Protein G, Protein L, Protein Z, Protein A / G, and Protein A / G / L. Protein A and Protein G are bacterial proteins originally obtained from the bacteria Staphylococcus aureus and Group G Streptococcus, respectively, and have high affinity for the Fc region of IgG-type antibodies. Protein A / G combines the binding domains of Protein A and Protein G. Protein A / G / L combines the binding domains of Protein A, Protein G, and Protein L. Protein A, Protein G, Protein L, Protein Z, Protein A / G, and Protein A / G / L share structural similarities.

[0171] The term "instructional material" includes publications, records, diagrams, links, or any other medium of expression that can be used to communicate the utility of one or more compositions of the invention for their designated use. The instructional material of the kits of the invention can, for example, be affixed to a container holding the composition or component or shipped together with a container holding the composition or component. Alternatively, the instructional material can be shipped separately from the container with the instructional material and the composition or component intended to be used cooperatively by the recipient.

[0172] The term "label" refers to a molecule that produces or can be induced to produce a detectable signal. In some embodiments, the label produces a signal for detecting adjacent biomolecules. Examples of labels that can be used include avidin labels, NeutrAvidin labels, and streptavidin labels for detecting biotin tags.

[0173] The term "linker" refers to a structure that connects two or more substructures. A linker has at least one uninterrupted chain of atoms that extends between the substructures. The atoms of the linker are connected by chemical bonds, typically covalent bonds.

[0174] The phrases "bonded to," "coupled / connected to," "conjugated to," "capable of being conjugated to," "attached to," and "linked to" mean directly or indirectly bonded / conjugated / attached / connected. For example, a bait molecule can be directly bonded to a photoreactive moiety with no intervening atoms, groups, or moieties between them; alternatively, it can be indirectly bonded to a photoreactive moiety by one or more intervening atoms, groups, moieties, or linkers between them. Intervening atoms, groups, moieties, or linkers can include, for example, one or more non-carbon atoms, groups, or moieties that can include amine, amide, ether, ester, or thioester bonds, or unsubstituted or substituted alkylene or alkenylene groups, which can be optionally interrupted by one or more heteroatoms and / or rings, including optionally substituted aromatic rings.

[0175] The term "mass spectrometer" refers to an instrument that measures the mass-to-charge ratio of one or more molecules in a sample. A mass spectrometer typically comprises an ion source and a mass analyzer. Examples of mass spectrometers include matrix-assisted laser desorption ionization (MALDI), continuous or pulsed electrospray (ES) ionization, ionspray, magnetic sector, thermospray, time-of-flight, and massive cluster bombardment mass spectrometry.

[0176] The term "mass spectrometry" refers to the detection of gas phase ions using a mass spectrometer.

[0177] The term "mass spectrometry analysis" includes linear time-of-flight (TOF), reflectron time-of-flight, single quadrupole, multiple quadrupole, single magnetic sector, multiple magnetic sector, Fourier transform, ion cyclotron resonance (ICR), or ion trap.

[0178] The terms "photoactivated" or "light activated" refer to the excitation of atoms by radiant energy (e.g., by light of a particular wavelength or range of wavelengths, UV light, etc.). In some instances, the light-activated catalyst promotes covalent bond formation between a tagged phenol and an amino acid.

[0179] The term "peptide" refers to a polymer in which the monomers are amino acids and the monomers are linked together through amide bonds. Peptides are typically at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, or at least 500 or more amino acids in length.

[0180] The term "photoreactive site" refers to a functional site that is activated upon exposure to light (e.g., light of a particular wavelength or range of wavelengths, UV light, etc.). In some instances, the photoreactive site can facilitate covalent bond formation between a subject probe and an amino acid or biomolecule.

[0181] The term "proximity molecule" or neighboring molecule refers to a molecule that is near another molecule (typically a molecule of interest). A proximity molecule or neighbor molecule may be bound (e.g., covalently or non-covalently) to the molecule of interest, or it may be in close proximity to the molecule of interest and not bound to it.

[0182] The term "prey" refers to the binding partner of a bait molecule. For example, if an antibody is a bait, the corresponding protein that the antibody can bind to is the corresponding prey. In some embodiments, a bait can bind to a single prey. In some embodiments, a bait can bind to two or more preys.

[0183] The term "protein tag" refers to a peptide sequence of amino acids. Protein tags can usually be conjugated to a label. An example of a protein tag is a "self-labeling" tag. Examples of self-labeling tags include BL tag, CLIP tag, covalent TMP tag, HALO tag, and SNAP tag. SNAP tag is a 20 kDa variant of the DNA repair protein O6-alkylguanine-DNA alkyltransferase that specifically recognizes and reacts rapidly with benzylguanine (BG) derivatives. During the labeling reaction, the benzyl moiety is covalently attached to the SNAP tag, releasing guanine. CLIP tag is a variant of SNAP tag that is configured to specifically react with O2-benzylcytosine (BC) derivatives rather than benzylguanine (BG).

[0184] The term "secondary antibody" refers to an antibody that specifically recognizes a region of another antibody. Generally, a secondary antibody recognizes the Fc region of an antibody of a particular isotype. A secondary antibody may also recognize the Fc region from one or more particular species.

[0185] The term "small molecule" refers to a molecule with low molecular weight, including carbohydrates, drugs, enzyme inhibitors, lipids, metabolites, monosaccharides, natural products, nucleic acids, peptides, peptidomimetics, second messengers, small organic molecules, and xenobiotics. Typically, a small molecule has a molecular weight of less than (about) 1000 or less than about 500. A small molecule may be a drug molecule. Drug molecules that can be used herein are molecules originally intended for use in the diagnosis, cure, mitigation, treatment, or prevention of a device.

[0186] The term "tag" refers to a functional group, compound, molecule, or substituent, etc., that can enable detection of a target or other molecule. A tag can enable a detectable biological or physiochemical signal that can be detected by any means, such as absorbance, chemiluminescence, colorimetry, fluorescence, luminescence, magnetic resonance, phosphorescence, radioactivity. The detectable signal provided by the tag can be directly detectable by the biochemical or physiochemical properties of the tag site (e.g., fluorophore tag) or indirectly detectable by the tag interacting with another compound or agent. Typically, the tag is a small functional group or a small organic compound, such as biotin, desthiobiotin, etc. In some embodiments, the tag employed has a molecular weight of less than about 1,000 Da, less than 750 Da, less than 500 Da, or even lower.

[0187] The term "tagging" refers to the process of adding a tag to a functional group, compound, molecule, or substituent, etc. Typically, tagging allows for the detection of a target molecule.

[0188] The term "tyramide signal amplification" (TSA) refers to catalyzed reporter deposition (CARD), an enzyme-mediated detection method that utilizes the catalytic activity of an enzyme (e.g., horseradish peroxidase) to catalyze inactive tyramides to highly active tyramides. This amplification can occur in the presence of low concentrations of hydrogen peroxide (H2O2). In some instances, the tyramide can be labeled with a detectable label, such as a fluorophore (e.g., biotin or 2,4-dinitrophenol (DNP)).

[0189] Unless otherwise indicated, the practice of the techniques described herein may employ techniques of chemistry, biochemistry, cell biology, immunology, molecular biology (including cell culture, recombinant technology, sequencing technology), and organic chemistry as described in the prior art and in references in the field (e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, Green and Sambrook, eds., Spring Harbor Laboratory Press, Cold Spring Harbor 2014, and corrections and updates thereto; John D. Roberts and Marjorie C. Caserio (1977), Basic Principles of Organic Chemistry, 2nd Edition, WA Benjamin, Menlo Park, Calif.).

[0190] (kit)

[0191] Described herein are kits that may be useful for practicing the methods described herein, for example, for analyzing, tagging, and labeling biomolecules, identifying and / or isolating one or more biomarkers (e.g., proximal or adjacent molecules), predicting biomarkers, and identifying biomarker lists. The kits may include photoreactive probes and / or subject probes. The kits may include additional components that facilitate the particular application for which the kit is designed. Thus, for example, the kit may further include materials useful for detecting the sample and / or for detecting the label (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, an enzyme, or associated detection reagents including reagents for performing catalyzed reporter deposition (CARD) or signal amplification (e.g., biotin-binding proteins (e.g., avidin, neutravidin, streptavidin, etc.), HRP, tyramide, hydrogen peroxide, etc.). The kit may also include wash solutions for one or more steps (e.g., after sample fixation), such as blocking agents, detergents, salts (e.g., sodium chloride, potassium chloride, phosphate buffered saline (PBS)). The kit may include variations of wash solutions, such as concentrates of wash buffers configured to be diluted prior to use or components used to make one or more wash solutions, as well as other reagents routinely used for the performance of a particular method. The kit may include fixatives and other sample preparation materials (e.g., ethanol, methanol, formalin, paraffin, etc.).

[0192] The kit can include instructional materials disclosing methods for making or modifying one or more probes, such as methods for attaching a bait molecule to a photoreactive site to prepare a photoreactive probe, methods for applying the photoreactive probe to a sample, methods for conjugating the bait molecule of the photoreactive probe to a prey molecule (in the sample), methods for removing (washing away) unconjugated photoreactive probe from the sample, methods for applying a subject probe having a tag (also referred to herein as a main subject probe) to the sample, methods for photoactivating the photoreactive probe to allow the subject probe having a tag to bind to a molecule of interest, methods for removing (washing away) unbound subject probe, and methods for applying a label to the sample, etc. The kit can additionally or instead include instructional materials teaching the use of the photoreactive probes, subject probes, labels, wash solutions, and the like.

[0193] The photoreactive kits described herein can be advantageously used with a microscope system, such as the systems described herein and in US Patent Publication No. 2018 / 0367717 A1, that allow for automated labeling of cellular biomolecules proximal to a biomolecule of interest. The labeled molecule may be directly adjacent to the biomolecule of interest, or may be in close proximity but not directly adjacent, such as when an intervening molecule for capture or analysis is present between the biomolecule of interest and the cellular biomolecule. Molecules that are in close proximity but not adjacent to the molecule of interest may be part of a cellular structure or may otherwise contribute to the cellular microenvironment of interest. Figure 1 shows a schematic diagram of a system useful for photoselective spatial tagging and labeling of biomolecules. The bottom part of Figure 1 shows a substrate 106, such as a microscope stage, and a monolayer of a plurality of cells 108 disposed on the substrate 106. In some embodiments, the surface of the entire substrate or a portion of the substrate can be analyzed using an automated microscope system to identify regions of interest. For example, the sample can be stained or labeled to identify regions of interest. The top of FIG. 1 shows a close-up of a cell 108a, one of a plurality of cells 108. The cell 108a has a nucleus 116 and a plurality of different types of organelles 112, such as a cell membrane, mitochondria, ribosomes, and vacuoles. The microscope system 102 selectively illuminates a region of interest (ROI) 118 with narrowband light 104 for analysis of the region of interest 118. Illumination can be in a selected area, while other (and larger) regions 114 of the cell and substrate are not illuminated. As described in more detail below, the narrowband light 104 activates photoreactive probes to enable binding of the subject probes to the biological sample only in the region of interest 118.

[0194] The photoreactive probe has the formula (I): [ka] It can be expressed as:

[0195] wherein the C portion comprises a chemical bond or linker, the B portion comprises a photoreactive moiety bound to the C portion; and the G portion comprises a bait molecule bound to the C portion and is configured to conjugate with a biological sample.

[0196] In some embodiments, the photoreactive probe has formula (IA): [ka]

[0197] (Wherein, the linker C of the photoreactive probe has K in the proximal region of the linker. 1 Part; and The distal region of the linker is 2 moiety, and photoreactive site B is located in the proximal region of the linker (K 1 ) and the bait molecule G is linked to the distal region of the linker (K 2 ) may have the following structure:

[0198] FIG. 2A shows a schematic of a photoreactive probe 205 having a bait molecule 251 conjugated to a photoreactive moiety 253. FIG. 2B shows a schematic of a subject probe 206 (sometimes referred to herein as a primary subject probe) having a tag moiety 261 and a subject moiety 263. FIG. 2C shows a subject probe having a desthiobiotin tag and a phenol subject moiety. Although FIGS. 2A and 2C (collectively) show the bait molecule 251, tag moiety 261, and subject moiety 263 as an antibody (such as a secondary antibody), desthiobiotin, and phenol, respectively, any of the bait molecules, tag moieties, and subject moieties described herein can be used. FIG. 2D shows a schematic of a subject probe 236 having a biotin tag 226 and an activated phenolic group 243′. FIG. 2D shows the subject probe after photoirradiation of the photoreactive probe (probe 205), which subsequently activates the phenolic group of the subject probe, such as the phenolic group shown in FIG. 2C, such that the activated subject probe has a phenolic group (e.g., a reactive group) with a free radical. The free radical may be particularly useful for conjugation with nearby proteins. The biotin tag 226 (or other tag) can later be used to isolate or enrich the tagged adjacent protein to which it is attached. For example, if the tag 226 is biotin, the adjacent protein can be isolated or enriched by using biotin-avidin affinity technology (biotin-biotin binding protein affinity technology).

[0199] Figure 2E shows the interaction of a ruthenium photoreactive probe with a desthiobiotin tag and a subject probe with a phenol subject moiety, resulting in labeling of an adjacent protein. After entering the reactive range of the photoreactive moiety 253' / dashed circle 228 shown in Figure 2G, the phenol becomes a tyrosyl radical, which reacts specifically with and conjugates to the amino acid tyrosine residue of a nearby protein.

[0200] 2F-2G show schematic steps of biomolecular proximity tagging in target region 228. As shown in FIG. 2F, after photoreactive probe 205 is delivered to a sample, such as a cell or organelle sample, and is able to detect and bind to a primary antibody or a target biomolecule 301, such as a carbohydrate, lipid, nucleic acid, or protein, therein (e.g., via bound photoreactive probe 205a), a subject probe 206 is added to the sample. FIG. 2G shows photoexcitation of photoreactive probe 205 to form photoexcited photoreactive probe 205' having photoexcited photoreactive site 253. Photoexcited photoreactive probe 205' is conjugated to target biomolecule 301, allowing subject probe 206 to bind to an adjacent molecule upon photoexcitation of photoreactive probe 205 conjugated to target biomolecule 301 (arrow 254). (FIG. 2G shows the tag portion 261 of the subject probe 206 bound to a molecule such as adjacent molecule 261; for clarity, the remainder of the probe 206 (including subject portion 263) is not shown but is present). While this example shows a primary antibody recognizing and binding to a target biomolecule of interest 301, other photoreactive probes 205 having other bait molecules 251 can also detect and bind to any target as described herein. The subject probe 206 includes a tag portion 261 that can be detected using the methods described herein. For example, if the tag portion 261 is a biotin derivative (including biotin), it can be detected using at least one biotin-binding protein (avidin, streptavidin, and / or neutravidin) detection method. In some instances, advantageously, this light-assisted enzymatic labeling method can significantly reduce background noise with selective photocatalytic probes (probes 205), label cytoplasmic organelles with higher resolution and greater detail, and achieve subcellular proteomics without tyramide signal amplification.

[0201] FIG. 2H shows an example of a labeling system 207 that can be used with the subject probe 206 shown in FIG. 2B to label a biomolecule adjacent to a target biomolecule of interest. The labeling system 207 includes a labeling complex 271 having a connector 272, and an enzyme or catalyst 274, and an additional subject probe 278. FIG. 2H also shows a fluorescent connector site 284. The fluorescent connector site 284 can be useful to identify which areas are labeled by the subject probe 206 if the tag portion 261 of the subject probe 206 has a high affinity for the connector. Although this example shows a fluorescent connector site 284, any connector with or without a fluorophore can be used. In some embodiments, the connector 272 can be a biotin-binding protein-dye conjugate (avidin-dye conjugate, streptavidin-dye conjugate, neutravidin-dye conjugate), or the like, and the enzyme or catalyst 274 can be a tag-peroxidase, utilizing peroxide (not shown) for activity. FIG. 2H also shows a tag 282, such as biotin, that can be used to conjugate the enzyme or catalyst 274 to the connector 272 (e.g., a biotin-binding protein such as avidin). After the tag (biotin) 282-enzyme 274 complex is conjugated to the connector 272, which is then conjugated onto the protein that has been labeled by the subject probe 206, the enzyme or catalyst 274 can be used to catalyze a proximal reaction, allowing additional subject probes to be activated to label proteins near the biotin 282-enzyme 274 complex. In this case, the subject probe 206 and the additional subject probe 278 may be the same or different molecules.

[0202] Figure 2H shows that connector 272 and tag moiety 261 (Figure 2B) recognize each other and conjugate together. An enzyme or catalyst 274 can activate additional subject probe 278, and once activated, the activated subject probe 278 (e.g., a tyramide probe) can bind to and detectably label a biomolecule in its vicinity. Additional subject probe 278 can include a tag moiety and a subject moiety, which can be the same as or different from subject probe 206. Any tag moiety and / or subject moiety described herein with respect to primary subject probe 206 can be used in additional subject probe 278.

[0203] FIG. 2I shows a comparison between direct photochemical labeling (top, named Process II) and photoassisted enzymatic labeling (bottom, named Process III) for a specimen containing a biomolecule (I) using the photoreactive kits and systems described herein to label the biomolecule in a small region of interest (ROI). Before performing either Process II or Process III, a sample (e.g., a cell or tissue sample) containing the biomolecule of interest 210 (herein, a protein is used as an example, but other biomolecules can be analyzed instead) is analyzed and a region of interest is identified. The sample can be pre-treated, such as fixed and stained. For example, the sample can be fixed and stained with a cell stain (e.g., hematoxylin and eosin (H&E); Masson's trichrome stain), and the protein of interest can be identified using an immunofluorescently labeled antibody that recognizes the protein of interest or by other methods. Once the region of interest is identified, the complex of adjacent biomolecules within the region of interest can be analyzed using the methods and systems described herein. As illustrated in process II of FIG. 2I, a sample can be treated with a direct photoreactive probe 212, and light (also referred to as pattern light) directed to an area of ​​interest in the sample can activate the direct photoreactive probe 212 to generate an activated direct photoreactive probe 212'. The activated direct photoreactive probe 212' can form a complex with other molecules in close proximity (indicated by a dotted circle in process II). The activated direct photoreactive probe 212' can diffuse to label neighboring molecules 211 near the molecule of interest 210. However, the labeling diameter (300-600 nm) of photoactivation of the direct photoreactive probe is spatially limited by the diffraction limit of the light source used. Furthermore, because the direct photoreactive probe is free to diffuse, any proteins in the path of the direct photoreactive probe (e.g., in the path of the pattern light) can be labeled. Process II also shows that it can label biomolecules 231 that are further away. The region or labeling precision labeled with the activated direct photoreactive probe 212' ranges from about 300 to 600 nm.This region may contain biomolecules that are not in close proximity to the protein of interest, potentially leading to confusing, misleading, or unhelpful results.

[0204] In contrast, in process III shown at the bottom of FIG. 2I, the photoreactive site 253 is pre-conjugated with the bait molecule 251 to form the photoreactive probe 205 (see FIG. 2A). The photoreactive site 253 can be pre-conjugated with the bait molecule 251, for example, via covalent bond with or without a linker, such as the linker shown in FIG. 4. As illustrated in process III of FIG. 2I, the photoreactive probe 205 is delivered to the sample (analyte) on the substrate 209, and the bait molecule 251 (as part of the photoreactive probe 205) recognizes the corresponding biomolecule of interest (e.g., target or prey). As illustrated in step (i) of FIG. 2I, again, a patterned light is directed to the sample in a selected location (region of interest). Here, however, the pattern light activates the photoreactive sites 253 of the photoreactive probes 205' currently bound to the biomolecules of interest (e.g., target or prey), and the activated photoreactive probes 205' promote the subject sites 263 of the primary subject probes 206 to form covalent bonds with nearby molecules or sites in the specimen. In addition to the pattern light being directed only to a limited area of ​​photoactivation, the readily accessible subject probes 206 are only activated within a limited catalytic radius (e.g., within the reaction range of the photoreactive sites, such as that represented by the target region 228), and are sufficiently reactive that they cannot diffuse widely after activation, and thus the primary subject probes 206 become covalently bound to the neighboring molecules 211 and 214. Step (i) also illustrates how background or undesired labeling can be reduced using the probes and methods described herein. In step (i), the photoreactive probe 205a is bound to a biological molecule; however, because the photoreactive probe 205 is outside the light delivery area (pattern light area), the photoreactive probe 205a is not activated and does not bind to the subject probe 206 and molecules outside the light delivery area.

[0205] Unbound subject probes are washed off with a wash solution. Steps (ii) and (iii) of FIG. 2I show the labeling of molecules near the molecule of interest 210 using the labeling system 207 shown in FIG. 2H. Other labeling systems can also be used. As an example, the connector 272 of the labeling system 271 is conjugated with the tag moiety 261 of the biomolecule (211) labeled in 206, and an enzyme or catalyst 274 activates the additional subject probe 278 to form an activated subject probe 278'. The activated additional subject probe 278' binds to the neighboring molecule 211 (see additional subject probe 278'' in FIG. 2I). Because the photoreactive probe 205' is bound to the molecule of interest 210, and the subject probe 206 and the additional subject probe 278 do not diffuse too far before reacting, the neighboring molecule 214 is labeled, while the more distant molecule 231 is not labeled.

[0206] By photoselectively localizing an enzyme or catalyst 274, such as peroxidase, near the molecule of interest (e.g., as part of the labeling system 207) and labeling neighboring molecules 214 within the region of interest using the tagging and labeling just described, the reaction catalyzed at 271 described in FIG. 2I can be localized to a small region of <100 nm. In some variations, larger regions (e.g., up to about 200 nm, up to about 300 nm, up to about 400 nm, up to about 500 nm, up to about 1 μm, up to about 2 μm, up to about 5 μm) can be labeled. Furthermore, some molecules of interest in a sample may have multiple small regions of localization, so that the photoreactive probe can simultaneously interact with different molecular complexes containing the same molecule of interest at different locations. Photolabeling can be used sequentially at two or more locations. For example, after applying light as shown in process III of Figure 2I, light can be selectively applied to a second (third, fourth, etc.) location in the sample, and this process can be repeated as many times as desired. In addition to labeling (placing labels on) a relatively small number of adjacent molecules in a very small area of ​​the sample, for example, thanks to the use of light and probe-guided microscopic analysis as described herein and below, this process can also be performed with sufficiently light or gentle treatment that cellular structures remain intact during use of the methods herein, advantageously allowing for the detection of naturally occurring biomolecular interactions.

[0207] Non-limiting examples of bait molecules that can be used herein can include one or more of an antibody, a CLIP tag, a HaloTag, a SNAP tag, a functional protein (e.g., Protein A, Protein G, Protein L, Protein A / G, Protein A / G / L, or a protein drug), an immunoglobulin binding peptide, a biotin-binding protein (including avidin, streptavidin, and / or neutravidin), an RNA molecule, a small molecule (e.g., erlotinib), a nucleic acid molecule, a fluorescent in situ hybridization (FISH) probe, an antigen-binding (Fab) region, a nanobody, a biological drug, and the like. Examples of biological drugs that can be used as baits include abatacept (Orencia); abciximab (ReoPro); abobotulinumtoxinA (Dysport); adalimumab (Humira); adalimumab-at (Amjevita); ado-trastuzumab emtansine (Kadcyla); aflibercept (Eylea); agalsidase beta (Fabrazyme); albiglutide (Tanzeum); aldesleukin (Proleukin); alemtuzumab (Campath, Lemtrada); alglucosidase alfa (Myozyme, Lumizyme); alirocumab (Praluent); alteplase, cassofloactivase (Activase); anakinra (Kineret); asfotase alfa (Strensiq); asparaginase (Elspar); blackleg disease (Erwinia chrysanthemi-derived asparaginase (Erwinaze); atezolizumab (Tecentriq); basiliximab (Simulect); becaplermin (Regranex); belatacept (Nulojix); belimumab (Benlysta); bevacizumab (Avastin); bezlotoxumab (Zinplava); blinatumomab (Blincyto); brentuximab vedotin (Adcetris); canakinumab (Ilaris); capromab pendetide (ProstaScint); certolizumab pegol (Cimzia); cetuximab (Erbitux); collagenase (Santyl);Clostridium histolyticum histolyticum-derived collagenase (Xiaflex); daclizumab (Zenapax); daclizumab (Zinbryta); daratumumab (Darzalex); darbepoetin alfa (Aranesp); denileukin diftitox (Ontak); denosumab (Prolia, Xgeva); dinutuximab (Unituxin); dornase alfa (Pulmozyme); dulaglutide (Trulicity); ecallantide (Kalbitor); eculizumab (Soliris); elosulfase alfa (Vimizim); elotuzumab (Empliciti); epoetin alfa (Epogen / Procrit); etanercept (Enbrel); etanercept-szzs (Erelzi); evolocumab (Repatha); filgrastim (Neupogen); filgrastim-sndz (Zarxio); follitropin alfa (Gonal f);galsulfase (Naglazyme);glucarpidase (Voraxaze);golimumab (Simponi);golimumab injection (Simponi Aria);ibritumomab tiuxetan (Zevalin);idarucizumab (Praxbind);idursulfase (Elaprase);incobotulinumtoxinA (Xeomin);infliximab (Remicade);infliximab-dyyb (Inflectra);interferon alfa-2b (Intron A);interferon alfa-n3 (Alferon N injections); interferon beta-1a (Avonex, Rebif); interferon beta-1b (Betaseron, Extavia); interferon gamma-1b (Actimmune); ipilimumab (Yervoy); ixekizumab (Taltz); laronidase (Aldurazyme); mepolizumab (Nucala); methoxypolyethylene glycol-epoetin beta (Mircera); metreleptin (Myalept); natalizumab (Tysabri); necitumumab (Portrazza); nivolumab (Opdivo); obiltoxaximab (Anthim); obinutuzumab (Gazyva);Ocriplasmin (Jetrea);Ofatumumab (Arzerra);Olaratumab (Lartruvo);Omalizumab (Xolair);OnabotulinumtoxinA (Botox);Oprelvekin (Neumega);Palifermin (Kepivance);Palivizumab (Synagis);Panitumumab (Vectibix);Parathyroid hormone (Natpara);Pegaspargase (Oncaspar);Pegfilgrastim (Neulasta);Peginterferon alfa-2a (Pegasys);Peginterferon alfa-2b (PegIntron, Sylatron);Peginterferon beta-1a (Plegridy);Pegloticase (Krystexxa);Pembrolizumab (Keytruda);Pertuzumab (Perjeta);Ram These include silumab (Cyramza); ranibizumab (Lucentis); rasburicase (Elitek); raxibacumabresulizumab (Cinqair); reteplase (Retavase); rilonacept (Arcalyst); rimabotulinumtoxinB (Myobloc); rituximab (Rituxan); romiplostim (Nplate); sargramostim (Leukine); sebelipase alfa (Kanuma); secukinumab (Cosentyx); siltuximab (Sylvant); tbo-filgrastim (Granix); tenecteplase (TNKase); tocilizumab (Actemra); trastuzumab (Herceptin); ustekinumab (Stelara); vedolizumab (Entyvio); ziv-aflibercept (Zaltrap);

[0208] Non-limiting examples of photoreactive moieties include aryl azides, benzophenones, riboflavin, flavin, lumiflavin and / or derivatives thereof, fluorescein or derivatives thereof, KillerRed (photosensitizer protein), miniSOG (photosensitizer protein), another photosensitizer protein (e.g., configured to generate reactive oxygen species (ROS) upon irradiation with light), methylene blue or derivatives thereof, phenols, pterin derivatives, derivatives, ruthenium-based photocatalysts, and rose bengal derivatives. Fluorescein is a fluorescent organic dye with four negatively charged carboxylic acid groups, and derivatives thereof are generally configured to preserve this active center and may have, for example, additions such as hydrocarbon tails, isothiocyanates, carboxylic acids, or amines. Derivatives of other molecules (unless otherwise stated or clear from the context) generally preserve the characteristic functionality of the molecule, but may be modified otherwise. In some embodiments, multiple photoreactive moieties are attached to an antibody (or other bait molecule) to form a photoreactive probe. The number of photoreactive sites attached to an antibody (or other bait molecule) can range from 1 to 50 (eg, 1, 1 to 5, 1 to 10, 1 to 20, etc.).

[0209] 3A-3G and 3Y-3AI show examples of photoreactive moieties (e.g., 253 photoreactive moieties) that can be used in the photoreactive probes described herein (e.g., photoreactive probe 205). 3A-3N show ruthenium-based photocatalysts. In 3Y-3AI, the shaded areas indicate the attachment locations of linkers (covalent bonds). 3O shows a rose bengal derivative. 3P-3Q show fluorescein derivatives. 3R shows a methylene blue derivative. 3S-3T show lumiflavin derivatives. 3U-3V show riboflavin and flavin derivatives. 3W-3X show pterin derivatives. The selection of a particular photoreactive moiety may depend on the desired wavelength and type of bait molecule. For example, the components of the photoreactive probe and the components for the pre-probe analysis may be selected so that they do not interfere (or interfere minimally) with each other.

[0210] In some embodiments, a ruthenium-based compound represented by formula (II) may be used in the B portion (photoreactive site) of a photoreactive probe represented by formula (I) or formula (IA):

[0211] [ka]

[0212] In the formula, L1, L2, L3, and L4 are each independently a ligand; and X 1 and X 2 are each independently a ligand having a reactive site, where the reactive site can be attached to the C portion of formula (I) listed above. For example, X 1 and X 2 may each independently be 3-ethynylpyridine, 3-(bromomethyl)pyridine, maleimide, 4'-methyl-4-carboxybipyridine-N-succinimidyl ester, nicotinaldehyde, l-(4-(pyridin-3-yl)-lH-1,2,3-triazol-l-yl)ethanone, 4-pentynenitrile, or 4-aminobutyne, etc.; L 1 and L 2 may be linked to form a first bidentate ligand, L 3 and L 4may be linked to form a second bidentate ligand, wherein the first bidentate ligand and the second bidentate ligand are each independently selected from 2,2'-bipyridyl (bpy), 4'-methyl-4-carboxybipyridine-N-succinimidyl ester, 4,4'-dicyano-5,5'-dimethyl-2,2'-bipyridine (CN-Me-bpy), 4,4'-dimethyl-2,2'-bipyridine (dmb), 4,4'-di-tert-butyl-2,2'-bipyridine (dbpy), 4,4',5,5'-tetramethyl-2,2'-bipyridine (dmb), 4,4',5,5'-tetramethyl-2,2'-bipyridine (dbpy ... , 2'-bipyridine (tmb), 2-phenylpyridine (ppy), 6-bromo-2,2'-bipyridine, 6,6'-dibromo-2,2'-bipyridine, 5-bromo-2,2'-bipyridine, 6-amino-2,2'-bipyridine, 6,6'-diamino-2,2'-bipyridine, 2,2'-bipyridine-6-carbonitrile, 2,2'-bipyridine-6,6'-bis(carbonitrile), 2,2'-bipyridine-6-carboxylic acid, 2,2'-bipyridine-6,6'-dicarboxylic acid, or biquinoline. Thus, the ruthenium-based compound of formula (II) may be [Ru(bpy) 2 (methyl-bpy-NHS)] 2+ , [Ru(bpy) 2 (bpy-(NHS)2)] 2+ , [Ru(bpy) 2 (methyl-bpy-(CH2)3-NHS)] 2+ , [Ru(bpy) 2 (bpy-((CH2) 3 -NHS) 2 ] 2+ , sulfo-tag NHS ester, [Ru(bpy) 2 (methyl-bpy-COOH)] 2+ , [Ru(bpy) 2 (bpy-(COOH) 2 )] 2+ , [Ru(bpy) 2 (Methyl-bpy-(CH 2 ) 3 COOH)] 2+ , [Ru(bpy) 2 (bpy-((CH2)3COOH)) 2 ] 2+, their salts (e.g., bis(hexafluorophosphate)), or any combination thereof. The NHS ester group may be replaced with a chemical group such as a maleimide group, an iodoacetyl group, a cysteine / thiol group, or a click chemistry group. The carboxyl group may be replaced with a chemical group such as a maleimide group, an iodoacetyl group, a cysteine / thiol group, or a click chemistry group. The click chemistry group may be an alkyne, BCN, DBCO, or N 3 In one embodiment, the ruthenium-based compound of formula (II) can be [Ru(bpy) 2 (isothiocyanato-phenanthroline)] 2+ , [Ru(bpy) 2 (aminophenanthroline)] 2+ , [Ru(bipyridine) 2 (3-ethynyl-pyridine) 2 ]+, Ru(bipyridine) 2 (3-Ethynylpyridine) 2 Cl 2 , Ru(bipyridine) 2 (3-Ethynylpyridine) 2 (PF 6 ) 2 , [Ru(biquinoline) 2 (4-Pentynenitrile) 2 ]+, Ru(biquinoline) 2 (4-Pentynenitrile) 2 Cl 2 , Ru(biquinoline) 2 (4-Pentynenitrile) 2 (PF6) 2 , [Ru(bipyridine) 2 (4-Aminobutyne) 2 ] + , [Ru(bipyridine) 2 (4-Pentynenitrile) 2 ] + , [Ru(bipyridine) 2 (Nicotinaldehyde) 2 ] + , [Ru(bipyridine)2(1-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethanone) 2 ]+ , [Ru(bipyridine) 2 (3-(Bromomethyl)pyridine) 2 ] + , [Ru(bipyridine)2(maleimide) 2 ] + , Ru(4,4-dicarboxylic acid-2,2'-bipyridine)(4,4'-bis(p-hexyloxystyryl)-2,2-bipyridine)(NCS) 2 , cis-bis(isothiocyanato)bis(2,2'-bipyridyl-4,4'-dicarboxylato)ruthenium(II), cis-bis(2,2'-bipyridine)dichlororuthenium(II) hydrate, bis(4,4-dicarboxy-2,2-bipyridine)dichlororuthenium(II), di-tetrabutylammonium cis-bis(isothiocyanato)bis(2,2'-bipyridyl-4,4'-dicarboxylato)ruthenium(II), a salt thereof, a stereoisomer thereof, or a tautomer thereof, and any combination thereof.

[0213] 4A to 4L show exemplary sites for the C (linker) portion in the photoreactive probe represented by the above-listed formula (I). In some embodiments, the C portion of the photoreactive probe may be, for example, NHS-BCN, NHS-(PEG), n -BCN, NHS-DBCO, NHS-(PEG)n-DBCO, NHS-alkyne, NHS-(PEG)n-alkyne, NHS-N 3 , NHS-(PEG) 3 , NHS-maleimide, NHS-(PEG) n -Maleimide, NHS-iodoacetyl group, NHS-(PEG) n -Iodoacetyl group, NHS-cysteine / thiol group, NHS-(PEG) n -Cysteine / thiol groups, Maleimide-peptides / amino acids, Maleimide-(PEG) n -Peptide / amino acid, maleimide-oligonucleotide, iodoacetyl-peptide / amino acid, iodoacetyl-(PEG) n-peptide / amino acid, or iodoacetyl-oligonucleotides, etc., can be used, where each n can be independently an integer from 1 to 20. In some embodiments, the linker is (PEG) n , a peptide, an amino acid, or an oligonucleotide, and where each n can independently be an integer from 1 to 20. Further examples of polymeric linkers include other polyethylene glycols (PEGs), polypropylene glycols, polyethylenes, polypropylenes, polyamides, and polyesters. The linkers can be linear molecules with at least one or two chain atoms, but can contain many more.

[0214] 5A-5K show examples of tag moieties that can be used in the subject probes described herein. The tag moieties (sometimes referred to herein as "tags" or "tags") are configured to interact with a detectable label to label a biomolecule adjacent to a molecule of interest (first molecule) to produce a detectable label. 5A-5E show examples of click chemistry tags that can be used with the probes. The click chemistry tag can be, for example, an azide moiety or an alkyne moiety. 5F-5H show examples of biotin derivatives that can be used as probe tags in the subject probes. 5I shows a digoxigenin moiety tag. 5J shows a peptide tag. In particular, 5J shows a poly-His tag (SEQ ID NO: 1) that contains six histidines. However, the histidine tag could instead contain fewer or more histidines, such as five histidines or seven to ten histidines, or more than ten histidines. Figure 5K shows a SNAP tag. In certain instances, CLIP tags, oligonucleotides, or HaloTags could also be used.

[0215] 6A-6L show examples of subject moieties that can be used in the subject probes described herein, such as in the subject probe 206 shown in FIG. 2B (e.g., with a tag moiety). After activation of a photoreactive probe (e.g., photoreactive probe 205) with light, the activated photoreactive probe can generate a reactive intermediate (e.g., a free radical) from the subject moiety 263 of the subject probe 206, which can form a covalent bond with an amino acid or other biomolecule adjacent thereto (e.g., an adjacent molecule, such as adjacent molecule 211). The amino acid can be alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, etc. In some variations, the subject moiety can form a bond with an adjacent carbohydrate, lipid, or nucleic acid.

[0216] In the implementation of the photocatalytic ruthenium complex-antibody conjugate as the photoreactive probe 205, bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium can be specifically conjugated to a secondary antibody via an NHS-amide bond, and the secondary antibody can selectively hybridize to the primary antibody in the region of interest (ROI) (see, for example, FIG. 2E-FIG. 2E). [Ru(bpy) 3 ] 2+ is a photocatalyst that can be excited by either single-photon or two-photon irradiation at about 425 nm and 780 nm, respectively. The resulting oxidized [Ru(bpy) 3 ] 3+can steal an electron from the phenolic hydroxyl group of biotin-phenol (see, e.g., FIG. 2B) as a subject probe, generating a phenoxyl radical and a proton at a nearby tyrosine residue. To achieve higher labeling density for omics studies, e.g., proteomic profiling, HRP amplification can be utilized to further covalently bind tyramide radicals to nearby tyrosine residues of nearby proteins, followed by streptavidin enrichment and on-bead digestion. Finally, a subcellular / localized proteomic profile can be obtained by performing quantitative proteomic analysis on the specimen, e.g., after removing the specimen from the microscope slide. Implementations include the use of antibody-based ruthenium complexes ([Ru(bpy)) as selective and photocatalytic probes for spatial and localized proteomic analysis. 3 ] 2+ )-antibody conjugation, identification of novel proteins in organelles that cannot be fractionated or isolated by conventional methods, and size- and morphologically distinguishable labeling by single- or two-photon irradiation.

[0217] In some embodiments, described herein is a photoreactive kit that includes at least one of a photoreactive probe and a primary subject probe, or both a photoreactive probe and a primary subject probe. In this and other embodiments, the photoreactive probe has the formula (I): [ka] wherein the C moiety is a single chemical bond or linker; the B moiety comprises a photoreactive moiety attached to the C moiety; and the G moiety comprises a bait molecule attached to the C moiety. In this and other embodiments, the primary subject probe comprises a detectable tag and a subject moiety. The primary subject probe can comprise a detectable tag bound to a photoexcitable subject moiety. In some embodiments, the bait molecule of the photoreactive probe is configured to conjugate with a first molecule in a biological sample, and the photoreactive probe is configured to form a covalent bond with a target molecule in the biological sample when the photoreactive probe is photoactivated and the primary subject probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe. In some embodiments, the first molecule and the target molecule are different entities. In some embodiments, the first molecule and the target molecule are the same entity. The disclosure of a first molecule herein may also refer to or be applicable to a target molecule, unless the context indicates otherwise.

[0218] Photoselective tagging and labeling as described herein can be performed in various types of samples, such as samples from tissues, cells, or particles, such as samples from entities (e.g., human subjects, mouse subjects, rat subjects, insect subjects, plants, fungi, microorganisms, viruses), or non-biological tissue or cell samples, such as cell culture samples or artificial tissue scaffold samples (e.g., cultured laboratory cells, in vitro developed cardiac tissue, 3d printed tissue, etc.). Samples for analysis using the probes, materials, and methods described herein can be living (living cells) or non-living (e.g., fixed). Samples for tagging and labeling can include monolayer samples, multilayer samples, samples fixed to a substrate (e.g., microscope slides), samples not fixed to a substrate, suspensions of cells, or extracts such as in vitro cell extracts, reconstituted cell extracts, or synthetic extracts. In some embodiments, the sample is not fixed (unfixed). Examples of probes useful for tagging live cells include those that utilize small molecules or are sometimes referred to as self-labeling molecules (e.g., CLIP tags, HALO tags, SNAP tags). In some embodiments, a large number of cells can be automatically analyzed using the methods and materials described herein (e.g., at least about 1,000 cells, at least 10,000 cells, at least 100,000 cells, at least 1,000,000 cells). In some embodiments, a smaller number of cells can be analyzed, such as 1,000 cells or less, 100 cells or less, or only a few cells, or a single cell. In some embodiments, the sample is fixed. For example, cell or tissue samples can be fixed with, for example, acetic acid, acetone, formaldehyde (4%), formalin (10%), methanol, glutaraldehyde, or picric acid. The fixative can be a relatively strong fixative and can crosslink molecules, or it can be weaker and can not crosslink molecules. Cell or tissue samples for analysis may be frozen, for example using dry ice, or flash frozen, prior to analysis.The cell or tissue sample may be embedded in a solid or semi-solid material, such as paraffin or resin, prior to analysis. In some embodiments, the cell or tissue sample for analysis may be fixed and subsequently embedded, such as formalin-fixed and paraffin-embedded (FFPE). The bait moiety (of the photoreactive probe) may be one or more of a nucleic acid, a protein, and a small molecule. The nucleic acid bait moiety may be DNA, cDNA, or RNA. The nucleic acid bait moiety may be an in situ hybridization probe, such as a fluorescent in situ hybridization probe, or a non-fluorescent in situ hybridization probe, such as a chemiluminescent in situ hybridization probe (CISH). An in situ hybridization probe is one or more nucleic acid strands composed of DNA, cDNA, or RNA that may contain or be modified to contain a fluorescent or other detectable moiety. Fluorescent or non-fluorescent in situ hybridization probes can be shorter or longer, but are typically 15 to 2,000 bases in length (e.g., 15 to 30 bp in length, 15 bp to 100 bp in length, 30 bp to 100 bp in length, 100 bp to 1000 bp in length, 500 bp to 200 bp in length, etc.). The in situ hybridization bait portion (of the photoreactive probe) can be useful for hybridization with a prey or target (can be configured to hybridize with a prey or target). After the in situ hybridization bait portion of the (photoreactive) probe binds to the prey, light activation can be used to photoactivate the photoreactive probe, and the primary subject probe can be acted upon by the photoactivated probe to form a photoexcited primary subject probe, which can form (or is configured to form) a covalent bond with a second molecule (a third molecule, a fourth molecule, etc.), such as a biomolecule (such as a carbohydrate, lipid, nucleic acid, and protein) in a biological sample.In some embodiments, the bait molecule can include a reactive ligand. The reactive ligand can be configured to react with a corresponding molecule in a sample of interest. Such a reaction can result in rapid and irreversible bond formation between the reactive ligand and the corresponding molecule. In some embodiments, the reactive ligand bait molecule can utilize self-labeling protein (SLP) technology. Self-labeling protein technology is based on the formation of a specific covalent bond between a pair of reactants: a reactive ligand and a fusion protein configured (engineered) to bind to the ligand. The fusion protein for use in the self-labeling protein technology can be a self-labeling protein tag (typically derived from an enzyme) that is fused to a protein of interest (POI). In contrast to enzyme reactions that increase the rate of a chemical reaction without being permanently altered by the reaction, the self-labeling protein technology can result in the formation of a specific covalent bond between a ligand and a corresponding fusion protein. As with other enzyme reactions, the rate of the reaction using the self-labeling protein technology is fast and the reaction is highly specific. In the self-labeling protein technology, the ligand can label the fusion protein without requiring an additional enzyme for its labeling. Examples of reactive ligands useful as baits (in photoreactive probes) include, but are not limited to, CLIP tag™ ligands (also referred to as CLIP tag™ substrates or CLIP tags), HaloTag® ligands (also referred to as HaloTag® substrates or HaloTag®), and SNAP tag® ligands (also referred to as SNAP tag® substrates or SNAP tags). An example of a SNAP tag® ligand useful as a bait is a benzylguanine (BG) derivative, and an example of a corresponding fusion protein is a derivative of the 20kDa DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATG) that reacts specifically and rapidly with the benzylguanine (BG) derivative and is fused to a protein of interest (e.g., a self-labeling protein SNAP tag protein portion).The SNAP-tag® reaction can release a chemically inert guanine, making it (and other self-labeling protein systems) safe to use in live cells, in addition to being useful in other samples, such as non-living (fixed) cells, cell extracts, or other tissue extracts. An example of a CLIP-tag™ ligand useful as a bait is a benzylcytosine (BC) derivative, such as O2-benzylcytosine, which has a cytosine leaving group via a benzyl linker. An example of a corresponding fusion protein is a derivative of the 20 kDa DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATG), which reacts specifically and rapidly with benzylcytosine (BC) derivatives and is fused to a protein of interest (e.g., the self-labeling protein CLIP-tag protein portion). Examples of HaloTag® ligands useful as baits are reactive haloalkane (e.g., chloroalkane) derivatives, and examples of corresponding fusion proteins are modified versions of bacterial dehalogenases that remove halogens from aliphatic hydrocarbon molecules, such as by fused nucleophilic aspartic acid residues, and are fused to a protein of interest (e.g., a self-labeling protein HaloTag protein moiety). As part of some photoreactive probes, a reactive ligand bait molecule is attached to the photoreactive site of the photoreactive probe via a photoreactive probe linker (or a single chemical bond) (e.g., as part of the "G" in GCB photoreactive probes, G-K2-K1-B photoreactive probes, etc.). The protein of interest can be an endogenous protein or a non-endogenous protein (antibody, structural protein, etc.). Fusion proteins with a protein of interest can be tagged with a self-labeling protein moiety (CLIP tag protein moiety, HaloTag protein moiety, SLIP tagged protein moiety), expressed using standard recombinant protein expression techniques, and used as described herein. Cells can be genetically modified with a nucleic acid comprising a nucleotide sequence encoding the fusion protein and analyzed directly, or the fusion protein can be expressed and added to a sample, e.g., a cell sample, a cell extract sample, a tissue extract sample, etc.In some variations, the self-labeled protein is not a fusion protein and can include a post-translationally modified tag moiety or protein moiety non-covalently bound to the protein of interest.

[0219] The prey of the in situ hybridization bait moiety can be a target DNA, target cDNA, or target RNA, such as a portion of a genome (e.g., a chromosome, expressed RNA, rRNA, etc.) in a cell, a cell extract, or other tissue extract.

[0220] The concentration of the photoreactive probe can range from 0.1 ug / mL to 100 ug / mL, and the concentration of the subject probe can range from 1 uM to 20 mM. The wavelength of light for activation of the photoreactive probe or photoselective tagging and labeling is, in some embodiments, about 200 nm to about 1600 nm, for example, about 200 nm to about 250 nm, about 250 nm to about 300 nm, about 300 nm to about 350 nm, about 350 nm to about 400 nm, about 400 nm to about 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 550 nm to about 600 nm, about 600 nm to about 650 nm, about 650 nm to about 700 nm, about 750 nm to about 800 nm, about 800 nm to about 900 nm, about 900 nm to about 1000 nm, about 1000 nm to about 1100 nm, about 1000 nm to about 1200 nm, about 1000 nm to about 1300 nm, about 1000 nm to about 1400 nm, about 1000 nm to about 1500 nm, about 1000 nm to about 1600 nm, about 1000 nm to about 1700 nm, about 1000 nm to about 1800 nm, about 1000 nm to about 1900 nm, about 1000 nm to about 2000 nm, about 1000 nm to about 2100 nm, about 1000 nm to about 2200 nm, about 1000 nm to about 2300 nm, about 1000 nm to about 2400 nm, about 1000 nm to about nm to about 700 nm, about 700 nm to about 750 nm, about 750 nm to about 800 nm, about 800 nm to about 850 nm, about 850 nm to about 900 nm, about 900 nm to about 950 nm, about 950 nm to about 1000 nm, about 1000 nm to about 1100 nm, about 1100 nm to about 1200 nm, about 1200 nm to about 1300 nm, about 1300 nm to about 1400 nm, about 1400 nm to about 1500 nm, or about 1500 nm to about 1600 nm. In some embodiments, the wavelength of light for photoselective tagging and labeling ranges from about 700 nm to about 1600 nm (near infrared light) (e.g., 780 nm) for a two-photon light source; or from about 300 nm to about 650 nm or to about 700 nm (visible light) for a single-photon light source (e.g., 360 nm, 405 nm, 425 nm). The wavelength of light used for photoactivation of the photoreactive probe is generally different from the wavelength of light used for imaging. In some embodiments, activation of the photoreactive probe utilizes optical radiation (light) of about 300-450 nm, 550 nm for single-photon activation, or >720 nm for multiphoton activation. The particular wavelength depends on the particular photoreactive site of the photoreactive probe.

[0221] (method)

[0222] Also disclosed herein are methods for photoselectively tagging and labeling biological molecules and methods of analysis. The methods may be used to tag and / or label carbohydrates, lipids, nucleic acids, proteins, either alone or in combination. The methods may include treating (exposing) a biological sample with a bait molecule and a photoreactive probe having a photoreactive moiety and allowing the bait molecule to bind to a first molecule (i.e., prey) in the biological sample. In some embodiments, the biological sample comprises a plurality of cells. In some embodiments, the biological sample comprises a plurality of live cells. In some embodiments, the biological sample comprises at least 1, at least 100, at least 1000, or at least 10,000 live cells. In some embodiments, the biological sample comprises a cell extract. In some embodiments, the photoreactive moiety is coupled to the bait molecule via a chemical bond or a linker. In some embodiments, the photoreactive probe and the first molecule form a non-covalently conjugated probe-first molecule complex or molecule. In some embodiments, the bait molecule comprises an antibody, a CLIP tag, a HaloTag, a SNAP tag, protein A, protein G, protein L, protein A / G, protein A / G / L, an immunoglobulin-binding peptide, a biotin-binding protein(s) (such as avidin, streptavidin, neutravidin, etc.), an RNA molecule, a small molecule, a nucleic acid molecule, a fluorescent in situ hybridization (FISH) probe, an antigen-binding (Fab) region, or a nanobody. In some embodiments, the photoreactive site or photoreactive probe comprises a ruthenium-based photocatalyst, an iridium-based photocatalyst, a rose bengal derivative, a fluorescein derivative, a methylene blue derivative, a flavin derivative, a lumiflavin, a riboflavin, a pterin derivative, a photosensitizing protein, a miniSOG, a Killer Red, a phenol, an aryl azide, or a benzophenone.

[0223] The method may include illuminating a biological sample with an imaging light source of an image-guided microscope system. The method may include imaging the illuminated sample with a camera. The method may include acquiring with the camera at least one image of a subcellular morphology of the sample in a first field of view. The method may include processing the at least one image and determining a region of interest in the sample based on the processed image. The method may include obtaining coordinate information of the region of interest.

[0224] The method may include delivering a subject probe to the biological sample. The method may include selectively irradiating the region of interest with optical light based on the obtained coordinate information, thereby activating a photoreactive site and resulting in covalent bond formation between the subject probe and the adjacent molecule or molecule of interest in the biological sample within the region of interest in the biological sample. The method may include removing unconjugated photoreactive probes from the biological sample to allow the photoreactive probe to direct the selective irradiation to the selected region of interest. In some embodiments, the selectively irradiating step includes irradiating a region with 25 us / pixel to 400 us / pixel, 50 us / pixel to 300 us / pixel, 75 us / pixel to 200 us / pixel, or 400 us / pixel to 5000 us / pixel. In some embodiments, the primary subject probe includes a tag portion and a subject portion. In some embodiments, the tag portion of the primary subject probe comprises a biotin derivative, a click chemistry tag, a HaloTag, a SNAP tag, a CLIP tag, a digoxigenin, a nucleic acid tag, or a peptide tag. In some embodiments, the click chemistry tag comprises an alkyne-based or azide-based moiety. In some embodiments, the subject moiety of the subject probe is configured to generate (in the biological specimen) a reactive intermediate (e.g., a phenoxyl radical or a carbene, etc.) that is responsible for covalent bond formation with an amino acid of a protein adjacent to the molecule of interest. In some embodiments, the activated photoreactive moiety can promote the formation of a free radical in the subject probe, thereby generating a covalent bond between the photoreactive moiety and an amino acid or biomolecule of the biological sample within the selected region of interest.In some embodiments, the amino acid (e.g., of the protein of interest or an adjacent protein molecule) can be alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, etc.

[0225] The method may further comprise conjugating a detectable label to the subject probe and detectably proximity labeling a proximal neighbor to the first molecule (i.e., the prey) with a detectable labeling activity. In some embodiments, the detectable label comprises a catalytic label. In some embodiments, the detectably proximity labeling comprises photoselective proximity labeling an area less than 5 μm, less than 2 μm, less than 1 μm, less than 500 nm, less than 300 nm, less than 200 nm, or less than 100 nm in diameter (or longest dimension). Some embodiments comprise conjugating a connector to the subject probe. In some embodiments, the connector is conjugated to the subject probe by affinity between the connector and the main subject probe to locate the biological sample covalently bound to the subject probe. Some embodiments further comprise conjugating a tag-peroxidase to the connector. In some embodiments, the tag-peroxidase is configured to catalyze an additional subject probe (e.g., a tyramide probe) to form a covalent bond between the additional subject probe and the biological sample. (The subject probe may be a first subject probe, and the additional subject probe may be a second subject probe.) More specifically, the tag-peroxidase can activate the additional subject probe to form a covalent bond between the additional subject probe and a tyrosine of the biological sample with a free radical. Some embodiments further include removing at least the region of interest from the microscope stage. Some embodiments further include performing mass spectrometry or sequence analysis on the selectively irradiated sample.

[0226] Some methods include contacting a biological sample having a target biomolecule with a photoreactive probe described herein to non-covalently conjugate the photoreactive probe of the kit with the target biomolecule, washing away unconjugated photoreactive probe, spatially selectively activating the photoreactive probe of the kit using optical radiation, thus inducing bond formation between a tagged subject probe of the kit and a nearby molecule in close proximity to the target biomolecule, washing away unbound subject probe, and further including the steps of labeling the tagged biomolecule / probe complex with a label and selectively proximity-labeling proximal neighbors to the target biomolecule.

[0227] Also disclosed herein is a method implemented in mass spectrometry for processing photolabeled samples to predict biomarkers. The method may include separating a plurality of biological samples into a photolabeled sample group and an unlabeled sample group. The method may include delivering a photoreactive kit as described herein to the photolabeled sample group and the unlabeled sample group. In some embodiments, the bait molecule is non-covalently conjugated with a first molecule in the biological sample. The method may include selectively illuminating a selected region of interest of the photolabeled sample group and keeping the unlabeled sample group in the dark, where the illuminating step allows the main subject probe to form a covalent bond with the biological sample. In some embodiments, the selectively illuminating the selected region of interest further includes activating the photoreactive site in the selected region such that the activated photoreactive site allows the main subject probe to form a covalent bond with the biological sample in the selected region of interest. The method may include removing unconjugated photoreactive probes from the biological sample to allow the photoreactive probes to direct the selective irradiation to the selected region of interest. In some embodiments, the activated photoreactive site activates the main subject probe to have a free radical and form the covalent bond with an amino acid or biomolecule of the biological sample in the selected region of interest. In some embodiments, the amino acid may be alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0228] The method may include extracting a plurality of proteins bound to the subject probe from the light-labeled sample group and the unlabeled sample group by affinity precipitation between the proteins bound to the main subject probe and a plurality of affinity beads (e.g., streptavidin magnetic beads). The method may include performing mass spectrometry on the extracted proteins. The method may include calculating a relative quantitative value of each protein in the identified protein list between the light-labeled sample group and the unlabeled sample group according to an intensity value of a peptide fragment of the each protein. The method may include determining a threshold value of the relative quantitative value between the light-labeled sample group and the unlabeled sample group. The method may include predicting at least one biomarker that corresponds to the relative quantitative value of the each protein exceeding the threshold value once the threshold value is determined.

[0229] Also disclosed herein is a method implemented in mass spectrometry to process photolabeled samples to identify a biomarker list. The method may include delivering a photoreactive kit as described herein to a biological sample. In some embodiments, the bait molecule is non-covalently conjugated to a first molecule in the biological sample. The method may include selectively illuminating a selected region of interest of the biological sample to allow the main subject probe to label the proteins of the biological sample in the selected region of interest. In some embodiments, selectively illuminating the selected region of interest further includes activating the photoreactive site in the selected region to allow the activated photoreactive site to allow the main subject probe to form the covalent bond with the biological sample in the selected region of interest. The method may include removing unconjugated photoreactive probe from the biological sample to allow the photoreactive probe to guide the selective illumination to the selected region of interest. In some embodiments, the activated photoreactive site can activate the main subject probe to have a free radical and form the covalent bond with an amino acid or biomolecule of the biological sample in the selected region of interest. In some embodiments, the amino acid can be alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0230] The method may include extracting a plurality of the probe-labeled proteins from the biological sample by affinity precipitation between proteins bound to the main subject probe and a plurality of affinity beads (e.g., streptavidin magnetic beads). The method may include performing mass spectrometry on the extracted proteins. The method may include identifying the extracted proteins of the biological sample. The method may include calculating an intensity value of a peptide fragment of each protein in a list of identified proteins of the biological sample. The method may include ranking the list of identified proteins according to the intensity value of each protein.

[0231] The method can include identifying and / or purifying one or more biomarkers (eg, proximal or adjacent molecules).

[0232] To predict a biomarker or identify a list of biomarkers, the method may further comprise the step of delivering a connector to the biological sample and conjugating the connector to a protein or biomolecule bound to the primary subject probe due to the affinity between the connector and the primary subject probe. In some embodiments, the connector may be a fluorescent connector to identify the location of the biological sample covalently bound to the primary subject probe. The method may further comprise the step of delivering a tag-peroxidase to the biological sample and conjugating the tag-peroxidase to the connector so that the tag-peroxidase can catalyze an additional subject probe (e.g., a tyramide probe) to form a covalent bond between the additional subject probe and the biological sample. In some embodiments, the tag-peroxidase activates the additional subject probe to have a free radical and form a covalent bond between the additional subject probe and a tyrosine of the biological sample.

[0233] Figure 22 shows the workflow for the use of the photoreactive kit described herein and subsequent mass spectrometry. Workflow of the photoreactive kit combined with a microscopic photolabeling system and subsequent mass spectrometry. Photolabeled nucleoli were collected for LC-MS / MS analysis (bottom left). True positive protein distribution of the nucleolar proteome. Proteins were ranked in order of ratio (2-photon labeling / control) and 97 of the top 100 proteins were annotated as nucleolar proteins (bottom center). Gene ontology analysis of the nucleolar proteome (bottom right). Protocol: See below [LC-MS / MS analysis] and [Protein identification and label-free quantification]. EXAMPLES

[0234] (Experimental and Methods)

[0235] (Ruthenium-based antibody conjugation) Ru(bpy) NHS ester (bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) [CAS No.: 136724-73-7] was conjugated to donkey anti-rabbit / donkey anti-mouse IgG antibody by amide coupling reaction as shown in FIG. 7. 300 μg of antibody was diluted with 0.5 mg / mL antibody and 0.35 mM Ru(bpy) in 100 mM borate buffer (pH 8.0). 3 In the final condition of NHS ester, Ru(bpy) 3 The reaction was allowed to proceed for 2 hours at room temperature in the dark. Glycine was added to 100 mM to inactivate the reaction, and then antibody-Ru(bpy) was added. 3 The conjugate was purified by offline size exclusion column (PD midiTrap G-25, Cytiva) using gravity flow. Purified antibody-Ru(bpy) 3 The concentration of the conjugate was measured by the Pierce™ BCA Protein Assay (Thermo Fisher Scientific) using unconjugated antibody as a standard.3 To confirm that the molecule was conjugated onto the antibody, the purified product was further detected at 455 nm and 280 nm wavelengths by a NanoDrop spectrophotometer (Thermo Fisher Scientific).

[0236] (Cell preparation) Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS at 37°C in a 5% CO2 humidified environment. 2 × 105 cells were seeded into glass-bottom chambers and incubated for approximately 16 hours until 80-90% confluency. Cells were then washed with PBS and fixed with 2.4% paraformaldehyde (PFA) or methanol. Fixed cells were incubated with PBS / 0.5% Triton X-100 to permeabilize the cell membrane and blocked with 3% BSA in PBS / 0.1% Triton X-100 for 1 hour, followed by 0.002% streptavidin blocking for 30 minutes and 40 μM biotin blocking for 15 minutes.

[0237] (Ruthenium-based antibody hybridization) Cells were incubated with primary antibodies in blocking buffer [PBS / 0.1% Triton X-100 containing 3% BSA] for 2 h at room temperature using the following antibodies: rabbit anti-NCL, mouse anti-G3BP1, mouse anti-NPC. After washing with PBS / 0.1% Triton X-100, 10 μg / mL of antibody-Ru(bpy) was added. 3 The conjugates were hybridized to the primary antibodies overnight at 4° C. Subsequently, the cells were stained with a fluorescent marker (goat anti-rabbit AlexaFluor 647 or goat anti-mouse AlexaFluor 647) for 1 hour at room temperature.

[0238] Two-photon labeling of intracellular samples for quantitative LC-MS / MS analysis The cells were incubated with two-photon (2P) labeling reagent containing 5-7 mM desthiobiotin-phenol and 0.005% methyl viologen. A two-photon laser coupled to a microscope system was used with a laser power of 100-200 mW for spatially resolved photolabeling, and the cells were exposed to laser exposure times of 100-1000 microseconds. To quench the photochemical reaction, the labeled cells were washed with a buffer containing 10 mM sodium ascorbate, 5 mM Trolox, and 0.02% sodium azide, and the cells were finally washed three times with PBST.

[0239] (Verification of the performance of two-photo labeling by fluorescence microscopy) Labeled cells / signals were probed with neutrAvidin-DyLight 488 for 1 h using blocking buffer containing 3% BSA / PBS / 0.1% Triton X-100 and antibody-Ru(bpy) 3 The conjugates were hybridized with a fluorescent marker (goat anti-donkey TexasRed) or anti-primary antibodies (goat anti-rabbit-568 or goat anti-mouse-568) at room temperature for 1 h. Subsequently, cells were stained with a nuclear marker (Hoechst 33258) for 30 min at room temperature. A Zeiss LSM 880 confocal microscope was applied to verify that the labeling signals were within the boundaries of the organelles in xy and xz sections.

[0240] FIG. 8 shows the experimental results of antibody-based ruthenium (Ab-Ru) targeted photolabeling (confocal imaging of nucleolus markers (magenta), antibody-based ruthenium (red), 2P labeling signal: desthiobiotin (green); scale bar: 20 μm). FIG. 8 shows the experimental results of antibody-based ruthenium (Ab-Ru) targeted photolabeling (confocal imaging of nucleolus markers (magenta), antibody-based ruthenium (red), 2P labeling signal: desthiobiotin (green); scale bar: 20 μm).

[0241] (Protein extraction and on-bead digestion) The labeled cells were harvested by scraping with a buffer containing 10 mM Tris (pH 8.0), 1% Triton X-100, 1x protease inhibitor cocktail, 10 mM sodium ascorbate, 5 mM Trolox, and 1 mM sodium azide. The harvested cells were sonicated for 2 min at 60% power with a Q125 sonicator (Qsonica) at 1 sec on / 2 sec off intervals, and the scraping buffer was then evaporated for 2 h by a SpeedVac system. 160 μL of lysis buffer containing 4% sodium dodecyl sulfate (SDS), 1% Triton X-100, 100 mM Tris (pH 8.0), and 20 mM dithiothreitol (DTT) was added to the harvested cells, and the mixture was vortexed for 5 cycles at 1 min on / 2 min off intervals. To activate crosslinked amide groups resulting from PFA fixation, lysed cells were further heated at 99°C for 45 min, followed by another 5 cycles of vortexing at 1 min on / 2 min off intervals. Lysates were centrifuged at 16,000g for 20 min at 20°C, and the supernatants were collected. Protein concentrations were measured using a Pierce™ 660 nm protein assay (Thermo Fisher Scientific), and immunoprecipitations were performed on 240 μg protein. Streptavidin magnetic beads were washed three times with dilution buffer [0.5% Triton X-100 / PBS], protein lysates were diluted 10-fold to reduce SDS concentration to less than 0.4%, and the diluted lysates were added to the washed beads and incubated for 16 h at 2-8°C with rotation. The biotin-protein bound beads were then washed with the following washing buffers: Buffer A [2% SDS, 50 mM Tris (pH 8.0)]; Buffer B [0.5 M NaCl, 0.1% deoxycholate, 0.1% SDS, 1% Triton X-100, 50 mM HEPES]; Buffer C [0.5% deoxycholate, 0.5% Triton X-100, 10 mM Tris (pH 8.0), 250 mM LiCl] to maximally reduce non-specific binding.For on-bead digestion, the beads were further washed three times with 100 μL of 50 mM triethylammonium bicarbonate buffer, and then the biotin-protein-bound beads were mixed with 0.2 μg of trypsin / Lys-C (V5071, Promega) in a final volume of 20 μL for 100 min at 37° C. for the first digestion. The supernatant was then collected and digested overnight without adding any further enzymes. Finally, the digest was acidified by adding 2 μL of 10% formic acid and desalted by C18 Ziptip. The desalted peptides were dried by Speedvac and stored at −20° C. prior to LC-MS / MS analysis.

[0242] (LC-MS / MS analysis) Detection of immunoprecipitation products by data-dependent acquisition mass spectrometry. LC-MS / MS analysis was performed using an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Desalted peptides were resuspended in 0.1% formic acid in water and loaded onto a PepMap™ 100 C18 HPLC column (2 μm, 100 Å, 75 μm×25 cm; Thermo Fisher Scientific), and peptides were eluted during a 160 min gradient for nucleus-irradiated samples and a 120 min gradient for nucleolus-, SG-irradiated samples. Complete MS spectra ranging from m / z 375 to 1500 were acquired in the Orbitrap at a resolution of 120,000, an AGC target of 4×105, and a maximum injection time of 50 ms. Fragment ion spectra were recorded at a resolution of 30,000 in top speed mode in an Orbitrap using data-dependent methods. Monoisotopic precursor ions were selected by the quadrupole with isolation windows of 1.2, 0.7, and 0.4 Th for ions with charge states of 2+, 3+, and 4-7, respectively. An AGC target of 5 × 104, a maximum injection time of 54 ms, higher energy collisional dissociation (HCD) fragmentation at 30% collision energy, and a maximum cycle time of 3 seconds were all applied. Dynamic exclusion was set to 60 seconds with an exclusion window of 10 ppm. Precursor ions with unassigned, 1+, or charge states greater than 8+ were excluded from fragmentation selection.

[0243] (Protein Identification and Label-Free Quantification) Raw data from the same batch of two-photon irradiation were collectively processed against the UniProtKB / Swiss-Prot human protein database (version 2020.02, 20,365 entries) by the Sequest HT algorithm using Proteome Discoverer (Thermo Fisher Scientific) for feature extraction, peptide identification, and protein prediction. Database searches were performed as follows: tryptic peptides with up to three missed cleavages; mass tolerance of 10 ppm for peptide ions and 0.05 Da for fragment ions; static carbamidomethylation (+57.0215 Da) for Cys residues; dynamic deamidation (+0.9840 Da) for Asp and Gln, oxidation (15.9949 Da) for Met residues, acetylation (+42.0106 Da) for protein N-termini, and desthiobiotin phenol modification (+331.1896) for Tyr residues. The minimum peptide length was set to 6 residues. The false discovery rate (FDR) for both peptides and proteins was set at 1%.

[0244] For label-free quantification, the time window of chromatographic peak alignment was set to 20 min. Peptide-level data were then normalized to the total peptide intensity, and the quantitative value for a given protein was derived from the sum of the normalized intensities of the top three most intense unique peptides belonging to that protein.

[0245] Figure 9 shows the results of the nucleolus labeling experiment. Figure 9 shows the total proteins identified in the nucleolus labeling experiment. 91% of the top 100 proteins are nucleolar proteins, suggesting that the labeling of the small ROI (nucleolus) with Ab-Ru and the subject probe (biotin-phenol) is accurate, and the extraction step and identification of the tag (biotin) labeled proteins results in good labeling.

[0246] Proteins are ranked by their fold change ratio (log labeled cells / unlabeled cells). Figure 10 shows that proteome composition reveals the accuracy and performance of novel protein discovery for stress granules (SGs). 62% of the enriched proteins were SG proteins, and at least 6 proteins among the enriched proteins not included in the current SG database were verified to colocalize with the SGs by immunocytochemistry, suggesting that antibody-based ruthenium-targeted photolabeling can discover novel proteins in the labeled area.

[0247] Figure 11A-C show three commercially available photoactivatable probes: 2+ Structural description of Figure 11B: riboflavin, and Figure 11C: rose bengal (upper panel). Direct scanning photolabeling of U-2 OS cells with biotinyl tyramide was performed over the entire FOV (wavelength (λ) = 780 nm, power = 100 mW). Figure 11D shows that the highest fluorescence signal was observed for Ru(bpy)3. 2+The experimental protocol in Figure 11 shows that U-2 OS cells were detected using 500 μM desthiobiotin-phenol and free photoreactive moieties in the range of 100-1000 μM: Ru(bpy)32+, riboflavin, and rose bengal at room temperature for 10 min. A two-photon microscope was used for spatially resolved photochemical labeling over the entire field of view (FOV). A two-photon microscope was used with a laser power of 10-200 mW for spatially resolved photolabeling, and cells were exposed to a laser exposure time of 100-200 microseconds. To quench the photochemical reaction, the labeled cells were washed with a buffer containing 10 mM sodium ascorbate, 5 mM trolox, and 0.02% sodium azide, and finally, the cells were washed three times with PBST. To quench the photochemical reaction, the labeled cells were washed with a buffer containing 10 mM sodium ascorbate, 5 mM Trolox, and 0.02% sodium azide, and finally the cells were washed three times with PBST. The labeled cells / signals were probed with neutrAvidin-DyLight 488 for 1 h using a blocking buffer containing 3% BSA / PBS / 0.1% Triton X-100. Subsequently, the cells were stained with a nuclear marker (Hoechst 33258) for 30 min at room temperature.

[0248] Figure 12 shows successful antibody-photoreactive moiety conjugation. 1 mg of antibody was reacted with Ru(bpy)-NHS at a 100-fold molar ratio for 2 or 24 hours, and the result was detected by SEC-HPLC to determine the Ru(bpy) moiety on the antibody. 3 2+ The conjugation of the molecule was verified. This SEC-HPLC confirmed that Ru(bpy) 3 2+ The antibody conjugated with Ru(bpy) (160 kDa) was shown to have characteristic peaks at both 280 nm and 455 nm at 10.4 min, indicating that Ru(bpy) 3 2+ and indicates successful conjugation of the antibody.

[0249] Figures 13A-B show the results of successful antibody-photoreactive moiety conjugation. 3 2+ Antibody conjugation followed by desalting of unconjugated Ru(bpy) using a 40 kDa zeba spin desalting column 3 2+ Molecular removal. Primary Ru(bpy) 3 2+ The conjugated antibody peak was observed at both 280 nm and 455 nm absorbance. Free Ru(bpy) was observed at 16 min at 455 nm absorbance. 3 2+ No chromatographic analysis was performed. See protocols: 1) "Ruthenium-based antibody conjugation" (above) and 2): "SEC-HPLC protocol (verification of Ab-Ru conjugates)" in Figure 12 and Figure 13A-B. The purity of unconjugated and Ru(bpy)-conjugated antibodies (Ab-Ru) under native conditions was determined by SEC-HPLC. The mobile phase was sodium phosphate buffer, pH 7.4. Test samples were diluted to 1 mg / mL with the mobile phase, and 100 μL of the resultant was loaded onto a TSK gel. The samples were injected into a HPLC system equipped with a G3000SWXL column, 7.8 mm (ID) x 30 cm. An isocratic flow of the mobile phase at 1.0 mL / min was applied for the protein separation. The monitored eluent characteristics were unconjugated antibody (160 kDa), Ab-Ru (160 kDa), aggregates (>160 kDa), and free Ru(bpy) molecules (628 Da). Unconjugated antibody and aggregates were detected by 280 nm, and Ab-Ru and free Ru(bpy) molecules by 455 nm. All measurements were performed by a photodiode array detector.

[0250] 14A-C show the results of specific photolabeling of induced cells using conjugation of an antibody with a ruthenium-based photoreactive moiety. FIG. 14 shows the results of an antibody-based ruthenium (Ab-Ru) targeted photolabeling experiment (confocal imaging of stress granule marker (green), photolabeled signal: desthiobiotin (red); scale bar: 10 μm). FIG. 14A shows illumination at 470 nm. FIG. 14B shows the control (no light). FIG. 14C shows the Z-axis (side view) of the cells shown in FIG. 14A. Ru(bpy) conjugated to antibody 3 2+ Specificity and high resolution of radiation effects on (Ab-Ru). U-2 OS cells were induced with arsenite and hybridized with rabbit anti-G3BP1 (stress granule marker: green) to indicate regions of interest for labeling. A donkey anti-mouse secondary antibody was added to the Ru(bpy) 3 2+ (Ab-Ru photoactive probe) and irradiated with 470 nm LED wavelength together with desthiobiotin-phenol and methyl viologen. A clear stress granule pattern was observed after Dy-550 neutravidin staining. The no light control group did not show stress granule pattern after Dy-550 neutravidin staining. Z-axis images also showed the specificity and high resolution of photolabeling action with Ab-Ru photoactive probe in z-plane. Protocols of Figures 14, 17, 18, 20, 22, 23, and 24: "Hybridization of Ruthenium-Based Antibodies" (see above), followed by "Labeling and Analysis": Cells were incubated with photolabeling reagent containing 5 mM desthiobiotin-phenol and 0.005% methyl viologen. A 470 nm LED laser coupled to a microscope system was used with a laser power of 10-200 mW for spatially resolved photolabeling, and cells were exposed to laser exposure times of 37-1000 μs. To quench the photochemical reaction, the labeled cells were washed with a buffer containing 10 mM sodium ascorbate, 5 mM Trolox, and 0.02% sodium azide, and finally, the cells were washed three times with PBST.

[0251] Labeled cells / signals were probed with neutrAvidin-DyLight550 for 1 h using blocking buffer containing 3% BSA / PBS / 0.1% Triton X-100, and mouse anti-G3BP1 antibody was hybridized with a fluorescent marker (goat anti-mouse 488) for 1 h at room temperature. A Zeiss LSM 880 confocal microscope was applied to verify that the labeling signals were within the organelle boundaries in xy and xz sections.

[0252] FIG. 14 shows experimental results of antibody-based ruthenium (Ab-Ru) targeted photolabeling (confocal imaging of stress granule marker (green), photolabeled signal: desthiobiotin (red); scale bar: 10 μm).

[0253] FIG. 15 shows selective photolabeling of mouse brain tissue samples using antibodies and a probe with a ruthenium-based photoreactive moiety. The control (no light) shows no labeling. 20 μM frozen mouse brain tissue sections were stained with rabbit anti-nucleolin (a nuclear marker) and then hybridized with an anti-rabbit Ab-Ru photoactive probe (a donkey anti-rabbit secondary antibody was hybridized with Ru(bpy)3 2+ The tissue was conjugated with Dy-550 neutravidin and irradiated with 457 nm LED wavelength at powers of 10, 20, 30, 40, 50, and 60 mW together with desthiobiotin-phenol and methyl viologen. After Dy-550 neutravidin staining, a clear nuclear pattern was observed indicating photolabeled signal (desthiobiotin) on the tissue in selected areas. No-light control areas showed no labeling signal. Protocol for tissue preparation, primary antibody, and Ab-Ru staining in Figures 15, 16, and 19: "Tissue Preparation, Primary Antibody, and Ab-Ru Staining". PFA-fixed mouse brain tissue was embedded in optimal cutting temperature (OCT) compound and cryosectioned on cover slides. Mouse brain sections of 10 to 30 μm were incubated with rabbit anti-NCL antibody in blocking buffer [PBS / 0.1% Triton X-100 with 3% BSA] at 4°C overnight. After washing with PBS / 0.1% Triton X-100, 10 μg / mL of antibody-Ru(bpy) 3The conjugate was hybridized to the primary antibody overnight at 4°C. Following this, the tissue sections were stained with the fluorescent marker goat anti-rabbit AlexaFluor 647 for 1 hour at room temperature. Additional protocols for Figures 15 and 16: "457 LED light source": 10-30 μm tissue sections were incubated with a labeling reagent containing 5 mM desthiobiotin-phenol and 0.005% methyl viologen. A 457 nm LED laser coupled to a microscope system was used with a laser power of 10-200 mW for spatially resolved photolabeling, and the tissue sections were exposed to laser exposure times of 37-1000 microseconds. To quench the photochemical reaction, the labeled tissue sections were washed with a buffer containing 10 mM sodium ascorbate, 5 mM trolox, and 0.02% sodium azide, and then finally washed three times with PBST. Additional protocols for Figures 15, 16, 19: "Verification of photolabeling performance": Labeled tissues / signals were probed with NeutrAvidin-DyLight 550 for 1 hour using blocking buffer containing 3% BSA / PBS / 0.1% Triton X-100. Subsequently, tissue sections were stained with nuclear marker (DAPI) for 30 minutes at room temperature. Fluorescence or Zeiss LSM 880 confocal microscope was applied to verify that the labeling signal was within the nuclear borders in xy and xz sections.

[0254] FIG. 16 shows the results of an experiment demonstrating the photolabeling of mouse brain tissue samples using antibodies and a ruthenium-based photoreactive site probe and neutravidin stain. 20 μM frozen mouse brain tissue sections were stained with rabbit anti-nucleolin (a nuclear marker) and then hybridized with the anti-rabbit Ab-Ru photoactive probe (a donkey anti-rabbit secondary antibody was used with Ru(bpy)3 2+ The antibody was conjugated with Ru(bpy)3 in the z-plane and irradiated with a 457 nm LED wavelength together with desthiobiotin-phenol and methyl viologen. A clear nuclear pattern was observed after Dy-550 neutravidin staining. The Z-axis image shows the antibody-Ru(bpy)3 in the z-plane. 2+ The specificity and high resolution of photolabeling with photoactive probes were demonstrated.

[0255] FIG. 17 shows the results of an experiment demonstrating detection of nucleolar regions using a probe with a ruthenium-based photoreactive site and a secondary antibody bait. Photolabeled signals were labeled using two-photon technology. Photolabeled (ON) and unlabeled (OFF) regions of interest were located within the nucleolus (rabbit anti-NCL antibody: red) using the Ab-Ru photoactive probe (donkey anti-rabbit secondary antibody was probed with Ru(bpy)3). 2+ (conjugated with ). Photolabeled (PL) signals were stained with anti-desthiobiotin (Dy-488 NeutrAvidin). PL signals were labeled using two-photon at wavelength=780 nm. Protocols in Figures 17, 18, 20, 22, 23, and 24: 1) [Two-photon labeling of intracellular cells for quantitative LC-MS / MS analysis] (see above), and 2) [Verification of the performance of two-photon labeling by fluorescence microscopy] (see above).

[0256] Figures 18A-C show experimental results showing detection of photolabeled subcellular compartments using a probe with a ruthenium-based photoreactive site and an Alexa fluor 568 secondary antibody bait that binds to a primary antibody. Figure 18A shows nucleolus detection using a rabbit anti-nucleolin antibody. Figure 18B shows nuclear pore complex detection using a mouse anti-NPC antibody. Figure 18C shows stress granule detection using a mouse anti-Ras GTPase-activating protein-binding protein 1 antibody.

[0257] Figure 19 shows the results of an experiment demonstrating specific two-photon labeling on fixed mouse brain tissue. Protocol "2p labeling" in Figure 19: 10-30 μm mouse brain tissue sections were incubated with two-photon (2P) labeling reagent containing 5-7 mM desthiobiotin-phenol and 0.005% methyl viologen. A two-photon laser coupled to a microscope system was used with a laser power of 10-200 mW for spatially resolved photolabeling, and the tissue sections were exposed to laser exposure times of 10-1000 microseconds. To quench the photochemical reaction, the labeled tissue sections were washed with a buffer containing 10 mM sodium ascorbate, 5 mM trolox, and 0.02% sodium azide, and finally, the tissue sections were washed three times with PBST.

[0258] Figure 20 shows experimental results showing photolabeling stress granules with horseradish peroxidase-activated desthiobiotin covalently binding to tyrosine residues on and proximal to the enzyme site. Protocol for Figures 20 and 21: "Photolabeling followed by proximity labeling": Labeled cells / tissues / signals were probed with NeutrAvidin-DyLight488 or NeutrAvidin-DyLight550 for 1-2 hours at room temperature using blocking buffer containing 3% BSA / PBS / 0.1% Triton X-100, followed by anti-NeutrAvidin-HRP (biotin-HRP) hybridization for 1 hour at room temperature. 100 μm desthiobiotin was added and incubated in the cell / tissue sample for 30 minutes at room temperature. Hydrogen peroxide was added to catalyze the covalent binding of HRP-activated desthiobiotin to tyrosine residues on and proximal to the enzyme site. Proximity-labeled signals were probed with NeutrAvidin-DyLight 550 for 1 h and blocking buffer containing 3% BSA / PBS / 0.1% Triton X-100 for 1-2 h at room temperature. Fluorescence or Zeiss LSM 880 confocal microscopy was applied to verify that the labeled signals were within the organelle boundaries in xy and xz sections.

[0259] FIG. 21 shows the proximity labeling of photolabeled mouse liver tissue samples with horseradish peroxidase-activated desthiobiotin, which is covalently attached to tyrosine residues on and proximal to the enzyme site.

[0260] FIG. 22 shows an example of a workflow using a photoactivatable kit in combination with a microscopic photolabeling system as described herein and subsequent mass spectrometry.

[0261] FIG. 23 shows experimental results showing confocal micrographs demonstrating highly accurate and precise photolabeling (PL) stress granules with antibody-ruthenium photoactive probes using a two-photon labeling system at wavelength=780 nm.

[0262] Figures 24A-C show two-photon labeling with antibody-ruthenium photoactive probes. Figure 24A shows a Venn diagram of three biological replicates of two-photon labeled Ab-Ru photoactive probes. Figure 24B shows a LOG 2 Volcano plot of relative protein levels (PL / CTL ratio) in photolabeled samples versus control samples at scale. Over-represented (enriched) proteins are shown in the top right bounded by arrows a and b. Figure 24C shows that 74% of the true positive rate (annotated as stress granules (sg)) is identified in the top 50 proteins ranked by PL / CTL ratio. Protocol: See above [LC-MS / MS analysis] and [Protein identification and label-free quantification].

[0263] Figure 25A-B show that 37 proteins out of the top 50 proteins ranked by PL / CTL ratio were annotated as stress granule proteins (top). Validation of potential stress granule proteins by immunofluorescence detection. Confocal micrographs depicting stress granule formation of potential stress granule proteins in U-2 OS cells with and without arsenite stress. Potential stress granule proteins (green) are highly colocalized with the well-known G3BP1 SG marker (red). Proteome composition reveals the accuracy and power of using this method to discover novel stress granule biomarkers (bottom). Lens: 63x oil.

[0264] When a feature or element is referred to herein as being "on" another feature or element, it may be directly on the other feature or element, or intervening features and / or elements may be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that when a feature or element is referred to as being "connected," "attached," or "coupled" to another feature or element, it may be directly connected, attached, or coupled to the other feature or element, or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected," "directly attached," or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or illustrated with respect to one embodiment, the features and elements so described or illustrated may be applicable to other embodiments. It will also be recognized by those skilled in the art that a reference to a structure or feature being located "adjacent to" another feature may have portions that overlap or underlie the adjacent feature.

[0265] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, it will be understood that the terms "comprises" and / or "comprising", when used herein, specify the presence of the stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0266] Spatially relative terms such as "under," "below," "lower," "over," and "upper" may be used herein to facilitate the description of describing the relationship of an element or feature to another element(s) or feature(s) as depicted in the figures. It will be understood that the spatially relative terms are intended to encompass various orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures were turned upside down, an element described as "under" or "beneath" another element or feature would be positioned "over" that other element or feature. Thus, the exemplary term "under" can encompass both the "on" and "under" orientations. Additionally, the device may be otherwise oriented (rotated 90 degrees or to another orientation) and the spatially relative descriptors used herein interpreted accordingly. Similarly, terms such as "upward," "downward," "vertical," and "horizontal" are used herein for descriptive purposes only, unless expressly indicated otherwise.

[0267] Although the terms "first" and "second" may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context indicates otherwise. These terms may be used to distinguish one feature / element from another. Thus, a first feature / element described below may be termed a second feature / element, and similarly, a second feature / element described below may be termed a first feature / element, without departing from the teachings of the present invention.

[0268] Throughout this specification and the claims that follow, unless the context requires otherwise, the term "comprises" and variations such as "comprises" and "comprising" refer to various elements that may be employed jointly together in the methods and articles (e.g., compositions and methods, including apparatus and devices). For example, the term "comprising" will be understood to imply the inclusion of all described elements or steps, but without the exclusion of any other elements or steps.

[0269] In general, any apparatus and methods described herein should be understood to be inclusive, or all or a subset of the elements and / or steps may be exclusive, and may be expressed as "consisting of" or "consisting essentially of" the various elements, steps, sub-elements, or sub-steps.

[0270] As used herein in the specification and claims, including as used in the examples, and unless expressly stated otherwise, all numbers may be understood as if preceded by the word "about" or "approximately", even if the term is not explicitly stated. When describing a size and / or location, the phrase "about" or "approximately" may be used to indicate that the described value and / or location is within a reasonable range of expected values ​​and / or locations. For example, a numerical value may have a value that is + / -0.1% of the described value (or range of values), + / -1% of the described value (or range of values), + / -2% of the described value (or range of values), + / -5% of the described value (or range of values), + / -10% of the described value (or range of values), etc. All numerical values ​​given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range described herein is intended to include all subranges subsumed therein. It is also understood that when a value is disclosed, "less than or equal to" the value, "more than or equal to" the value, and possible ranges between the values ​​are also disclosed, as would be well understood by one of ordinary skill in the art. For example, when a value of "X" is disclosed, "less than or equal to" and "more than or equal to X" (e.g., where X is a numerical value) are also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that values ​​greater than, greater than, less than, less than, and equal to 10 and 15 are considered to be disclosed, as well as values ​​between 10 and 15. It is also understood that each units digit between two specific units digits is also disclosed. For example, when 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0271] Any of the methods (including user interfaces) described herein may be implemented as software, hardware, or firmware, which may be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (e.g., a computer, tablet, smartphone, etc.) that, when executed by the processor, cause the processor to control and perform any of the following steps including, but not limited to: displaying, communicating with a user, analyzing, changing parameters (including timing, frequency, intensity, etc.), making a decision, or alerting, etc.

Claims

1. Formula (I): 【Chemistry 1】 (In the formula, The C moiety is a single chemical bond or a linker; The B moiety comprises 1 to 50 photoreactive sites and is attached to the C moiety, where each of the 1 to 50 photoreactive sites has the formula (II): 【Chemistry 2】 (In formula (II): L 1 , L 2 , L 3 , and L 4 are each independently a ligand; and X 1 and X 2 are each independently a ligand having a reactive site, where the reactive site is configured for binding to the C moiety. Derived from a ruthenium-based compound represented by: The G portion comprises a bait molecule attached to the C portion, where the bait molecule is an antibody and is configured to conjugate with a first molecule in the sample. A photoreactive probe represented by: and A primary subject probe comprising a detectable tag moiety attached to a photoexcitable subject moiety. A photoreactive kit comprising: The photoreactive kit is configured such that the photoreactive probe is photoactivated at either a wavelength in the range of 700 nm to 1100 nm using a two-photon light source or a wavelength in the range of 200 nm to 1100 nm using a single light source, and when the primary subject probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe, the photoexcited primary subject probe forms a covalent bond with a target molecule in the sample.

2. X 1 and X 2 is independently selected from the group consisting of 3-ethynylpyridine, 3-(bromomethyl)pyridine, maleimide, 4'-methyl-4-carboxybipyridine-N-succinimidyl ester, nicotinaldehyde, l-(4-(pyridin-3-yl)-lH-1,2,3-triazol-l-yl)ethanone, 4-pentynenitrile, and 4-aminobutyne. The photoreactive kit of claim 1,

3. L 1 and L 2 are linked to form a first bidentate ligand, and L 3 and L 4 are linked to form a second bidentate ligand, wherein the first bidentate ligand and the second bidentate ligand are independently selected from 2,2'-bipyridyl (bpy), 4,4'-dicyano-5,5'-dimethyl-2,2'-bipyridine (CN-Me-bpy), 4,4'-dimethyl-2,2'-bipyridine (dmb), 4,4'-di-tert-butyl-2,2'-bipyridine (dbpy), 4,4',5,5'-tetramethyl-2,2'-bipyridine (tmb), 2-phenylpyridine (ppy), 6- The photoreactive kit according to claim 1, wherein the photoreactive compound is selected from the group consisting of bromo-2,2'-bipyridine, 6,6'-dibromo-2,2'-bipyridine, 5-bromo-2,2'-bipyridine, 6-amino-2,2'-bipyridine, 6,6'-diamino-2,2'-bipyridine, 2,2'-bipyridine-6-carbonitrile, 2,2'-bipyridine-6,6'-bis(carbonitrile), 2,2'-bipyridine-6-carboxylic acid, 2,2'-bipyridine-6,6'-dicarboxylic acid, and biquinoline.

4. The ruthenium-based compound of formula (II) is 【Chemistry 3】 【change】 2. The photoreactive kit of claim 1, wherein the photoreactive kit is one of the following:

5. The photoreactive moiety is 【Chemistry 4】 or a derivative thereof.

6. The linker is 【Chemistry 5】 The photoreactive kit according to any one of claims 1 to 5, comprising a moiety which is

7. The linker is an amino acid, (PEG) n 7. The photoreactive kit of claim 1, comprising at least one of: an oligonucleotide; or a peptide, wherein n is an integer from 1 to 20.

8. 2. The photoreactive kit of claim 1, wherein the photoreactive probe is photoactivated at a wavelength in the range of 700 nm to 1100 nm using a two-photon light source, and the primary subject probe is configured to form a photoexcited primary subject probe by being acted upon by the photoactivated probe, the photoexcited primary subject probe forming a covalent bond with a target molecule in the sample.

9. The photoreactive kit of claim 1, further comprising: a photoactivation kit configured to: photoactivate the photoreactive probe at a wavelength in the range of 300 nm to 800 nm using a single light source; and when the primary subject probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe, the photoexcited primary subject probe forms a covalent bond with a target molecule in the sample.

10. The photoreactive kit of any one of claims 1 to 6, wherein the detectable tag moiety is at least one of a biotin derivative, a digoxigenin tag, a CLIP tag, a HaloTag, a SNAP tag, an oligonucleotide, a peptide tag, and a click chemistry tag, and the click chemistry tag comprises an alkyne-based or azide-based moiety.

11. said photoexcitable subject moiety being 【Chemistry 6】 The photoreactive kit according to any one of claims 1 to 6, which is at least one of the following:

12. The photoreactive kit according to any one of claims 1 to 6, wherein the main subject probe is desthiobiotin-phenol or biotin-phenol.

13. said photoexcitable subject moiety being 【Chemistry 7】 The photoreactive kit according to any one of claims 1 to 6,

14. the detectable tag moiety is at least one of a biotin derivative, a click chemistry tag, a CLIP tag, a digoxigenin tag, a HaloTag, an oligonucleotide, a peptide tag, a SNAP tag, and the photoreactive site is 【Chemistry 8】 The photoreactive kit of claim 13, comprising a moiety which is

15. The photoreactive kit of any one of claims 1 to 6, further comprising a connector, the connector being capable of conjugating with the detectable tag portion of the main subject probe.

16. 16. The photoreactive kit of claim 15, wherein the connector is a fluorescent connector.

17. 16. The photoreactive kit of claim 15, further comprising a tag-enzyme conjugate capable of conjugating to the connector, and further comprising an enzyme of the tag-enzyme conjugate comprising a peroxidase.

18. a connector capable of conjugating to the detectable tag portion of the main subject probe; a tag-enzyme complex, wherein the tag of the tag-enzyme complex is capable of conjugating to the connector, and further wherein the enzyme of the tag-enzyme complex comprises peroxidase; and The photoreactive kit of any one of claims 1 to 6, further comprising an additional subject probe configured to form an additional subject probe covalent bond with the target molecule through the catalytic activity of the peroxidase of the tag-enzyme complex.

19. 20. The photoreactive kit of claim 18, wherein the additional subject probe is the same as or different from the primary subject probe and comprises an additional subject probe tag portion and an additional subject probe subject site.

20. 20. The photoreactive kit of claim 18, wherein the connector is a fluorescent connector.

21. The photoreactive kit according to any one of claims 1 to 6, wherein the concentration of the photoreactive probe is in the range of 0.1ug / mL to 100ug / mL, and the concentration of the main subject probe is in the range of 1uM to 20mM.

22. Formula (I): 【Chemistry 9】 (In the formula, The C moiety is a single chemical bond or a linker; the B moiety comprises at least one photoreactive site attached to said C moiety; and The G portion comprises a bait molecule bound to the C portion. A photoreactive probe represented by: A primary subject probe comprising a detectable tag moiety attached to a photoexcitable subject moiety. A photoreactive kit comprising: The bait molecule of the photoreactive probe is configured to conjugate with a first molecule in a sample; and The photoreactive kit is configured such that when the photoreactive probe is photoactivated and the primary subject probe is acted upon by the photoactivated probe to form a photoexcited primary subject probe, the photoexcited primary subject probe forms a covalent bond with a target molecule in the sample.

23. 23. The photoreactive kit of claim 22, wherein the bait molecule comprises at least one of an antibody, avidin, neutravidin, streptavidin, other biotin-binding proteins, CLIP tags, HaloTag, SNAP tags, other self-labeling protein tags, DNA or RNA fluorescent in situ hybridization (FISH) probes, other RNA molecules, other nucleic acid molecules, protein A, protein G, protein L, protein A / G, protein A / G / L, other immunoglobulin-binding peptides, drugs, or other small molecules.

24. 23. The photoreactive kit of claim 22, wherein the photoreactive moiety is at least one of riboflavin, lumiflavin, another flavin derivative, fluorescein or a derivative thereof, methylene blue or a derivative thereof, miniSOG photosensitizer protein, Killer Red photosensitizer protein, another photosensitizer protein, pterin or a derivative thereof, a ruthenium-based photocatalyst, and rose bengal or a derivative thereof.

25. 23. The photoreactive kit of claim 22, wherein the bait molecule is an antibody and a number of the photoreactive sites are attached to the antibody via the C moiety, wherein the number ranges from 1 to 50.

26. 23. The photoreactive kit of claim 22, wherein the photoreactive site is configured to enable the main subject probe to form the covalent bond with the molecule of the sample.

27. The photoreactive moiety has formula (II): 【Chemistry 10】 (In formula (II): L 1 , L 2 , L 3 , and L 4 are each independently a ligand; and X 1 and X 2 are each independently a ligand, 1 and X 2 at least one of the linking regions has a linking region, wherein the at least one linking region is attached to the C portion of the photoreactive probe.

26. The photoreactive kit of claim 25, derived from a ruthenium-based compound represented by:

28. X 1 and X 2 is independently selected from the group consisting of 3-ethynylpyridine, 3-(bromomethyl)pyridine, maleimide, 4'-methyl-4-carboxybipyridine-N-succinimidyl ester, nicotinaldehyde, l-(4-(pyridin-3-yl)-lH-1,2,3-triazol-l-yl)ethanone, 4-pentynenitrile, and 4-aminobutyne. The photoreactive kit of claim 27,

29. L 1 and L 2 are linked to form a first bidentate ligand, and L 3 and L 4 are linked to form a second bidentate ligand, wherein the first bidentate ligand and the second bidentate ligand are independently selected from 2,2'-bipyridyl (bpy), 4,4'-dicyano-5,5'-dimethyl-2,2'-bipyridine (CN-Me-bpy), 4,4'-dimethyl-2,2'-bipyridine (dmb), 4,4'-di-tert-butyl-2,2'-bipyridine (dbpy), 4,4',5,5'-tetramethyl-2,2'-bipyridine (tmb), 2-phenylpyridine (ppy), 6- 28. The photoreactive kit of claim 27, wherein the photoreactive compound is selected from the group consisting of bromo-2,2'-bipyridine, 6,6'-dibromo-2,2'-bipyridine, 5-bromo-2,2'-bipyridine, 6-amino-2,2'-bipyridine, 6,6'-diamino-2,2'-bipyridine, 2,2'-bipyridine-6-carbonitrile, 2,2'-bipyridine-6,6'-bis(carbonitrile), 2,2'-bipyridine-6-carboxylic acid, 2,2'-bipyridine-6,6'-dicarboxylic acid, and biquinoline.

30. The ruthenium-based compound of formula (II) 【Chemistry 11】 【change】 28. The photoreactive kit according to claim 27, which is any one of the above and derivatives thereof.

31. The photoreactive moiety is 【Chemistry 12】 23. The photoreactive kit of claim 22, comprising a moiety that is or a derivative thereof.

32. The linker is 【Chemistry 13】 23. The photoreactive kit of claim 22, comprising a moiety which is

33. The linker is an amino acid, (PEG) n 23. The photoreactive kit of claim 22, comprising at least one of: an oligonucleotide; or a peptide, wherein n is an integer from 1 to 20.

34. 23. The photoreactive kit of claim 22, wherein the photoreactive site is activatable using a light source at a wavelength in the range of 200 nm to 1100 nm to enable the main subject probe to form the covalent bond with the target molecule in the sample.

35. 23. The photoreactive kit of claim 22, wherein the photoreactive moiety is activatable using a two-photon light source at a wavelength in the range of 700 nm to 1100 nm to enable the main subject probe to form the covalent bond with the target molecule in the sample.

36. 28. The photoreactive kit of claim 27, wherein the photoreactive site is activatable using a light source at a wavelength in the range of 300 nm to 800 nm to enable the main subject probe to form the covalent bond with the target molecule in the sample.

37. 28. The photoreactive kit of claim 27, wherein the photoreactive moiety is activatable using a two-photon light source at a wavelength in the range of 700 nm to 1100 nm to enable the main subject probe to form the covalent bond with the target molecule in the sample.

38. 23. The photoreactive kit of claim 22, wherein the detectable tag moiety is at least one of a biotin derivative, a CLIP tag, a digoxigenin tag, a HaloTag, an oligonucleotide, a peptide tag, a SNAP tag, and a click chemistry tag, and the click chemistry tag comprises an alkyne-based or azide-based moiety.

39. The subject site is 【Chemistry 14】 and derivatives thereof.

40. 23. The photoreactive kit of claim 22, wherein the main subject probe is desthiobiotin-phenol or biotin-phenol.

41. said photoexcitable subject moiety being 【Chemistry 15】 28. The photoreactive kit of claim 27,

42. the detectable tag moiety is at least one of a biotin derivative, a click chemistry tag, a CLIP tag, a digoxigenin tag, a HaloTag, an oligonucleotide, a peptide tag, a SNAP tag; and The photoreactive moiety is 【Chemistry 16】 42. The photoreactive kit of claim 41, comprising a moiety which is

43. The photoreactive kit of any one of claims 22 to 42, further comprising a connector, the connector being capable of conjugating with the detectable tag portion of the main subject probe.

44. 44. The photoreactive kit of claim 43, wherein the connector is a fluorescent connector.

45. 44. The photoreactive kit of claim 43, further comprising a tag-enzyme conjugate capable of conjugating to the connector, and further comprising an enzyme of the tag-enzyme conjugate comprising a peroxidase.

46. a connector capable of conjugating to the detectable tag portion of the main subject probe; a tag-enzyme complex, wherein the tag of the tag-enzyme complex is capable of conjugating to the connector, and further wherein the enzyme of the tag-enzyme complex comprises peroxidase; and an additional subject probe configured to form a covalent bond with the target molecule by catalytic activity of the peroxidase of the tag-enzyme complex; The photoreactive kit of any one of claims 37 to 42, further comprising:

47. 47. The photoreactive kit of claim 46, wherein the additional subject probe is different from the primary subject probe and comprises an additional subject probe tag portion and an additional subject probe subject site.

48. 47. The photoreactive kit of claim 46, wherein the connector is a fluorescent connector.

49. 23. The photoreactive kit of claim 22, wherein the concentration of the photoreactive probe is in the range of 0.1 ug / mL to 100 ug / mL, and the concentration of the main subject probe is in the range of 1 uM to 20 mM.

50. Delivering a plurality of photoreactive probes of the photoreactive kit according to any one of claims 22 to 33 and 40 to 42 to a sample; non-covalently conjugating the bait molecules of the photoreactive probes of a first portion to a plurality of first molecules in the sample; delivering to said sample a plurality of main subject probes of the photoreactive kit according to any one of claims 22 to 33 and 40 to 42; selectively irradiating a selected region of interest of the sample with optical radiation, thereby activating the photoreactive sites of a plurality of photoreactive probes to generate a plurality of photoactivated photoreactive probes within the selected region; photoexciting the photoexcitable subject sites of the primary subject probe with the plurality of photoactivated photoreactive probes to generate a plurality of photoexcited primary subject probes having photoexcited subject sites; and forming covalent bonds between the plurality of photoexcited primary subject probes and a plurality of target molecules within the selected region of interest in the sample, thereby binding the plurality of primary subject probes to the plurality of target molecules.

23. A method for photoreactive labeling comprising:

51. 51. The method of claim 50, wherein non-covalently conjugating the bait molecules of the photoreactive probes of a first portion to a plurality of first molecules in the sample leaves the photoreactive probes of a second portion unconjugated, The method further comprising the step of removing the unconjugated photoreactive probe from the sample.

52. delivering a plurality of connectors to the sample; and conjugating said plurality of connectors to said detectable tag portions of said plurality of primary subject probes.

51. The method of claim 50, further comprising:

53. 53. The method of claim 52, wherein the plurality of connectors comprises fluorescent connectors, The method further comprises detecting the locations of the plurality of fluorescent connectors in the sample, thereby identifying the locations of the plurality of main subject probes and the plurality of target molecules covalently bound thereto.

54. the step of photoexciting further comprises photoexciting the primary subject probe to generate a plurality of photoexcited primary subject probes, each of which has a free radical; and 51. The method of claim 50, wherein the step of forming a covalent bond comprises forming a covalent bond between each of the plurality of photoexcited primary subject probes and an amino acid in each of the plurality of target molecules within the selected region of interest in the sample.

55. 51. The method of claim 50, wherein the step of forming a covalent bond comprises forming a covalent bond between each of the plurality of photoexcited primary subject probes and an amino acid in each of the plurality of target molecules within the selected region of interest in the sample.

56. 56. The method of claim 54 or 55, wherein the amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

57. delivering the connector of the photoreactive kit of claim 46 or 47 to the sample, the connector being capable of conjugating to the detectable tag portion of the main subject probe; delivering the tag-peroxidase conjugate of claim 46 or 47 to the sample, wherein the tag of the tag-enzyme conjugate is conjugable to the connector, and further wherein the enzyme of the tag-enzyme conjugate comprises a peroxidase; delivering to the sample an additional subject probe of the photoreactive kit of claim 46 or 47, wherein the additional subject probe is configured to form an additional subject probe covalent bond with the target molecule by catalytic activity of the peroxidase of the tag-enzyme complex; and conjugating the tag-peroxidase complex to the connector, wherein the tag-peroxidase catalyzes the additional subject probe to form a covalent bond between the additional subject probe and the sample.

54. The method of claim 52 or 53, further comprising:

58. 58. The method of claim 57, wherein the tag-peroxidase complex activates the additional subject probe to cause the subject site of the additional subject probe to have a free radical and form a covalent bond between the subject site of the additional subject probe and a tyrosine of the sample.

59. delivering a plurality of photoreactive probes of the photoreactive kit of any one of claims 22 to 33 and 40 to 42 to the sample; non-covalently conjugating the bait molecules of the photoreactive probes of a first portion to a plurality of first molecules in the sample; Delivering a plurality of main subject probes of the photoreactive kit according to any one of claims 22 to 33 and 40 to 42 to the sample; and illuminating the sample with an imaging light source of an image-guided system; imaging the illuminated sample with a camera; acquiring with the camera at least one image of a subcellular morphology of the sample in a first field of view; processing the at least one image and determining a region of interest in the sample based on the processed image; obtaining coordinate information of the region of interest; and selectively irradiating the region of interest with optical radiation in accordance with coordinate information to activate the photoreactive sites, the activated photoreactive sites enabling the primary subject probe to form a covalent bond with the sample in the region of interest. , an analytical method.

60. 60. The method of claim 59, wherein the selectively illuminating step comprises illuminating the area between 10 us / pixel and 200 us / pixel, between 25 us / pixel and 400 us / pixel, between 50 us / pixel and 300 us / pixel, between 75 us / pixel and 200 us / pixel, or between 400 us / pixel and 5000 us / pixel.

61. 60. The method of claim 59, wherein the selectively irradiating step comprises irradiating at a power intensity between 1 μW and 300 mW.

62. 60. The method of claim 59, wherein the selectively irradiating step comprises irradiating an area defined by a point spread function.

63. 60. The method of claim 59, further comprising conjugating a connector to said primary subject probe and detectably proximity labeling a proximal neighbor to said target molecule with a detectable labeling activity.

64. 64. The method of claim 63, wherein said detectably proximity-labeling comprises proximity-labeling an area less than 5 um, less than 2 um, less than 1 um, less than 500 nm, less than 300 nm, less than 200 nm, less than 100 nm, less than 50 nm, or less than 20 nm in diameter.

65. 64. The method of claim 63, wherein the connector comprises a catalytic label.

66. 60. The method of claim 59, wherein the sample comprises at least 1, at least 100, at least 1000, or at least 10,000 live or fixed cells.

67. 60. The method of claim 59, wherein the sample comprises fixed cells, fixed tissues, cell extracts, or tissue extracts.

68. 60. The method of claim 59, further comprising performing mass spectrometry or sequencing on the selectively irradiated sample.

69. 60. The method of claim 59, wherein the activated photoactive site activates the main subject probe to have a free radical and form the covalent bond with an amino acid of the sample within the selected region of interest.

70. 1. A mass spectrometry implemented method for processing a sample and predicting biomarkers, comprising: dividing the samples into a photolabeled sample group and an unlabeled sample group; delivering a photoreactive probe according to any one of claims 22 to 49 and a main subject probe to said photolabeled sample population and said unlabeled sample population; selectively illuminating selected regions of interest of the photolabeled samples and keeping the unlabeled samples in the dark, the illuminating step allowing the main subject probe to form a covalent bond with the samples; extracting proteins bound to the plurality of probes from the photolabeled sample population and the unlabeled sample population by affinity precipitation between the main subject probe and a plurality of affinity beads; performing mass spectrometry on the extracted proteins; Calculating the relative quantitative value of each protein in the identified protein list between the photolabeled sample group and the unlabeled sample group according to the intensity value of the peptide fragment of each protein; determining a threshold value of the relative quantification value between the photolabeled sample group and the unlabeled sample group; and Once the threshold is determined, predicting at least one biomarker that corresponds to the relative quantitative value of the individual protein that exceeds the threshold. The method comprising:

71. 71. The method of claim 70, further comprising the step of non-covalently conjugating said bait molecule to a target molecule in said sample.

72. 71. The method of claim 70, wherein selectively irradiating the selected region of interest further comprises activating the photoreactive sites in the selected region such that the activated photoreactive sites enable the primary subject probe to form the covalent bond with the sample in the selected region of interest.

73. 71. The method of claim 70, further comprising the step of delivering the bait molecule of the photoreactive probe to the sample.

74. 71. The method of claim 70, further comprising removing unconjugated photoreactive probes from the sample to allow the photoreactive probes to direct the selective irradiation to the selected region of interest.

75. The method of claim 70, further comprising the step of delivering the connector of the photoreactive kit of any one of claims 43 and 45 to 47 to the sample and conjugating the connector to the main subject probe due to the affinity between the connector and the main subject probe.

76. 76. The method of claim 75, wherein the connector is a fluorescent connector for identifying the location of the sample covalently bound to the main subject probe.

77. 73. The method of claim 72, wherein the activated photoreactive site activates the main subject probe to have a free radical and form the covalent bond with an amino acid of the sample within the selected region of interest.

78. 78. The method of claim 77, wherein the amino acid is selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

79. 77. The method of claim 75 or 76, further comprising the step of delivering the tag-peroxidase of the photoreactive kit of any one of claims 46 to 47 to the sample and conjugating the tag-peroxidase to the connector such that the tag-peroxidase is capable of catalyzing the additional subject probe to form a covalent bond between the additional subject probe and the sample.

80. 80. The method of claim 79, wherein the tag-peroxidase activates the additional subject probe to cause the subject site of the additional subject probe to have a free radical and form the covalent bond between the subject site of the additional subject probe and a tyrosine of the sample.

81. Obtaining samples; delivering to said sample a photoreactive kit according to any one of claims 22 to 49; selectively illuminating the selected region of interest of the biological sample to enable the main subject probe to label proteins of the sample in the selected region of interest; extracting a plurality of said probe-labeled proteins from said sample by affinity precipitation between said main subject probe and a plurality of affinity beads; performing mass spectrometry on the extracted proteins; and Identifying the extracted proteins of the sample. A mass spectrometry-implemented method for processing the light-labeled sample to identify a list of biomarkers, comprising:

82. 82. The method of claim 81, further comprising calculating intensity values ​​of peptide fragments of each protein in the identified protein list of said sample.

83. 82. The method of claim 81, further comprising the step of ranking said list of identified proteins according to said intensity value of each protein.

84. 82. The method of claim 81, further comprising the step of non-covalently conjugating said bait molecule to a target molecule in said sample.

85. 82. The method of claim 81 , wherein selectively irradiating the selected region of interest further comprises activating the photoreactive sites in the selected region such that the activated photoreactive sites enable the primary subject probe to form the covalent bond with the sample in the selected region of interest.

86. 82. The method of claim 81, further comprising the step of delivering the bait molecule of the photoreactive probe to the sample.

87. 82. The method of claim 81, further comprising removing unconjugated photoreactive probes from the sample to allow the photoreactive probes to direct the selective irradiation to the selected region of interest.

88. The method of claim 81, further comprising the step of delivering the connector of the photoreactive kit of any one of claims 43 and 45 to 47 to the sample and conjugating the connector to the main subject probe due to the affinity between the connector and the main subject probe.

89. 89. The method of claim 88, wherein said connector is a fluorescent connector for identifying the location of said sample covalently bound to said main subject probe.

90. 86. The method of claim 85, wherein the activated photoreactive site activates the main subject probe to have a free radical and form the covalent bond with an amino acid of the sample within the selected region of interest.

91. 91. The method of claim 90, wherein the amino acids are selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

92. 90. The method of claim 88 or 89, further comprising the step of delivering the tag-peroxidase of the photoreactive kit of any one of claims 46 to 47 to the sample and conjugating the tag-peroxidase to the connector such that the tag-peroxidase is capable of catalyzing the additional subject probe to form a covalent bond between the additional subject probe and the sample.

93. 93. The method of claim 92, wherein the tag-peroxidase activates the additional subject probe to cause the subject site of the additional subject probe to have a free radical and form the covalent bond between the subject site of the additional subject probe and a tyrosine of the sample.

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