Photocleavable mass-tags for multiplexed mass spectrometric imaging of tissues using biomolecular probes
Inventors
Lim, Mark J. • Yagnik, Gargey • Rothschild, Kenneth J.
Assignees
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Publication Number
US-11940447-B2
Publication Date
2024-03-26
Expiration Date
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).
Core Innovation
The invention provides nucleic acid probes conjugated to photocleavable mass-tags, including photocleavable peptide mass-tags, where the mass-tags comprise a core structure conjugated to a mass unit. The core structure includes a PC-Nucleus having a phenyl ring and a photocleavage site, and the nucleic acid probes have the general structure Mass Unit-Core Structure-Nucleic Acid.
A biological sample is contacted with the nucleic acid probes to effect binding to targets in the biological sample. The mass-tags are illuminated with light so as to photocleave at least a portion of the mass-tags prior to detection, and the detected mass-tags, or fragments thereof, are measured as molecular ions using mass spectrometric imaging, including MALDI-MSI.
The disclosure frames PC-MTs as enabling multiplex immunohistochemistry and multiplex in situ hybridization, including high-plex use on a single slide. It also supports multi-omic MSI on the same specimen by combining targeted PC-MT MSI with label-free MSI and/or in situ digestion for bottom-up multi-omic imaging.
Claims Coverage
The independent claims center on nucleic acid probes conjugated to photocleavable mass-tags with a PC-Nucleus phenyl-ring photocleavage core and a mass unit, target binding in a biological sample, photocleavage prior to detection, and mass spectrometric imaging detection of mass-tags or fragments as molecular ions. Dependent features specify aptamers or in situ hybridization, probe architectures that attach a plurality of photocleavable mass-tags, miRNA targeting, and carriers that increase the number of conjugated mass-tags for signal amplification.
Photocleavable PC-nucleus mass-tag architecture for nucleic acid probes
Nucleic acid probes are conjugated to photocleavable mass-tags comprising a core structure conjugated to a mass unit, where the core structure comprises a PC-Nucleus having a phenyl ring and a photocleavage site, arranged as Mass Unit-Core Structure-Nucleic Acid.
Target binding of nucleic acid probes in a biological sample
The biological sample is contacted with the nucleic acid probes to effect binding of the nucleic acid probes to the targets in the biological sample.
Photocleaving photocleavable mass-tags prior to detection
The mass-tags are illuminated with light so as to photocleave at least a portion of the mass-tags prior to detection.
Mass spectrometric imaging detection of mass-tags as molecular ions
Using mass spectrometric imaging, the mass-tags, or fragments thereof, are detected as molecular ions.
Aptamer nucleic acid probe for targeted binding
The nucleic acid probe is an aptamer.
In situ hybridization binding mode
Binding of the nucleic acid probe to the targets is performed by in situ hybridization.
Target-binding sequences plus non-target sequences enabling a plurality of mass-tags
Nucleic acid probes have target-binding sequences that hybridize to the nucleic acid targets and additional sequences not part of the target binding sequence that allow attachment of a plurality of photocleavable mass-tags.
MicroRNA target specificity
The RNA target is microRNA (miRNA).
Carriers increase number of conjugated photocleavable mass-tags for signal amplification
The probes include carriers that increase the number of conjugated photocleavable mass-tags and thereby facilitate further signal amplification.
Overall, the claimed coverage centers on nucleic-acid probe labeling with photocleavable PC-Nucleus phenyl-ring mass-tags, target binding in a biological sample, photocleaving prior to mass spectrometric imaging, and detecting mass-tags or fragments as molecular ions. Dependent refinements specify aptamers or in situ hybridization binding, probe architectures that attach multiple mass-tags, miRNA targeting, and carriers that increase the number of conjugated mass-tags to facilitate signal amplification.
Stated Advantages
Improves aqueous solubility and labeling efficiency through use of non-neutral/ionizable groups in the PC-MT core-structure design.
Increases sensitivity by attaching multiple PC-MTs per probe using carriers that increase the number of conjugated photocleavable mass-tags.
Facilitates signal amplification by increasing the number of conjugated photocleavable mass-tags via carriers.
Preferred 1-step PC-MT probe labeling reaction, contrasted with prior multi-step crosslinker-based labeling.
Reduces tissue loss during MIHC/MISH processing by using gold-coated conductive slides.
Supports improved MSI performance through photocleavage strategy and architecture/orientation considerations that reduce mass spectral byproducts and MS interference.
Enables multiplex detection using photocleavable peptide mass-tags with mass spectrometric imaging.
Supports high-plex multiplex immunohistochemistry and multiplex in situ hybridization on a single slide.
Supports multi-omic MSI on the same specimen by combining targeted PC-MT MSI with label-free MSI and/or in situ digestion for bottom-up multi-omic imaging.
Improves specificity through target-reactive probes and mass-coded detection of mass-tags or fragments as molecular ions.
Documented Applications
Multi-omic tissue imaging, including sequential/dual-MSI.
Dual-MSI for label-free lipids alongside targeted MIHC.
Lectin PC-MT probes for glycan targeting and glycan imaging (N- and O-glycans).
Bottom-up proteomic workflows using PC-MT probes with in situ digestion.
Non-imaging MS applications including MALDI-MS/ESI-MS and diagnostics.
Multiplex immunohistochemistry using photocleavable mass-tags and mass spectrometric imaging.
Multiplex in situ hybridization using photocleavable mass-tags and mass spectrometric imaging.
High-plex profiling on a single slide using PC-MT-tagged probes.
Tumor tissue biomarker analysis, including HER2, ER, PR, and Ki67.
Multi-omic MSI on the same specimen, combining targeted PC-MT MSI with label-free MSI and/or in situ digestion for bottom-up multi-omic imaging.
Detection targeting nucleic acid biomarkers including miRNA and DNA targets.
Immuno-oncology and immune biomarker targeting, including PD-1, PD-L1, CTLA-4, OX40, CD27, TIM3, and T-cell/B-cell/macrophage biomarkers such as CD3, CD4, CD8, CD45RO, CD20, and CD68.
Carbohydrate-binding protein and lectin targets, including WGA, PNA, and PHA-E4.
Use with diverse probe types including aptamers, receptors, ligands, carbohydrate-binding proteins, and nucleic-acid probes.
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