WO2025006872A2 - Procédé de photomarquage de biomolécules à l'aide de sondes photoréactives - Google Patents

Procédé de photomarquage de biomolécules à l'aide de sondes photoréactives Download PDF

Info

Publication number
WO2025006872A2
WO2025006872A2 PCT/US2024/036001 US2024036001W WO2025006872A2 WO 2025006872 A2 WO2025006872 A2 WO 2025006872A2 US 2024036001 W US2024036001 W US 2024036001W WO 2025006872 A2 WO2025006872 A2 WO 2025006872A2
Authority
WO
WIPO (PCT)
Prior art keywords
photoreactive
sample
probe
moiety
interest
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/036001
Other languages
English (en)
Other versions
WO2025006872A3 (fr
Inventor
Jung-Chi LIAO
Yi-De Chen
Chih-Wei Chang
Hoi Yin CHEUNG
Chia-Wen Chung
Hsiao-Jen CHANG
Yong-Da Sie
Weng Man Chong
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Syncell Taiwan Inc
Original Assignee
Syncell Taiwan Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Syncell Taiwan Inc filed Critical Syncell Taiwan Inc
Priority to EP24832997.1A priority Critical patent/EP4713681A2/fr
Publication of WO2025006872A2 publication Critical patent/WO2025006872A2/fr
Publication of WO2025006872A3 publication Critical patent/WO2025006872A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/32—Post-translational modifications [PTMs] in chemical analysis of biological material biotinylation

Definitions

  • Described herein are methods for proteomics discovery. Specifically described are methods of using photoreactive probes for tagging, analyzing, and identifying biomolecules in biological samples.
  • Spatial proteomics allows protein mapping of a biological sample to reveal geographic organization for underlying protein-protein interactions. Both cell biologists and histologists are largely benefited by recent development in spatial proteomics, enabling, e.g., disease-associated microenvironmental protein mapping, heterogeneous protein distributions on histological samples, or protein identification of specific organelles.
  • Targeted spatial proteomics aims to localize known proteins, whereas hypothesis-free spatial proteomics requires spatial protein identification without prior knowledge of what proteins to look for.
  • transcriptomics where PCR is capable of amplifying signals so that hypothesis-free transcriptomics like RNAseq is possible, no PCR-equivalent technology is yet available for proteomics.
  • the invention provides a photoreactive probe, which is easy to use, hypothesis-free, and has lower noise signal in fluorescence imaging. Therefore, such photoreactive probes are better tool to process a high content of proteins for tagging, analysis, isolation, and identification in a region of interest based on user-defined microscopic image features, widely useful for cell or tissue sample experiments.
  • the disclosure provides a method for photochemical labeling, comprising: delivering a photoreactive probe to a sample, and the photoreactive probe of formula (I): T — L — W (I), wherein the T portion is a biotin or another biotin-based moiety; the L portion is a chemical bond or a linker; and the W portion is a photoreactive moiety, wherein the photoreactive moiety can absorb UV light and become active to crosslink with an amino acid; selectively illuminating the sample with optical radiation to activate the photoreactive moiety of the photoreactive probe in a selected region of interest; and forming a non-specific crosslinking between the photoreactive moiety of the probe and a plurality of amino acids of different proteins of the sample in the selected region of interest.
  • the photoreactive probe delivered to the sample has a concentration of from 0.1 mM to 10 mM.
  • the region of interest comprises a plurality of illumination points and the exposure time of each illumination point in the step of selectively illuminating is in a range of 10 ps - 5000 ps.
  • the step of selectively illuminating comprises illuminating with light having a power intensity of from 1 mW to 600 mW.
  • the sample comprises fixed cells, fixed tissues, cell extracts, or tissue extracts.
  • the photoreactive moiety prior to crosslink with the protein, is converted into a radical moiety upon activation with the optical radiation.
  • the photoreactive moiety is one selecting from the group consisting of benzophenone, phenyl azide, phenyl diazirine, tetrafluorophenyl azide, hydroxyphenyl azide, and trifluoromethylphenyl diazirine.
  • the photoreactive moiety is activatable by single-photon excitation or two-photon excitation.
  • the photoreactive moiety is activatable at a wavelength ranging from 200 nm to 1600 nm with a single-photon excitation.
  • the photoreactive moiety is activatable at a wavelength ranging from 680 nm to 1600 nm with two-photon excitation.
  • the photoreactive probe is represented by formula (1-1) or (I- 2): wherein each of R and R' independently is hydrogen, an alkyl, or a nitrogen protecting group, and n is an integer of 1-20.
  • the sample is affixed to a microscope slide.
  • the non-specific crosslinking is to form a covalent bond between the active photoreactive moiety of the photoreactive probe and an alpha-carbon of an amino acid of the protein
  • the disclosure provides an image-guided photolabeling method comprising: (a) delivering a photoreactive probe to a sample, wherein the photoreactive probe comprises: a detectable tag portion which is biotin or another biotin-based moiety; and a photoreactive moiety which can absorb UV light and become active to crosslink with an amino acid, wherein the detectable tag portion and the photoreactive moiety are coupled by a linker or a chemical bond; (b) imaging the sample in a first field of view and acquiring at least one image in the first field of view of the sample with an imaging light source and a camera; (c) processing the at least one image and determining a region of interest in the first of view; (d) obtaining coordinate information of the region of interest; (e) according to the coordinate information, selectively illuminating the region of interest with optical radiation to activate the photoreactive moiety of the photoreactive probe and thereby form a non-specific crosslinking between the photoreactive moiety of the photore
  • the photoreactive moiety is one selected from the group consisting of benzophenone, phenyl azide, phenyl diazirine, tetrafluorophenyl azide, hydroxyphenyl azide, and trifluoromethylphenyl diazirine.
  • the photoreactive probe is represented by formula (1-1) or (I- 2): wherein each of R and R' independently is hydrogen, an alkyl, or a nitrogen protecting group, and n is an integer of 1-20.
  • the disclosure provides an analytical method for a probe- labeled protein, comprising: obtaining a sample; delivering a photoreactive probe to a sample, wherein the photoreactive probe is represented by formula (I): T — L — W (I), wherein the T portion is biotin or another biotin-based moiety; the L portion is a chemical bond or a linker; and the W portion is a photoreactive moiety which can absorb UV light and become active to crosslink with an amino acid; selectively illuminating a region of interest of the sample with optical radiation to activate the photoreactive moiety of the photoreactive probe in the region of interest and form a non-specific crosslinking between the photoreactive moiety and a plurality of the amino acids of different proteins of the sample in the region of interest; and isolating the plurality of probe-labeled proteins from the sample through an affinity precipitation between the T portion of the photoreactive probe and a plurality of affinity beads.
  • the method further comprises: subjecting the plurality of isolated proteins to mass spectrometry analysis; and identifying the plurality of isolated proteins of the sample.
  • the method further comprises removing the unbound photoreactive probes from the sample.
  • the photoreactive moiety is one selected from the group consisting of benzophenone, phenyl azide, phenyl diazirine, tetrafluorophenyl azide, hydroxyphenyl azide, and trifluoromethylphenyl diazirine.
  • the photoreactive probe is represented by formula (1-1) or (I- 2): wherein each of R and R' independently is hydrogen, an alkyl, or a nitrogen protecting group, and n is an integer of 1-20.
  • FIG. l is a schematic overview of the optoproteomics workflow according to some embodiments of the present disclosure.
  • FIG. 2 shows optical setup and the controlling system of an image-guided two- photon labeling microscope system according to some embodiments of the present disclosure.
  • a modulated femtosecond laser (780nm, 140fs pulses) was reflected off the galvanometer system (Galvo X, Galvo Y) and scanned onto the sample through the objective.
  • the scanning patterns (masks) can be generated in real-time based on a selected mask generator for automatically photolabeling interested targets in each field of view.
  • An all-in-one software with a user interface was developed to perform a fast high-content processing.
  • LED refers to light-emitting diode
  • CAM refers to camera
  • ILS illuminating light source
  • AOM refers to acousto-optic modulator
  • PFS refers to perfect focus system
  • Obj refers to objective
  • PRO refers to processor.
  • FIG. 3 shows a schematic depiction of a system useful for photoselective spatial tagging and proximity labeling of cells on a substrate according to some embodiments of the present disclosure.
  • FIG. 4A shows two chemical structures of one aspect of the photoreactive probe according to some embodiments of the present disclosure.
  • FIG. 4B shows a schematic illustration of another aspect of the photoreactive probe according to some embodiments of the present disclosure.
  • FIG. 4C shows a schematic illustration of illumination (IL) reaction between the photoreactive probe and alpha-carbon (Ca) of amino acid (AA) according to some embodiments of the present disclosure.
  • FIG. 4D schematically illustrates selectively photochemical labeling using the photoreactive probe described herein according to some embodiments of the present disclosure.
  • FIG. 4E shows two probes of biotin-PEG3 -benzophenone (B3-BzP, top panel) and desthiobiotin-PEG3 -benzophenone (DB3-BzP, bottom panel), respectively according to some embodiments of the present disclosure.
  • FIG. 5 shows biotin labeling efficiency significantly increased by labeling power and the concentration of B3-BzP according to some embodiments of the present disclosure.
  • FIG. 6B shows the measurement of photolabeling resolution using 40x magnification/0.95 numerical aperture (NA) objective lens by super-resolution structured illumination microscopy according to some embodiments of the present disclosure.
  • NA numerical aperture
  • FIG. 6C shows confocal images of top- (xy) and side- (z) views of photolabeled subcellular compartments, the ROIs were stained with Alexa fluor 568 secondary antibody, and photolabeled signals were stained with anti-biotin (neutravidin-488 fluorescent: green) according to some embodiments of the present disclosure.
  • NCL refers to nucleolin
  • NPC refers to nuclear pore complex
  • GM130 refers to Golgi matrix protein 130
  • G3BP1 refers to GAP SH3 domain-binding protein 1.
  • Scale bar 10 pm.
  • FIG. 6D shows the top- and side views of each labeled synapse (No: 1 to 4) in spreading assay, indicating a precise photolabeling according to some embodiments of the present disclosure.
  • the side view of photolabeling region was colocalized with CD3, a well- known marker of immune synapse on the bottom of cells. No biotin signal was found in a non-photolab eled cell (No: 5). Scale bar: 10 pm.
  • FIG. 6E shows photolabeled regions of immune synapse of Jurkat T cells and Raji B cells showed in green (neutravidin-488), as a precise and thin labeled layer according to some embodiments of the present disclosure. Scale bar: 10 pm.
  • FIG. 6F shows photolabeling on formalin-fixed and paraffin-embedded (FFPE) mouse brain tissue section according to some embodiments of the present disclosure.
  • FFPE paraffin-embedded
  • FIG. 7A shows overview of optoproteomics method used for proteomic profiling according to some embodiments of the present disclosure.
  • Cells were seeded on a glass chamber and photolabeled by optoproteomics system. Then, the photolabeled (PL) cells are lysed, enriched by streptavidin beads (STB) and digested by trypsin prior to LC-MS/MS measurement.
  • PL refers to photolabeled
  • Ctrl refers to control
  • LP refers to labeled protein
  • STB streptavidin bead
  • LF refers to lab el -free.
  • FIG. 7B shows Dot-blot assay of streptavidin-HRP detection according to some embodiments of the present disclosure. Biotin signals are observed in photolabeled (ON) cells but not in the control cells (OFF). SA-beads refer to streptavidin beads.
  • FIG. 7C shows protein distribution of true positive of three biological replicates of B3-BzP (dark gray) according to some embodiments of the present disclosure. Proteins that are not annotated as nuclear proteins are shown in light grey.
  • FIG. 7D shows the distribution of protein copy number ( ⁇ 10,000 copy number per cell: B3-BzP) according to some embodiments of the present disclosure.
  • FIG. 7E shows CORUM analyses of protein complexes by B3-BzP according to some embodiments of the present disclosure.
  • FIG. 8 shows a schematic route for synthesis of one aspect of the photoreactive probe DB3-BzP according to some embodiments of the present disclosure.
  • FIG. 9 shows results of selectively photochemical labeling from using the probes of DB3-BzP (right panel) and B3-BzP (left panel), respectively according to some embodiments of the present disclosure.
  • ranges set forth herein may be interpreted as being inclusive of their endpoints, and open-ended ranges may be interpreted to include only commercially practical values. Similarly, lists of values may be considered as inclusive of intermediate values unless the context indicates the contrary. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.
  • Microscopy-guided subcellular proteomic discovery in high sensitivity is exceedingly challenging due to the limited sensitivity of mass spectrometry and the lack of amplification tools for proteins.
  • optoproteomics an integrated technology termed optoproteomics, combining microscopy imaging, real-time deep learning-enabled image segmentation, pattern illumination-induced targeted photoaffinity labeling, affinity purification, and mass spectrometry to discover and/or capture proteins from a specific subcellular structure in cells or tissues.
  • the process may be considered spatial proteomic discovery.
  • millions of targeted spots across thousands of FOVs are illuminated fully automatically through real-time image analysis and mechatronic control of one FOV at a time to achieve high-volume protein labeling of analogous subcellular ROIs.
  • Affinity enrichment isolates tagged proteins, effectively collecting (e.g., microscooping) ROI proteins at the subcellular level.
  • the accumulated micro-scooped proteins are enough for mass spectrometric sensitivity to reveal the specific proteome in high sensitivity and specificity.
  • This brute-force protein accumulation to overcome the current technology gap in protein amplification requires speed optimization of millions of steps to finish the entire process in hours, photoreactive probe is further used to achieve super-resolution labeling precision.
  • optoproteomics can be applicable to widely diverse cell and tissue biology problems to enable hypothesis-free subcellular proteomic discovery and generate testable hypotheses in bulk.
  • Described herein are methods and compositions useful for identifying, tagging, obtaining, and analyzing target biomolecules and neighboring biomolecules interaction with the target biomolecules.
  • the methods utilize photoreactive probes particularly that can label different biomolecules, while largely maintaining naturally occurring molecular structure in the biomolecules.
  • the probes described herein may be particularly useful for labeling subsets of biomolecules in cellular regions of cells using an image guided microscope with precision illumination control such as the system described in U.S. Patent No. 11,265,449, to enable automatic labeling of cellular biomolecules of interest.
  • the probes can be used for in situ tagging of biomolecules such as proteins inside cells or tissues and that can be followed by proximity labeling such as using Tyramide Signal Amplification (TSA).
  • TSA Tyramide Signal Amplification
  • the biomolecules can be further analyzed by analytical techniques such as mass spectrometry and sequencing.
  • These compositions may be especially useful for performing omics studies, such as genomics, proteomics, and transcriptomics,
  • biotin-based moiety refers to a biotin and variations of biotin derivatives, such as biotin with an open ring or substitutions. Typically, a biotin moiety is readily detectable with a biotin-binding entity or protein, such as avidin or streptavidin. Examples of another biotin-based moieties include diaminobiotin, biotin carbonate 5, biotin carbamate 6, and iminobiotin. In a particular example, another biotin-based moiety is desthiobiotin.
  • linker a structure which connects two or more substructures.
  • a linker has at least one uninterrupted chain of atoms extending between the substructures.
  • the atoms of a linker are connected by chemical bonds, typically covalent bonds.
  • the term “mass spectrometry analysis” includes linear time-of-flight (TOF), reflectron time-of-flight, single quadruple, multiple quadruple, single magnetic sector, multiple magnetic sectors, Fourier transform, ion cyclotron resonance (ICR) or ion trap.
  • TOF linear time-of-flight
  • ICR ion cyclotron resonance
  • proximity molecule refers to a molecule that is near another molecule.
  • a proximity molecule or neighbor molecule may bound to the molecule (e.g., covalently or non-covalently) or may be close by and not bound to the molecule.
  • photoactivated or “light activated” refers to excitation of atoms by means of radiant energy (e.g., by a specific wavelength or wavelength range of light, UV light, etc.).
  • a photoactivated probe has a free radical group and can react with an alpha carbon of an amino acid.
  • alkyl group refers to a straight-chain (unbranched) or branched saturated group of hydrocarbons.
  • an alkyl group has 24 or fewer carbon atoms, such as having from 1 to 24 carbon atoms (“Ci-24 alkyl”).
  • an alkyl group has 1 to 6 carbon atoms (“Ci-6 alkyl”) or 2 to 6 carbon atoms (“C2-6 alkyl”).
  • C1-6 alkyl groups include methyl (Ci), ethyl (C2), propyl (C3) (e.g., n-propyl, isopropyl), butyl (C4) (e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C5) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (Ce) (e.g., n-hexyl).
  • alkyl groups include n-heptyl, n-octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like.
  • the alkyl group can be cyclic (e.g., cycloalkyl) or acyclic.
  • cycloalkyl is meant a monovalent saturated or aromatic cyclic hydrocarbon group of from 3 to 24 carbon atoms, such as from 3 to 10 carbon atoms, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl, and the like.
  • An alkyl group can be unsubstituted or substituted.
  • An unsubstituted alkyl group is composed only of carbon and hydrogen atoms.
  • a substituted alkyl group has a special molecule or group bonded to a carbon atom (in place of a H atom) and can have one or more than one special molecules or groups attached.
  • the alkyl group of a substituted alkyl group can include haloalkyl, in which the alkyl group is substituted with one or more halo atoms (e.g., F, Cl, Br, or I).
  • the alkyl group can be substituted with one, two, three or, in the case of alkyl groups of two carbons or more, four or more substituents independently.
  • nitrogen protecting group refers to a substituent present on a nitrogen atom. Nitrogen can be chemically reactive and a nitrogen protecting group in a molecule can be configured to block reactivity under conditions configured for making modifications elsewhere in the molecule. Nitrogen protecting groups are well known in the art, and include, but are not limited to amide groups, carbamate groups, sulfonamide groups, and others.
  • crosslink refers to the process of chemically joining two or more molecules by a covalent bond, forming a three-dimensional network of connected polymers.
  • the crosslinking is photochemical crosslinking that occurs through a photochemical reaction, which is initiated by the absorption of light.
  • the photoreactive moiety described herein can absorb UV light and generate reactive species to form a covalent bond between polymer chains.
  • non-specific crosslinking refers to the process where crosslinking agents form covalent bonds between biomolecules in a manner that is not selective for specific sites or interactions. This means that the crosslinking occurs randomly or broadly across various accessible sites within the biomolecules.
  • the non-specific crosslinking means when the photoreactive moiety absorbs light energy and being activated in the selected region of interest, each photoreactive moiety can form a covalent bond with an amino acid, and different photoreactive moieties can crosslink with different amino acids, either on the same protein or different proteins.
  • Step 1 Epifluorescence imaging of a FOV.
  • a biological sample i.e. tissues, or cultivated cells
  • the fluorescence imaging allows visualization of cell morphology, cellular/subcellular compartments. For example, as shown in FIG.l, in a FOV 102 (circle), cells 101 (gray patches) were observed. According to the fluorescence signal, the researchers are able to assume target proteins existing in region of interests 103 (dark spot).
  • the photoreactive probes of the invention can be delivered to the biological sample before Step 1, or between Step 1 and Step 2.
  • Step 2 ROI mask generation of the FOV by traditional image processing or deep learning.
  • the mask 104 defines all the region of interests 103 to be pattered illumination in the next step.
  • Step 3 Selectively illumination toward the ROIs for photochemical labeling (activation of the photoreactive probes described herein).
  • Step 4 Moving the stage of the microscope to the next FOV and repeating the Step 2 and Step 3, until all the FOVs were illuminated.
  • the optical system contained a microscope 110 with a drift-free focusing setup, an sCMOS camera (CAM) 120 and LED 130 for imaging, an illuminating light source (ILS) 140 (e.g. 780-nm femtosecond laser) for two-photon illumination that triggers a photochemical reaction, an acousto-optic modulator (AOM) 150 as the illuminating light source shutter, and a pair of galvanometer scanning mirrors 160 (e.g., galvo X 162 and galvo Y 164) to direct the light illumination.
  • the microscope 110 includes the stage 118 and the PFS 116 by a USB interface.
  • the on/off switching of the laser was modulated by the AOM 150.
  • the laser beam was then steered by galvo X 162 and galvo Y 164, and passed through a scan lens (SL) 113, and a tube lens (TL) 114, which are the parts of the microscope 110, to be merged in the microscope 110 through dichroic mirrors (DMs, e.g., DM1 111 and DM2 112), then focused onto the sample plane by an objective 115.
  • DMs dichroic mirrors
  • the two-photon labeling can be guided from the imaging results, which can be generated by attaching a LED 130 illumination source on the microscope 110 to make epifluorescence imaging.
  • the fluorescence generated within the specimen can be collected by the objective 115, and the fluorescence signals were guided with the dichroic mirrors (e.g., DM1 111 and DM2 112) and cleaned via an emission filter 117 to be projected onto a sCOMS camera 120, able of frame rates down to 19 ms.
  • the dichroic mirrors e.g., DM1 111 and DM2 112
  • field programmable gate array FPGA
  • FPGA field programmable gate array
  • the photoreactive probe can be represented by formula (I):
  • T— L— W (I) in which the T portion is a biotin or another biotin-based moiety; the L portion is a chemical bond or a linker; and the W portion is a photoreactive moiety for covalent bond formation with a biomolecule in a sample upon application of light energy.
  • FIG. 3 shows a schematic depiction of a system used for photoselective spatial tagging and labeling.
  • substrate 206 such as a sample slide on a microscope stage, and a monolayer of plurality of cells 208 disposed on the substrate.
  • the surface of an entire substrate, or a portion of the substrate can be analyzed using an automated microscope system to identify a region of interest. For example, a sample can be stained or labeled to identify a region of interest.
  • the top part of FIG. 3 shows an expanded view of cell 208a, one of a plurality of cells 208.
  • the cell 208a has a nucleus 216 and a plurality of different types of organelles 212, such as cell membranes, mitochondria, ribosomes, and vacuoles.
  • Microscope system 202 selectively shines narrow band of light 204 onto region of interest (ROI) 218 for analysis of the region of interest 218.
  • ROI region of interest
  • the illumination can be selective, and large regions 214 of the cell and substrate are not illuminated.
  • narrow band of light 204 activates a photoreactive probe in only the region of interest 218.
  • FIG. 4A show an example of photoreactive probe that can be used in the compositions and for practicing the methods described herein.
  • the probe of formula (1-1) and (1-2) include a benzophenone as the photoreactive moiety W and a polyethylene glycol-based (PEG-based) linker as the linker L for linkage between the photoreactive moiety W and the biotin or desthiobiotin moiety T.
  • the linker L is not limited to the PEG-based linker, and may be, for example, peptide, amino acid, oligonucleotide, other polymeric linker, or a combination thereof.
  • Other examples of polymeric linkers include polypropylene glycol, polyethylene, polypropylene, polyamides, and polyesters.
  • the linker can be linear molecules in a chain of at least one or two atoms and can include more.
  • n is an integer of 1-20, and preferably is 1-6.
  • R and R' each independently are hydrogen, an alkyl, or a nitrogen protecting group.
  • the linker L may be replaced with a chemical bond, and the biotin or desthiobiotin moiety T may be bonded to the photoreactive moiety W without the linker L therebetween, as illustrated in FIG. 4B.
  • FIG. 4C illustrates the reaction between the benzophenone of the photoreactive probe and alpha-carbon of an amino acid AA of the biomolecule upon illumination.
  • the general structure of the amino acid 40 comprises alpha-carbon AA, a hydrogen, a carboxyl group 41, an amine group 42, and an R-group 43.
  • the benzophenone group is activated by the application of light energy and converted into an excited state.
  • the excited singlet state rapidly undergoes intersystem crossing to form a more stable triplet state.
  • benzophenone can abstract a hydrogen atom from C-H bonds (the alpha-carbon of amino acids AA) of nearby amino acids, particularly tyrosine, tryptophan, methionine, or cysteine residues.
  • the abstraction results in the formation of a benzophenone ketyl radical and an amino acid radical.
  • the benzophenone ketyl radical and the amino acid radical can then recombine to form a covalent bond, crossing-linking the benzophenone to the amino acid residue.
  • FIG. 4D shows selectively photochemical labeling using the photoreactive probe and the system described herein on a specimen to label biomolecules in a selected ROI.
  • a sample e.g., a cell or tissue sample
  • a biomolecule of interest 310 protein will be used herein by way of example, but other biomolecules could instead be analyzed
  • the sample can be pretreated, such as fixed and stained.
  • a sample can be fixed and stained with a cell stain (e.g., hematoxylin and eosin (H&E); Masson's trichrome stain), identified with an immunofluorescent labeled antibody recognizing a biomolecule of interest 310 or by other methods.
  • a cell stain e.g., hematoxylin and eosin (H&E); Masson's trichrome stain
  • the sample on substrate 309 is treated with a plurality of photoreactive probes 312 and patterned light (PLT) selectively illuminated the sample in the user-defined region of interest (grey area) so as to activate the photoreactive probes 312 to form activated photoreactive probe 312' (i.e. photoactivated probes or light activated probes).
  • PLT patterned light
  • the activated photoreactive probe 312' (showed by the dotted circle) is able to form complexes with any amino acid residues by the photocrosslinking mechanism described in FIG. 4C.
  • the activated photoreactive probes 312' can diffuse, thus not only neighbor molecules 311 near the biomolecule of interest 310, but also more distant biomolecules 331 can be labeled.
  • the area labeled by activated photoreactive probe 312', or labeled precision, can cover a region of about 250-1000 nm.
  • the region of interest can be determined according to the biomolecule of interest 310 in situ.
  • the patterned light provides light energy to active photoreactive probe 312 so as activated photoreactive probe 312’ can non-specifically crosslink with a plurality of amino acids of different proteins within entire region of interest.
  • unreacted probes 312 i.e. unbound probe
  • the biomolecules labeled by the activated photoreactive probes 312' can be detected by avidin, streptavidin or NeutrAvidin, carrying a reporter group, e.g. horseradish peroxidase (HRP) or a fluorescent label.
  • HRP horseradish peroxidase
  • FIG. 4E illustrates the practical chemical structure of the two photoreactive probes using in the following embodiments.
  • One is a biotin-containing probe (biotin-benzophenone, BBzP, such as B3-BzP) (top panel), and another is a desthiobiotin-containing probe (desthiobiotin-benzophenone, DBBzP, such as DB3-BzP) (bottom panel).
  • biotin-containing probe biotin-benzophenone, BBzP, such as B3-BzP
  • DBBzP desthiobiotin-benzophenone
  • Photoselective tagging and labeling as described herein can be performed in various types of samples, such as samples obtained from tissues, cells, or particles, such as from an entity (e.g., a human subject, a mouse subject, a rat subject, an insect subject, a plant, a fungi, a microorganism, a virus) or tissues samples or cell samples that are not from an organism, such as cell culture samples or artificial tissue scaffold samples (e.g., cultured laboratory cells, in vitro developed heart tissue, 3-d printed tissue, etc.). Samples for analysis using the probes, materials, and methods described herein can be living (live cells) or can be not living (e.g., fixed).
  • an entity e.g., a human subject, a mouse subject, a rat subject, an insect subject, a plant, a fungi, a microorganism, a virus
  • tissue samples or cell samples that are not from an organism such as cell culture samples or artificial tissue scaffold samples (e.g., cultured laboratory
  • a sample for tagging and labeling can include a monolayer sample, a multi-layer sample, a sample fixed to a substrate (e.g., a microscope slide), a sample not fixed to a substrate, a suspension of cells, or an extract, such as an in vitro cell extract, a reconstituted cell extract, or a synthetic extract.
  • a sample is not fixed (unfixed).
  • a sample is fixed.
  • a cell or tissue sample may be fixed with e.g., acetic acid, acetone, formaldehyde (4%), formalin (10%), methanol, glutaraldehyde, or picric acid.
  • a fixative may be a relatively strong fixative and may crosslink molecules or may be weaker and not crosslink molecules.
  • a cell or tissue sample for analysis may be frozen, such as using dry ice or flash frozen, prior to analysis.
  • a cell or tissue sample may be embedded in a solid material or semisolid material such as paraffin or resin prior to analysis.
  • a cell or tissue sample for analysis may be subject to fixation followed by embedding, such as FFPE.
  • the concentration of the photoreactive probe treated with the sample can range from 0.1 mM to 10 mM.
  • the wavelength of light for activation of the photoreactive probe or photo selective tagging and labeling ranges in some embodiments from about 200 nm to about 1600 nm. In some embodiments, the wavelength of light for performing photoselective tagging and labeling ranges from about 680 nm to about 1600 nm at two-photon light source; or ranges from about 300 nm to about 650 nm (e.g. 365 nm) at single-photon light source.
  • the wavelengths used for photoactivation of the probe are different from the wavelengths used for imaging.
  • the activation of the photoreactive probe utilizes optical radiation (light) at from around 300-450 nm, 550 nm for single-photon activation or >650 nm for multiphoton activation.
  • the particular wavelength used in some embodiments in this disclosure is 780 nm at two-photon light source.
  • the methods may be used to tag and/or label carbohydrates, lipids, nucleic acids, proteins, either alone or in combination.
  • the methods may include the step of treating a biological sample with a photoreactive probe having a biotin or another biotin-based moiety and a photoreactive moiety, and covalently binding the photoreactive moiety to a biomolecule in the biological sample.
  • this disclosure provides a method for photochemical labeling a biomolecule in a sample, comprising the following steps: delivering a photoreactive probe above to a sample; and selectively illuminating a selected region of interest of the sample with optical radiation to activate the photoreactive moiety of the photoreactive probe in the selected region of interest for covalent bond formation between the photoreactive moiety and a biomolecule in the sample in the selected region of interest.
  • the illumination of the ROIs is achieved by point scanning.
  • the output of imaging processing after ROI mask generation is an XY-coordinate array (2D array) of targeted points to be illuminated covering the interested regions defined by the users.
  • the number of illumination points per FOV can vary depending on the user’s criteria.
  • the exposure time of each illumination point is in a range of 100-500 ps to make sure enough photochemical reaction was achieved within reasonable working hours.
  • the efficiency of photochemical reaction is also related to laser power intensity. Higher power intensity may shorten working time (fast photochemical reaction) but may damage sample as well. Therefore, when performing the step of selectively illumination, the power intensity is in a range of 1 mW to 600 mW. Preferably, the optimal power intensity that can be measured under a microscope is in a range of 10 mW to 300 mW.
  • this disclosure provides an image-guided photolabeling method comprising following steps: (a) delivering a photoreactive probe to a sample; (b) imaging the sample in a first field of view and acquiring at least one image in the first field of view of the sample with an imaging light source and a camera; (c) processing the at least one image and determining a region of interest in the first field of view; (d) obtaining coordinate information of the region of interest; (e) according to the coordinate information, selectively illuminating the region of interest with optical radiation to activate the photoreactive moiety of the photoreactive probe and thereby form a non-specific crosslinking between the photoreactive moiety of the photoreactive probe and a plurality of amino acids of different proteins of the sample in the region of interest; and (f) after the region of interest of the first field of view has been illuminated, moving to a second field of view of the sample, and repeating the steps (b) to (e) until all the field of views of the sample have been fully
  • this disclosure provides an analytical method for a probe- labeled protein comprising the following steps: obtaining a sample; delivering a photoreactive probe above to the sample, selectively illuminating a region of interest of the sample with optical radiation to activate the photoreactive moiety of the photoreactive probe in the region of interest and form a non-specific crosslinking between the photoreactive moiety and a plurality of the amino acids of different proteins of the sample in the region of interest; and isolating the plurality of probe-labeled proteins from the sample through an affinity precipitation between the T portion of the photoreactive probe and a plurality of affinity beads .
  • Example 1 Photoactivable amino acid crosslinking enables spatial photo-induced [0100] To achieve photo-induced labeling of proteins at a microscopic illumination point, the sample was added with molecules with three elements: a photocatalysis, a photoactivable amino acid linker to covalently bind to a protein, and a probing tag for protein pulldown.
  • BBzP (Fig. 4E) was used as the photoreactive probe in the following Examples 1-3.
  • Benzophenone at the illumination point was excited to become 1,2-diradical, which reacted with the C-H bond of a-carbon, forming a covalent bond of the corresponding amino acid. That is, a plurality of amino acids of different proteins within the entire patterned light area can be biotinylated.
  • U-2OS cells (HTB-96, ATCC, VA, USA) were cultivated at 37°C in a 5% CO 2 humidified environment in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS. 2* 10 5 cells were seeded in glass bottom chambers (80287, ibidi) and incubated for approximately 16 h to 80-90% of confluency. Afterwards, cells were washed with phosphate buffered saline (PBS) then fixed with 2.4% paraformaldehyde solution (PF A, 15710, Electron Microscopy Sciences) or 100% ice-cold methanol.
  • PBS phosphate buffered saline
  • PF A paraformaldehyde solution
  • Example 2 Subcellular spatial biotinylation by BBzP is achieved for cell and tissue samples
  • the labeling resolution should be at a submicron level.
  • a line scan illumination was performed on U-2OS cells and superresolution structured illumination microscopy (SR-SIM) was used to measure the biotinylation labeling width by staining with B3-BzP and anti-biotin (FIG. 6A). 0.39 pm labeling resolution was able to reach when using a 40x magnification/0.95 NA objective (FIG. 6B).
  • SR-SIM superresolution structured illumination microscopy
  • the segmented region was illuminated to induce targeted biotinylation.
  • the in situ biotinylated regions matched well with the corresponding subcellular structures in lateral (xy) and axial (z) directions, suggesting a high spatial labeling specificity (FIG. 6C).
  • Immune synapse induction was performed as follows: Raji B cells (CCL-86, ATCC) were used as the antigen presenting cells (APCs). To activate the APCs for immune synapse (IS) formation, 1 * 10 6 Raji B cells were pelleted to resuspend with 100 pL serum-free RPMI (SH30027, HyClone) containing 0.1 pg Staphylococcal Enterotoxins (ET404, Toxin Technology Inc) and incubated at 37°C for 1 h. On the last 20 min of the incubation, the Raji B cells were stained with 20 pM cell tracker Red CMTPX
  • Calbindin-D28K staining of a FFPE mouse brain tissue section revealed a high abundant of Purkinje cells in cerebellum with clear axon and dendrite architecture and a distinctive neuron cell body layer. Deep learning-enabled segmentation and following targeted illumination resulted in biotinylation at cell bodies, demonstrating photolabeling specificity for FFPE tissue samples.
  • Example 3 Optoproteomics enables subcellular spatial proteomics in high sensitivity and specificity by BBzP
  • Photolabeling on subcellular structures was performed as follows: cells were incubated with photon labeling reagent containing 0.1-2.0 mM BBzP. Two-photon laser coupled with a microscopic system was used for photolabeling at a laser power of 100-200 mW, and the cells were subjected to an image-guided laser-exposure time at 100-1000 ps. Verification of the performance of two-photon labeling by fluorescent microscopy was performed as follows: labeled cells were washed with PBS-T three times.
  • Protein extraction and on-bead digestion was performed as follows: labeled cells were harvested by scraping with buffer containing 10 mM Tris (pH 8.0), 1% Triton X-100, 1-fold protease inhibitor cocktail, 10 mM sodium ascorbate, 5 mM Trolox, and 1 mM sodium azide. Harvested cells were sonicated at 60% power using a Q125 sonicator (Qsonica) with Is on/ 2s off interval for 2 min, then subjected to evaporate the scraping buffer for 2 h by SpeedVac system.
  • Qsonica Q125 sonicator
  • PierceTM 660nm Protein Assay was used to measure the protein concentrations, and 240 pg of proteins were subjected to the immunoprecipitation. Streptavidin magnetic beads were washed with dilution buffer (0.5% Triton X-100/PBS) three times, and the protein lysates were diluted 10- fold to reduce the SDS concentration to be less than 0.4%, and the diluted lysates were added to the washed beads and incubated at 2-8°C for 16 h with rotation.
  • the biotinylated-protein bonded beads were washed with the following washing buffers to reduce the non-specific binding maximally: Buffer A (2% SDS, 50 mM Tris, pH 8.0); Buffer B (0.5M NaCl, 0.1% deoxycholic acid, 0.1% SDS, 1% Triton X-100, 50 mM HEPES); Buffer C (0.5% deoxycholic acid, 0.5% Triton X-100, 10 mM Tris, 250 mM LiCl, pH 8.0).
  • Buffer A 2% SDS, 50 mM Tris, pH 8.0
  • Buffer B 0.5M NaCl, 0.1% deoxycholic acid, 0.1% SDS, 1% Triton X-100, 50 mM HEPES
  • Buffer C (0.5% deoxycholic acid, 0.5% Triton X-100, 10 mM Tris, 250 mM LiCl, pH 8.0).
  • the beads were further washed with 100-pL 50 mM TEABC three times, and the biotin-protein bonded beads were then mixed with 0.2 pg of Trypsin/ Lys-C (V5071, Promega) in a final volume of 20 pL at 37°C for 100 min for an initial digestion. After that, the supernatant was collected and subjected to the overnight digestion without adding further enzyme. Finally, the digests were acidified by adding 2 pL of 10% formic acid and were desalted by C18 Ziptip Desalted peptides were dried by Speedvac and stored at -20°C prior to LC-MS/MS analysis.
  • Trypsin/ Lys-C V5071, Promega
  • LC-MS/MS analysis was performed as follows: detection of immunoprecipitated product 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).
  • the desalted peptides were resuspended in 0.1%formic acid in water and loaded onto a PepMapTM 100 C18 HPLC column (2 pm, 100 angstrom, 75 pm x 25 cm; Thermo Fisher Scientific), and peptides were eluted over 160 min gradients for nuclei-illuminated samples, over 120 min gradients for nucleoli-, SG-illuminated samples.
  • the full MS spectra ranging from m/z 375- 1500 were acquired at a resolving power of 120,000 in Orbitrap, an AGC target value of 4 x 10 5 , and a maximum injection time of 50 ms.
  • Fragment ion spectra were recorded in the topspeed mode at a resolving power of 30,000 in Orbitrap using a data-dependent method.
  • Monoisotopic precursor ions were selected by the quadrupole using an isolation window of 1.2, 0.7, 0.4 Th for the ion with 2+, 3+, 4-7 charge states, respectively.
  • An AGC target of 5 x 10 4 maximum injection time of 54 ms, higher-energy collisional dissociation (HCD) fragmentation with 30% collision energy, and a maximum cycle time of 3s were all applied.
  • Dynamic exclusion was set to 60s with an exclusion window of 10 ppm.
  • Precursor ions with the charge state of unassigned, 1+, or superior to 8+ were excluded from fragmentation selection.
  • Streptavidin dot-blot analysis was performed as follows: photolabeled U-2OS cells and control U-2OS cells were lysed and immunoprecipitated with streptavidin beads as described above. Dot-blot analysis was performed in a PVDF membrane. PVDF membrane was activated with 100% MEOH, and soaked in PBS-T for 2 min, then 8 pL of each sample were spot on the membrane. After dry, the membrane (blot) was rinsed with PBS-T and blocked with 5% BSA for 30 min at RT, and washed with PBS-T. Then the membrane was incubated with Streptavidin-HRP (1 : 1000) in 5% BSA at 4°C overnight on a seesaw shaker.
  • the blot was washed with PBS-T before to development with ECL substrate (Bio-Rad) using iBright FL1500 image system (Invitrogen).
  • the blot was stripped with NaOH for 5 min at 60°C 3 times, blocked with 2.5% BSA, and re-probed with mouse anti-tubulin 1 : 500 (GTX112141, GeneTex) in 2.5% BSA at 4°C overnight on a seesaw shaker, and washed with PBS-T. Then the blot was incubated with anti-mouse-HRP (1 : 500) in 2.5% BSA at RT for 2 h. The blot was washed with PBS-T prior to development with ECL substrate (Bio-Rad) using iBright FL 1500.
  • biotinylated proteins by BBzP were enriched by immunoprecipitation using streptavidin beads, tryptically digested on beads, and subject to LC-MS/MS measurement.
  • Dot-blot analysis showed that biotinylated proteins by BBzP were enriched in the protein lysate or streptavidin beads pull-down when photolabeling was turned on (FIG. 7B), demonstrating effective photobiotinylation of the sample.
  • a distribution of overall protein abundances quantified in PL to control (CTL) was calculated (FIG. 7C).
  • CTL PL to control
  • the sensitivity and specificity of the spatially labeled nuclear proteome were determined by calculating the percentage of the true positive proteins from a final protein list. A total of 1,150 proteins showed differentially enriched, where 1,032 were annotated as nuclear proteins, accounting for 90% of true positive rate.
  • Protein identification and label-free quantification was performed as follows: raw data from the same batch of two-photon illumination were processed together with Proteome Discoverer (Thermo Fisher Scientific) by Sequest HT algorithm against the UniProtKB/Swiss-Prot human protein database (version 2020.02, 20,365 entries) for feature extraction, peptide identification, and protein inference.
  • Peptide level data was then normalized to the total peptide intensity, and the quantification value for a given protein was derived from the sum of normalized intensities of the top three intense unique peptides belonging to that protein.
  • Label-free quantification (LFQ) was performed to extract enriched proteome from background signals. After streptavidin beads enrichment and identification by mass spectrometry, protein IDs from both control group and experimental group were analyzed by comparing their peak intensities. The distribution of true positive and false positive proteins was separated in the axis of log2(PL/Control) and gave the PL labeled protein IDs by applying a cutoff of log2(PL/Control).
  • Example 4 Probe synthesis of desthiobiotin-benzophenone (DBBzP) [0120] The probe of desthiobiotin-PEG3 -benzophenone (DB3-BzP) was synthesized according to synthesis scheme illustrated in FIG. 8. The illustrated synthesis scheme is given as an example and not for limiting purposes.
  • Example 5 Photochemical labeling by DBBzP on subcellular structures of cells [0121] Cells were cultivated at 37°C in a 5% CO2 humidified environment in Dulbecco's Modified Eagle Medium supplemented with 10% FBS. 2* 10 5 cells were seeded in glass bottom chambers and incubated for approximately 16 h to 80-90% of confluency.
  • the cells were subjected to a laser-exposure time at 100-1000 microseconds at a power of 50-400 mW to activate the benzophenone group to converted into an active diradical, forming a covalent bond with the alpha-carbon of amino acids in the selected regions of interest.
  • a laser-exposure time at 100-1000 microseconds at a power of 50-400 mW to activate the benzophenone group to converted into an active diradical, forming a covalent bond with the alpha-carbon of amino acids in the selected regions of interest.
  • PBS phosphate buffered saline
  • Quality control was performed as follows: the labeling (desthiobiotinylation) efficiency and labeling reagent residues can be observed and evaluated by staining cells with 500-fold diluted NeutrAvidin-488 (488nm fluoresce dye conjugated NeutrA vidin, N488) in 0.1% PEST supplemented with 3% BSA for Ih at RT on a seesaw shaker. A signal-to-noise ratio was used as the procedure of quality control:
  • N488 intensity of nonROIs - Blank where N488 intensity is the average intensity of the N488 channel from a fluorescent microscope; ROI, region of interest; Blank, average intensity of N488 channel at the areas without cells.
  • nucleoli of U-2OS cells were labeled by B3-BzP (left) and DB3-BzP (right) and imaged by fluorescent microscopy, the signal-to-noise ratios are 5.8 and 17.8 respectively.
  • the total time to photo-label proteins of a 2 cm x 2 cm sample well using a 40x objective was 3 to 16 hours.
  • One to ten sample wells were needed to collect enough proteins for mass spectrometry analysis.
  • the nonspecific labeling background both were lower, demonstrating that both photoreactive probes have better labeling efficiency and spatial specificity.
  • first”, “second”, “third” and “fourth” 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 feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Est proposé un procédé de marquage photochimique comprenant les étapes consistant à administrer une sonde photoréactive à un échantillon, et la sonde photoréactive de formule (I) : T─L─W, la partie T étant de la biotine ou une autre fraction à base de biotine, la partie L comprenant une liaison chimique ou un agent de liaison, la partie W étant une fraction photoréactive, la fraction photoréactive pouvant absorber de la lumière ultraviolette et devenir active à des fins de réticulation avec un acide aminé ; et éclairer sélectivement l'échantillon avec un rayonnement optique pour activer la fraction photoréactive de la sonde photoréactive dans la région d'intérêt sélectionnée ; et former une réticulation non spécifique entre la fraction photoréactive de la sonde et une pluralité d'acides aminés de différentes protéines de l'échantillon dans la région d'intérêt sélectionnée.
PCT/US2024/036001 2023-06-29 2024-06-28 Procédé de photomarquage de biomolécules à l'aide de sondes photoréactives Ceased WO2025006872A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP24832997.1A EP4713681A2 (fr) 2023-06-29 2024-06-28 Procédé de photomarquage de biomolécules à l'aide de sondes photoréactives

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363511010P 2023-06-29 2023-06-29
US63/511,010 2023-06-29
US202363517437P 2023-08-03 2023-08-03
US63/517,437 2023-08-03

Publications (2)

Publication Number Publication Date
WO2025006872A2 true WO2025006872A2 (fr) 2025-01-02
WO2025006872A3 WO2025006872A3 (fr) 2025-05-08

Family

ID=93940040

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/036001 Ceased WO2025006872A2 (fr) 2023-06-29 2024-06-28 Procédé de photomarquage de biomolécules à l'aide de sondes photoréactives

Country Status (3)

Country Link
EP (1) EP4713681A2 (fr)
TW (1) TWI904733B (fr)
WO (1) WO2025006872A2 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020164649A1 (en) * 2000-10-25 2002-11-07 Rajendra Singh Mass tags for quantitative analysis
KR20220119066A (ko) * 2019-12-19 2022-08-26 스키노시브 접착성 광보호성 화합물 및 이의 용도
WO2021188978A1 (fr) * 2020-03-20 2021-09-23 Academia Sinica Sondes photoréactives et clivables permettant le marquage de biomolécules

Also Published As

Publication number Publication date
TWI904733B (zh) 2025-11-11
EP4713681A2 (fr) 2026-03-25
TW202500977A (zh) 2025-01-01
WO2025006872A3 (fr) 2025-05-08

Similar Documents

Publication Publication Date Title
US20220155316A1 (en) Protein sequencing methods and reagents
US12535669B2 (en) Microscope-based system and method for image-guided microscopic illumination
US20200064340A1 (en) Quantitative dna-based imaging and super-resolution imaging
US20080220434A1 (en) Detection Of Molecule Proximity
CN109661439A (zh) 发荧光的水溶剂化共轭聚合物
CZ20033143A3 (en) Methods and compositions for analyzing proteins
CN111094462A (zh) 深紫外线可激发的水溶剂化聚合物染料
JP2023548149A (ja) 生体分子プローブを使用した組織のマルチプレックス質量分析イメージングのための新規な光切断可能な質量タグ
WO2023049177A1 (fr) Séquençage de protéine et de peptide à molécule unique
Chen et al. Microscopy-guided subcellular proteomic discovery by high-speed ultra-content photo-biotinylation
EP4713681A2 (fr) Procédé de photomarquage de biomolécules à l'aide de sondes photoréactives
Müller et al. Developing a new cleavable crosslinker reagent for in-cell crosslinking
Magrassi et al. Single-molecule localization to study cytoskeletal structures, membrane complexes, and mechanosensors
Yan et al. Nonprotein based enrichment method to analyze peptide cross-linking in protein complexes
Wang et al. Chip-Based 3D Interferometric Nanoscopy
WO2024092194A2 (fr) Sondes réactives et procédés d'abaissement et de purification pour marquer des biomolécules
Pelicci et al. Correlative Multi-Modal Microscopy: A Novel Pipeline for Optimizing Fluorescence Microscopy Resolutions in Biological Applications. Cells, 2023. 12 (3): p. 354 doi: 10.3390/cells12030354. PMID: C737FDCE-95FA-4F60-AC22-041E9A60D8BE
Wang et al. Effective and transient mapping of protein–protein interactions: Application of novel releasable photoaffinity linkers
Melder Development of a Compound Interaction Screen on a photoactivatable Cellulose Membrane (CISCM) to identify drug targets
Taban et al. Nanoscale imaging of expanded cells and proteins with spontaneously blinking dyes
Faulkner Characterising nanoscale organisation of repair foci during repair of double strand breaks using expansion microscopy.
Jiang et al. Electron Acceptive Mass Tag for Mass Spectrometric Imaging-Guided Synergistic Targeting to Mice Brain Glutamate Receptors
Kuzmich Metal Labeling for Low Affinity Binding Biomolecules
HK40105305A (zh) 单分子蛋白质和肽测序
Chang et al. Spatial Mapping of Proteins in Nuclear and Nucleolar Compartments

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2024832997

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2024832997

Country of ref document: EP

Effective date: 20251218

ENP Entry into the national phase

Ref document number: 2024832997

Country of ref document: EP

Effective date: 20251218

ENP Entry into the national phase

Ref document number: 2024832997

Country of ref document: EP

Effective date: 20251218

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2024832997

Country of ref document: EP

Effective date: 20251218

ENP Entry into the national phase

Ref document number: 2024832997

Country of ref document: EP

Effective date: 20251218

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24832997

Country of ref document: EP

Kind code of ref document: A2

WWP Wipo information: published in national office

Ref document number: 2024832997

Country of ref document: EP