# US 12,181,482 B2 · Photocleavable mass-tags for multiplexed mass spectrometric imaging of tissues using biomolecular probes

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- Number: US 12,181,482 B2
- Document ID: US12181482B2
- Status: Granted
- Kind: B2
- Application number: 18/212,456
- Filed: 2023-06-21
- Published: 2023-11-02
- 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/US12181482B2

## 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. 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, and also encompasses multi-omic MSI procedures, where MSI of photocleavable mass-tag probes is combined with other modes of MSI.

## Claims

### Claim 1

A method, comprising:
a) providing i) a compound having the structure
wherein X is a protecting group and wherein A and B are chemical linkers; and ii) amino acids including isotopes, analogs, derivatives or modifications thereof, and natural and unnatural amino acids; and
b) subjecting said compound and said amino acids to solid-phase peptide synthesis.

### Claim 2 (depends on 1)

The method of claim 1, wherein step b) results in a plurality of amino acids attached to one or both ends of the compound of step a).

### Claim 3 (depends on 2)

The method of claim 2, wherein the amino acids attached comprise the sequence APRLRFYSL.

### Claim 4 (depends on 2)

The method of claim 2, wherein the amino acids attached comprise the sequence GSGGK, wherein K is lysine comprising an ε-amino group.

### Claim 5 (depends on 4)

The method of claim 4, further comprising c) converting said ε-amino group of lysine to a probe reactive moiety.

### Claim 6 (depends on 5)

The method of claim 5, wherein the probe reactive moiety is selected from the group consisting of amine-reactive N-hydroxysuccinimidyl (NHS) esters, sulfo-N-hydroxysuccinimidyl (sulfo-NHS) esters, succinimidyl esters (SE), sulfo-succinimidyl esters (SSE), aldehydes, tetrafluorophenyl (TFP) esters, sulfhydryl-reactive maleimides or iodoacetamides, poly-reactive epoxy moieties, azides and alkynes.

### Claim 7 (depends on 5)

The method of claim 5, wherein said probe reactive moiety is reacted with a probe.

### Claim 8 (depends on 7)

The method of claim 7, wherein said probe is selected from the group consisting of proteins and nucleic acids.

### Claim 9 (depends on 8)

The method of claim 8, wherein said protein is an antibody.

### Claim 10 (depends on 9)

The method of claim 9, where 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 11 (depends on 8)

The method of claim 8, wherein said nucleic acid probe is an aptamer.

### Claim 12 (depends on 8)

The method of claim 8, wherein said protein is a lectin.