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US-12618852-B2 - Photocleavable mass-tags for multiplexed mass spectrometric imaging of tissues using biomolecular probes

US12618852B2US 12618852 B2US12618852 B2US 12618852B2US-12618852-B2

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).

Inventors

  • Mark J. Lim
  • Gargey Yagnik
  • Kenneth J. Rothschild

Assignees

  • AMBERGEN, INC.

Dates

Publication Date
20260505
Application Date
20210811

Claims (20)

  1. 1 . A multiplex method for co-detecting 3 or more different types of carbohydrates in a tissue sample, said method comprising: a. providing i) a tissue sample, ii) a mass spectrometry imaging (MSI) instrument, iii) probes comprising carbohydrate-binding proteins conjugated to a mass-tag having the general structure: Mass Unit-Core Structure-Probe, wherein said core structure has the structure: and iv) a UV light source that is exogenous to the MSI instrument; b. contacting said tissue sample with 3 or more different probes comprising carbohydrate-binding proteins to effect binding of said carbohydrate-binding proteins to said tissue sample to create a probed tissue sample, each of said carbohydrate-binding proteins reactive with a different carbohydrate, and each of said carbohydrate-binding proteins conjugated to a unique photocleavable mass-tag; c. photocleaving at least a portion of said mass-tags before step d by illuminating said mass-tags with said exogenous source of UV light source and not using the MSI instrument's laser beam if present in the MSI instrument so as to photocleave at least a portion of said mass-tags before step d by illuminating said mass-tags with UV light; and d. detecting, using mass spectrometric imaging of said probed tissue sample, said mass-tags, or fragments thereof, as molecular ions.
  2. 2 . The method of claim 1 , wherein said tissue sample is a thin tissue section.
  3. 3 . The method of claim 1 , wherein said tissue sample was frozen and thin-sectioned before step b).
  4. 4 . The method of claim 1 , where said tissue sample was formalin-fixed and thin-sectioned before step b).
  5. 5 . The method of claim 1 , wherein said tissue sample was formalin-fixed, paraffin embedded and thin-sectioned before step b).
  6. 6 . The method of claim 1 , wherein said tissue sample is mounted on a slide before step b).
  7. 7 . The method of claim 6 , wherein said slide comprises gold.
  8. 8 . The method of claim 7 , wherein said slide is a glass slide with a gold layer.
  9. 9 . The method of claim 1 , wherein said tissue sample is mounted on a slide before step d).
  10. 10 . The method of claim 9 , wherein said slide comprises gold.
  11. 11 . The method of claim 10 , wherein said slide is a glass slide with a gold layer.
  12. 12 . The method of claim 1 , wherein said tissue sample is from a tumor.
  13. 13 . The method of claim 12 , wherein said tumor is a breast tumor.
  14. 14 . The method of claim 1 , wherein said 3 or more carbohydrate-binding proteins are in a mixture and said tissue sample in step b) is contacted with said mixture to create said probed tissue sample.
  15. 15 . The method of claim 14 , wherein said mixture additionally contains 1 or more probes, each of said probes reactive with a different target within said tissue, and each of said probes conjugated to a unique mass-tag.
  16. 16 . The method of claim 1 , wherein at least 1 of said carbohydrate-binding proteins comprises a fluorescent moiety in addition to said mass-tag.
  17. 17 . The method of claim 1 , wherein at least 1 of said carbohydrate-binding proteins is a lectin.
  18. 18 . The method of claim 1 , wherein said mass-tags are non-rare-earth-metal mass-tags.
  19. 19 . The method of claim 1 , wherein said mass-tags comprise a plurality of amino acids.
  20. 20 . The method of claim 1 , wherein said exogenous source of UV light is an LED UV light.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application Ser. No. 63/106,990 entitled “Novel Photocleavable Mass-Tags for Multiplexed Mass Spectrometric Imaging of Tissues using Antibody and Nucleic Acid Probes”, filed Oct. 29, 2020, hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. CA236097 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION 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). BACKGROUND OF THE INVENTION Immunohistochemistry (IHC) and in situ hybridization (ISH) are widely used to determine the structural organization of biomolecules at the tissue, cellular and subcellular level [Katikireddy and O'Sullivan (2011) Methods Mol Biol 784:155-67; Howat and Warford (2014) Methods 70:1-2; Stack, Wang et al. (2014) Methods 70:46-58]. For example, IHC is the preferred method for studying extracellular amyloid plaques and intracellular tau-based neurofibrillary tangles in neurodegenerative disorders [Deng, Bigio et al. (2011) Methods Mol Biol 793:259-72; Dugger and Dickson (2017) Cold Spring Harb Perspect Biol 9]. In oncology, IHC and ISH can be used to diagnose, classify into subtypes and determine optimal treatment of various cancers [Renwick, Cekan et al. (2013) J Clin Invest 123:2694-702; Zaha (2014) World J Clin Oncol 5:382-92], including the evaluation of tumor infiltrating lymphocytes (TILs) which are of prognostic value [Halse, Colebatch et al. (2018) Sci Rep 8:11158]. IHC and ISH analyses are generally performed on tissue samples, for example collected by biopsy or surgical resection of a tumor. Typically, tissue samples are fresh frozen (FF) or formalin-fixed and paraffin embedded (FFPE), and then thin-sectioned (e.g., 10 μm) and mounted onto glass microscope slides. Fluorophores or chromogenic agents conjugated to antibody or nucleic acid probes are the most common methods of visualizing the spatial distribution of targeted biomolecules using microscopy (e.g., protein antigens or genetic material such as miRNA) [Katikireddy and O'Sullivan (2011) Methods Mol Biol 784:155-67]. It is often vital to simultaneously determine the localization and potential co-localization of a number of biomarkers. This is critical in order to map, for example, the location of the hundreds of possible proteins and/or miRNAs involved in cell regulation and dysregulation in a highly heterogeneous tissue [Renwick, Cekan et al. (2014) Methods Mol Biol 1211:171-87; Blom, Paavolainen et al. (2017) Sci Rep 7:15580]. However, fluorescence microscopy is limited to the simultaneous detection of only a few biomarkers, since molecular fluorophores exhibit relatively broad excitation and emission bands, resulting in spectral overlap [Stack, Wang et al. (2014) Methods 70:46-58]. The multiplexing limit of standard fluorescence microscopy is generally 3-5, while hyperspectral/multispectral methods are limited to 8 [Tsurui, Nishimura et al. (2000) J Histochem Cytochem 48:653-62; Stack, Wang et al. (2014) Methods 70:46-58; Parra, Uraoka et al. (2017) Sci Rep 7:13380; Gorris, Halilovic et al. (2018) J Immunol 200:347-354]. Furthermore, these multiplexing methods often require cycling strategies (e.g., Perkin Elmer's OPAL multispectral platform) such as iterative staining followed by photobleaching or probe removal/denaturation [Wahlby, Erlandsson et al. (2002) Cytometry 47:32-41; Schubert, Bonnekoh et al. (2006) Nat Biotechnol 24:1270-8; Gerdes, Sevinsky et al. (2013) Proc Natl Acad Sci USA 110: 11982-7; Blom, Paavolainen et al. (2017) Sci Rep 7: 15580]. Such methods are