# US 12,181,481 B2 · Photocleavable mass-tags for multiplexed mass spectrometric imaging of tissues using biomolecular probes

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- Number: US 12,181,481 B2
- Document ID: US12181481B2
- Status: Granted
- Kind: B2
- Application number: 17/399,417
- Filed: 2021-08-11
- Published: 2022-05-05
- Granted: 2024-12-31
- Earliest claimed: 2020-10-29
- Assignees: AmberGen, Inc.
- Applicants: AmberGen, Inc.
- Inventors: Mark J. Lim; Gargey Yagnik; Kenneth J. Rothschild
- Web page: https://patentgrep.com/patent/US12181481B2

## CPC

- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G01N 33/6851: {Methods of protein analysis involving laser desorption ionisation mass spectrometry}
- C07C 271/20: to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
- C12Q 1/6841: In situ hybridisation
- G01N 33/5082: {Supracellular entities, e.g. tissue, organisms}
- G01N 33/533: with fluorescent label
- G01N 33/575: for cancer
- G01N 33/57515: of the breast
- G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins}
- G01N 2333/4724: Lectins
- G01N 2400/00: Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
- G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry
- G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material
- G01N 2570/00: Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
- Y02P 20/55: Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The field of this invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for the targeted detection and mapping of biomolecules (e.g., proteins and miRNAs) in tissues or cells for example, for research use and for clinical use such by pathologists (e.g., biomarker analyses of a resected tumor or tumor biopsy). In particular, the use of mass spectrometric imaging (MSI) as a mode to detect and map the biomolecules in tissues or cells for example. More specifically, the field of this invention relates to photocleavable mass-tag reagents which are attached to probes such as antibodies and nucleic acids and used to achieve multiplex immunohistochemistry and in situ hybridization, with MSI as the mode of detection/readout. Probe types other than antibodies and nucleic acids are also covered in the field of invention, including but not limited to carbohydrate-binding proteins (e.g., lectins), receptors and ligands. Finally, the field of the invention also encompasses multi-omic MSI procedures, where MSI of photocleavable mass-tag probes is combined with other modes of MSI, such as direct label-free MSI of endogenous biomolecules from the biospecimen (e.g., tissue), whereby said biomolecules can be intact or digested (e.g., chemically digested or by enzyme).

## Claims

### Claim 1

A multiplex method for co-detecting 3 or more different biomarkers in a tissue sample, said method comprising:
a. providing a tissue sample;
b. contacting said tissue sample with 3 or more different probes to create a probed tissue sample, each of said probes conjugated to a unique photocleavable mass-tag and at least 3 of said probes each binding to a different biomarker in said tissue sample, wherein said 3 or more probes are in a mixture and said tissue sample is contacted with said mixture to create said probed tissue sample;
c. illuminating said photocleavable mass-tags with an exogeneous source of UV light so as to photocleave at least a portion of said mass-tags before step d); and
d. detecting, using mass spectrometric imaging of said probed tissue sample, said unique mass-tags, or fragments thereof, from the at least 3 of said bound probes, wherein said mass-tags are detected as molecular ions.

### Claim 2 (depends on 1)

The method of claim 1, wherein said tissue sample is a thin tissue section.

### Claim 3 (depends on 1)

The method of claim 1, wherein said tissue sample was frozen and thin-sectioned before step b).

### Claim 4 (depends on 1)

The method of claim 1, where said tissue sample was formalin-fixed and thin-sectioned before step b).

### Claim 5 (depends on 1)

The method of claim 1, wherein said tissue sample was formalin-fixed, paraffin embedded and thin-sectioned before step b).

### Claim 6 (depends on 1)

The method of claim 1, wherein said tissue sample is mounted on a slide before step b).

### Claim 7 (depends on 6)

The method of claim 6, wherein said slide comprises gold.

### Claim 8 (depends on 7)

The method of claim 7, wherein said slide is a glass slide with a gold layer.

### Claim 9 (depends on 1)

The method of claim 1, wherein said tissue sample is mounted on a slide before step d).

### Claim 10 (depends on 9)

The method of claim 9, wherein said slide comprises gold.

### Claim 11 (depends on 10)

The method of claim 10, wherein said slide is a glass slide with a gold layer.

### Claim 12 (depends on 1)

The method of claim 1, wherein said tissue sample is from a tumor.

### Claim 13 (depends on 12)

The method of claim 12, wherein said tumor is a breast tumor.

### Claim 14 (depends on 1)

The method of claim 1, wherein said probes conjugated to a mass-tag have the following general structure: Mass Unit-Core Structure-Probe.

### Claim 15 (depends on 1)

The method of claim 1, wherein at least 1 of said probes comprises a fluorescent moiety in addition to said mass-tag.

### Claim 16 (depends on 1)

The method of claim 1, wherein said probes are selected from the group consisting of proteins and nucleic acids.

### Claim 17 (depends on 1)

The method of claim 1, wherein at least 1 of said probes is an antibody.

### Claim 18 (depends on 17)

The method of claim 17, wherein said antibody is selected from the group consisting of recombinant antibodies, nanobodies, single-chain fragment variable (scFv) antibodies, single domain antibodies, and VHH single domain antibodies.

### Claim 19 (depends on 1)

The method of claim 1, wherein at least 1 of said probes is selected from the group consisting of antibodies, receptors and ligands.

### Claim 20 (depends on 1)

The method of claim 1, wherein at least 1 of said probes is an aptamer.

### Claim 21 (depends on 1)

The method of claim 1, wherein at least 1 of said probes binds to RNA targets.

### Claim 22 (depends on 1)

The method of claim 1, wherein at least 1 of said probes binds to miRNA targets.

### Claim 23 (depends on 1)

The method of claim 1, wherein at least 1 of said probes binds to DNA targets.

### Claim 24 (depends on 1)

The method of claim 1, wherein said mass-tags are non-rare-earth-metal mass-tags.

### Claim 25 (depends on 1)

The method of claim 1, wherein said mass-tags comprise a plurality of amino acids.

### Claim 26 (depends on 1)

The method of claim 1, further comprising performing, after step a) but before step b), mass spectrometric imaging on said tissue sample.

### Claim 27 (depends on 1)

The method of claim 1, wherein a matrix compound is applied to said probed tissue sample before step d).

### Claim 28 (depends on 27)

The method of claim 27, wherein said matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-Dihydroxybenzoic acid (DHB) and 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid).

### Claim 29 (depends on 1)

The method of claim 1, wherein said tissue is contacted with 5 or more different probes.

### Claim 30 (depends on 1)

The method of claim 1, wherein said an exogeneous source of UV light is an LED UV source.