A new optically severable mass tag for tissue multipath mass spectrometry imaging using biomolecular probes

Abstract

Immunohistochemistry (INC) and in situ hybridization (ISH) methods are used for the targeted detection and localization of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for research applications and clinical applications such as those of pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies). These methods include mass spectrometry imaging (MSI) as a mode for detecting and localizing biomolecules in tissues or cells. The methods employ optically cleavable mass-tagged reagents linked to probes such as antibodies and nucleic acids and used to enable multiplexed immunohistochemistry and in situ hybridization, with MSI as the detection / readout mode. The methods also include multi-omics MSI procedures in which MSI of optically cleavable mass-tagged probes is combined with other MSI modes, such as direct label-free MSI of endogenous biomolecules from biological samples (e.g., tissues).

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 106,990, filed October 29, 2020, entitled “Novel Photocleavable Mass-Tags for Multiplexed Mass Spectrometric Imaging of Tissues using Antibody and NucleicAcid Probes,” which is hereby incorporated by reference in its entirety.

[0003] Statement regarding federally funded research or development

[0004] This invention was completed with government support under grant number CA236097 issued by the National Cancer Institute of the United States. The U.S. government holds certain rights to this invention. Invention Field

[0005] The field of this invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH), used for the targeted detection and localization of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for research applications and for clinical applications such as those of pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies). Specifically, mass spectrometry imaging (MSI) is used, for example, as a mode for detecting and localizing biomolecules in tissues or cells. More specifically, the field of this invention relates to optically cleavable mass-tagged reagents attached to probes such as antibodies and nucleic acids and used to enable multiplexed immunohistochemistry and in situ hybridization with MSI as the detection / readout mode. Probe types other than antibodies and nucleic acids are also included in the field of this invention, including but not limited to carbohydrate-binding proteins (e.g., lectins), receptors, and ligands. Finally, the field of this invention also includes multi-omics MSI procedures in which the MSI of optically cleavable mass-tagged probes is combined with other MSI modes, such as direct label-free MSI of endogenous biomolecules from biological samples (e.g., tissues), whereby said biomolecules may be intact or digested (e.g., chemically digested or by enzymatic digestion). Background of the Invention

[0007] Immunohistochemistry (IHC) and in situ hybridization (ISH) are widely used to determine the structure of biomolecules at the tissue, cellular, and subcellular levels [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 a 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 for the diagnosis, subtype classification, and determination of optimal treatment for 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) with prognostic value [Halse, Colebatch et al. (2018) Sci Rep 8:11158]. IHC and ISH analyses are typically performed on tissue samples, such as those 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 thinly sectioned (e.g., 10 μm) and fixed on a microscope glass slide. Fluoresceins or chromogenic agents conjugated to antibodies or nucleic acid probes are the most common method for visualizing the spatial distribution of target biomolecules (e.g., protein antigens or genetic material such as miRNA) using microscopy [Katikireddy and O'Sullivan (2011) Methods Mol Biol 784:155-67].

[0008] Simultaneous localization and potential co-localization of many biomarkers are often crucial. This is essential for, for example, locating the sites of hundreds of potential proteins and / or miRNAs involved in cellular regulation and aberration regulation in highly heterogeneous tissues [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 because molecular fluorophores exhibit relatively broad excitation and emission bands, leading to spectral overlap [Stack, Wang et al. (2014) Methods 70:46-58]. The multipath limit for standard fluorescence microscopy is typically 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 multiplexed methods often require cyclical 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 US A 110:11982-7; Blom, Paavolainen et al. (2017) Sci Rep 7:15580]. Such methods are complex, laborious, and incomplete loops can confound the results [Giesen, Wang et al. (2014) Nat Methods 11:417-22; Blom, Paavolainen et al. (2017) Sci Rep 7:15580].

[0009] In contrast, mass spectrometry imaging (MSI) facilitates high levels of multiplexing without the limitations of the aforementioned optical methods (limited only by mass resolution typically less than 1 Da). In short (see details...) Figure 1These methods use mass spectrometry to scan tissue samples, generating a complete mass spectrum at each “pixel,” thus allowing simultaneous imaging of any given mass of material within the spectrum [Arentz, Mittal et al. (2017) Adv Cancer Res 134:27-66]. This matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) technique was first introduced by the Caprioli research group [Caprioli, Farmer et al. (1997) Anal Chem 69:4751-60], and has since been widely used for direct, label-free imaging of biomolecules, including proteins, nucleic acids, lipids, metabolites, and even small drug compounds in complex tissues [Buchberger, DeLaney et al. (2018) Anal Chem 90:240-265]. This technique has also been extended to other mass spectrometry (MS) schemes, such as ESL-based DESI-MS imaging [Takats, Wiseman et al. (2004) Science 306:471-3]. While the MALDI and DESIMSI schemes currently do not match the spatial resolution of optical methods (e.g., 10 μm laser focusing with the newer Bruker rapifleX MALDI-MS instrument), improved resolution may be achieved using innovative designs such as transport geometry (2 μm) [Zavalin, Todd et al. (2012) J Mass Spectrom 47:i] or atmospheric pressure MALDI-MSI with laser focusing objectives (1.4 μm) [Kompauer, Heiles et al. (2017) Nat Methods 14:90-96].

[0010] However, due to insufficient mass resolution and poor sensitivity, it is generally impossible to perform MSI on intact macromolecules (such as proteins) [Buchberger, DeLaney et al. (2018) Anal Chem 90: 240-265]. The identification of specific biomolecules requires tandem MS / MS fragmentation, ultra-high mass resolution instruments, and / or bottom-up proteomics protocols (e.g., in situ proteolytic digestion of tissues). To overcome this limitation, several targeted MSI protocols have been introduced, which allow for multiplexing workflows similar to conventional IHC and ISH using labeled antibodies and nucleic acid probes. TAMSIM (Targeted Multiplexing Mass Imaging) is a matrix-free laser desorption / ionization (LDI) method that uses antibodies conjugated with small, organic, photocleavable mass tags that are cleaved and ionized during MSI [Thiery, Shchepinov et al. (2007) Rapid Commun Mass Spectrom 21: 823-9]. However, the mass tags are not easily synthesized and only display triple imaging [Thiery, Anselmi et al. (2008) Proteomics 8:3725-34]. Furthermore, those skilled in the art will recognize that co-crystallization of the analyte with an excess of exogenously added matrix compound, which facilitates the absorption of laser energy by the mass spectrometer and the transfer of the analyte to the analyte, is necessary for effective analyte evaporation / ionization and detection, known as matrix-assisted laser desorption / ionization (MALDI) mass spectrometry [Yao, Scott et al. (1998) J Am Soc Mass Spectrom 9:805-13; Duenas, Carlucci et al. (2016) J Am Soc Mass Spectrom 27:1575-8]. Therefore, the TAMSIM method will lack sensitivity.

[0011] In contrast, standard solid-phase synthesis readily generates peptide quality tags, the quality can be easily tuned by altering the sequence, and peptides are typically ionized with high efficiency. Lemaire et al. first introduced a photo-cleavable peptide-based MSI method for tissue-targeted imaging, known as Tag-Mass [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67]. However, quality tagging of probes (e.g., antibodies) is a complex multi-step process involving intermediate chemical linkers. Furthermore, the photo-cleavable core used in peptides provides suboptimal sensitivity. To date, these drawbacks have limited the widespread use of Tag-Mass, and thus only 2-way MSI has been implemented to date [Lemaire, Stauber et al. (2007) J Proteome Res6:2057-67; Franck, Arafah et al. (2009) Mol Cell Proteomics 8:2023-33; El Ayed, Bonnel et al. (2010) Med Sci Monit 16:BR233-45].

[0012] Imaging quality cytometry uses rare-earth metal-labeled antibodies in combination with inductively coupled plasma mass spectrometry (ICP-MS) [Giesen, Wang et al. (2014) Nat Methods 11:417-22]. This protocol has achieved the highest level of multiplexing to date, with at least 32-channel tissue staining. However, this method requires specialized MS instruments and is a destructive protocol that reduces molecules to elements (atomicization) for detection and analysis, and is therefore incompatible with performing non-targeted direct MSI analysis of biomolecules (in combination with targeted MSI using mass-labeled probes, as in Experimental Example 4 of this invention). For a review of an example of ICP-MS, see Wilschefski et al. [Wilschefski and Baxter (2019) Clin Biochem Rev 40:115-133].

[0013] Other drawbacks of the imaging quality cytometry protocol include the complexity of the probe labeling process, which involves preloading the polymer with metal ions, partially reducing the antibody and conjugating the two together, and multiple purifications of the polymer and antibody [Fluidigm, Quick Reference: "Maxpar X8 Antibody Labeling", accessed September 2020, www.fluidigm.com / binaries / content / documents / fluidigm / resources / maxpar-x8-antibody-labeling-quick-reference-fldm-00015-rev01 / maxpar-x8-antibody-labeling-quick-reference-fldm-00015-rev01 / fluidigm%3Afile]. Invention Overview

[0015] This invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for the targeted detection and localization of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for example, for research applications and for clinical applications such as those of pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies). Specifically, mass spectrometry imaging (MSI) is used, for example, as a mode for detecting and localizing biomolecules in tissues or cells. More specifically, the field of this invention relates to optically cleavable mass-tagged reagents attached to probes such as antibodies and nucleic acids and used to enable multiplexed immunohistochemistry and in situ hybridization with MSI as the detection / readout mode.

[0016] In one embodiment, the present invention provides a multiplexed method for co-detecting five or more different types of biomarkers in a tissue sample on a single slide, the method comprising: a) providing a tissue sample on a single slide; b) contacting the tissue sample with five or more different antibodies to achieve binding of the antibodies to the tissue sample, each of the antibodies being capable of reacting with a different biomarker, and each of the antibodies being conjugated to a unique mass tag; and c) detecting the mass tag or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, the method further comprises performing direct mass spectrometry imaging (i.e., tissue imaging on a single sample) on the tissue sample after step a) but before step b). In one embodiment, the five or more antibodies are in a mixture, and the tissue sample in step b) is contacted with the mixture. In one embodiment, the mass tag is a non-rare earth metal mass tag. In one embodiment, the mass tag comprises a plurality of amino acids. In one embodiment, the tissue sample is freshly frozen and sliced ​​before being fixed onto the single slide. In one embodiment, the slide comprises gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tissue sample is fixed in formalin, embedded in paraffin, and sliced ​​before being fixed onto the single slide. In one embodiment, the tissue sample is treated prior to the step of contacting the sample with the antibody, the treatment including deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further treated, the treatment including rehydration. In one embodiment, the rehydration is performed using a series of ethanol / water mixtures and aqueous saline buffers. In one embodiment, the tissue sample is further treated, the treatment including antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer at pH 6. In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the antibody conjugated to the mass tag has the following general structure, where X is a spacer group:

[0017]

[0018] In one embodiment, a matrix compound is applied to the mass tag prior to step c). In one embodiment, the matrix compound is selected from the group consisting of α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tissue sample is treated after the step of contacting the sample with the matrix compound, the treatment including matrix recrystallization. In one embodiment, the mass tag is photocuttable. In one embodiment, the method further includes irradiating the mass tag with light prior to step c) to photocut at least a portion of the mass tag. In one embodiment, the tissue is derived from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, at least one of the five or more antibodies comprises a fluorescent portion in addition to the mass tag. In one embodiment, the number of the different antibodies is eight or more. In one embodiment, a subset of said different antibodies reacts with: i) estrogen receptor (ER), ii) progesterone receptor (PR), iii) human epidermal growth factor receptor 2 (HER2), and iv) Ki67. In one embodiment, a subset of said different antibodies reacts with: i) CD3 (T cells), ii) CD4 (T helper cells), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, one of said different antibodies reacts with the B cell biomarker CD20. In one embodiment, one of said different antibodies reacts with the macrophage biomarker CD68. In one embodiment, one of said different antibodies reacts with an immune checkpoint molecule. In one embodiment, said immune checkpoint molecule is selected from the set of members: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0019] In one embodiment, the present invention provides a multiplexed method for co-detecting human epidermal growth factor receptor 2 (HER2) protein and estrogen receptor (ER) protein in a tissue sample on a single slide, the method comprising: a) providing a tissue sample on a single slide; b) contacting the tissue sample with a HER2 protein-specific antibody conjugated to a first non-rare earth metal mass tag comprising a plurality of amino acids; c) contacting the sample with an ER-specific antibody conjugated to a second non-rare earth metal mass tag comprising a second plurality of amino acids, wherein the second plurality of amino acids have a different mass than the first plurality of amino acids; and d) detecting the mass tag or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, steps b) and c) are performed simultaneously. In one embodiment, direct mass spectrometry imaging of the tissue sample is further included after step a) but before step b). In one embodiment, the tissue sample is freshly frozen and sliced ​​before being fixed onto the single slide. In one embodiment, the slide comprises gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tissue sample is fixed in formalin, embedded in paraffin, and sliced ​​before being fixed onto the single glass slide. In one embodiment, the tissue sample is treated prior to the step of contacting the sample with the antibody, the treatment including deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further treated, the treatment including rehydration. In one embodiment, the rehydration is performed using a series of ethanol / water mixtures and aqueous saline buffers. In one embodiment, the tissue sample is further treated, the treatment including antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer at pH 6. In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the antibody conjugated to the mass tag has the following general structure, where X is a spacer group:

[0020]

[0021] In one embodiment, a matrix compound is applied to the mass tag prior to step d). In one embodiment, the matrix compound is selected from the group consisting of α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tissue sample is treated after the step of contacting the sample with the matrix compound, the treatment including matrix recrystallization. In one embodiment, the mass tag is photocuttable. In one embodiment, the method further includes irradiating the mass tag with light prior to step d) to photocut at least a portion of the mass tag. In one embodiment, the tissue is derived from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the method further includes contacting the tissue with additional different antibodies prior to step d), each of the additional different antibodies being conjugated to a non-rare earth metal mass tag, the mass tag containing multiple amino acids. In one embodiment, the number of the different antibodies is eight or more. In one embodiment, one of the different antibodies reacts with a progesterone receptor (PR). In one embodiment, a subset of the different antibodies reacts with the following T-cell biomarkers: i) CD3 (T cells), ii) CD4 (T helper cells), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, one of the different antibodies reacts with the B-cell biomarker CD20. In one embodiment, one of the different antibodies reacts with the macrophage biomarker CD68. In one embodiment, one of the different antibodies reacts with an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the set of members: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0022] In one embodiment, the present invention provides a multiplexed method for co-detecting different types of tumor-infiltrating immune cells in a tumor on a single slide, the method comprising: a) providing a tumor tissue sample on a single slide; b) contacting the tissue sample with a first antibody specific for a first type of tumor-infiltrating immune cell to achieve binding of the antibody to the tissue sample, the antibody being conjugated to a first non-rare earth metal mass tag comprising a plurality of amino acids; c) contacting the tissue sample with a second antibody specific for a second type of tumor-infiltrating immune cell to achieve binding of the antibody to the tissue sample, the antibody being conjugated to a second non-rare earth metal mass tag comprising a plurality of amino acids; and d) detecting the mass tag or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, steps b) and c) are performed simultaneously. In one embodiment, direct mass spectrometry imaging of the tissue sample is further included after step a) but before step b). In one embodiment, the tissue sample is freshly frozen and sliced ​​before being fixed onto the single slide. In one embodiment, the slide comprises gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tissue sample is fixed in formalin, embedded in paraffin, and sliced ​​before being fixed onto the single slide. In one embodiment, the tissue sample is treated prior to the step of contacting the sample with the antibody, the treatment including deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further treated, the treatment including rehydration. In one embodiment, the rehydration is performed using a series of ethanol / water mixtures and aqueous saline buffers. In one embodiment, the tissue sample is further treated, the treatment including antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer at pH 6. In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the antibody conjugated to the mass tag has the following general structure, where X is a spacer group:

[0023]

[0024] In one embodiment, a matrix compound is applied to the mass tag prior to step d). In one embodiment, the matrix compound is selected from the group consisting of α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tissue sample is treated after the step of contacting the sample with the matrix compound, the treatment including matrix recrystallization. In one embodiment, the mass tag is photocuttable. In one embodiment, the method further includes irradiating the mass tag with light prior to step d) to photocut at least a portion of the mass tag. In one embodiment, the tumor is a lung tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the first antibody is specific for CD3 and the second antibody is specific for CD8. In one embodiment, the method further includes contacting the tissue with another different antibody prior to step d), each of the other different antibodies being conjugated to a non-rare earth metal mass tag, the mass tag containing a plurality of amino acids. In one embodiment, one of the additional different antibodies reacts with CD4. In one embodiment, one of the additional different antibodies reacts with the macrophage biomarker CD68. In one embodiment, one of the additional different antibodies reacts with an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the set of members: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3. In one embodiment, the present invention provides a composition comprising an antibody conjugated to i) a mass tag (via an optically cleavable linker) and ii) a fluorophore. In one embodiment, the antibody reacts with a biomarker selected from the set of members: i) estrogen receptor (ER), ii) progesterone receptor (PR), iii) human epidermal growth factor receptor 2 (HER2), and iv) Ki67. In one embodiment, the antibody reacts with a T-cell biomarker selected from the set of: i) CD3 (T cells), ii) CD4 (T helper cells), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, the antibody reacts with an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the set of: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0025] In one embodiment, the present invention provides a multiplexed method for detecting immune checkpoint molecules on tumor tissue on a single slide, the method comprising: a) providing a tumor tissue sample on a single slide; b) contacting the tissue sample with a mixture comprising a plurality of different antibodies, each different antibody being capable of reacting with a different immune checkpoint molecule, each of the antibodies being conjugated to a non-rare earth metal mass tag, the mass tag comprising a plurality of amino acids; and c) detecting the mass tag or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, wherein one of the plurality of different antibodies reacts with an immune checkpoint molecule selected from a set of members including PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3. In one embodiment, wherein the tumor tissue comprises a biopsy from a human patient. In one embodiment, wherein the biopsy tumor tissue reacts with an antibody targeting PD-L1. In one embodiment, further comprising treating the human patient with an immune checkpoint inhibitor specific to PD-L1. In one embodiment, the PD-L1 specific checkpoint inhibitor is selected from the group consisting of atezolizumab, avelumab, and duvalumab.

[0026] In one embodiment, the present invention provides a multiplexed method for co-detecting three or more (more preferably five or more) different biomarkers in a tissue sample, the method comprising: a) providing a tissue sample; b) contacting the tissue sample with three or more (more preferably five or more) different probes to generate a probed tissue sample, each of the probes being conjugated to a unique mass tag and at least three or more (more preferably five or more) of the probes each being conjugated to a different biomarker in the tissue sample; and c) using mass spectrometry imaging (e.g., tissue imaging performed on a single sample) of the probed tissue sample to detect the unique mass tag or fragments thereof from at least three or more (more preferably five or more) of the conjugated probes, wherein the mass tag is detected as a molecular ion. In one embodiment, the tissue sample is a thin tissue section. In one embodiment, the tissue sample is frozen and sliced ​​prior to step b). In one embodiment, the tissue sample is fixed with formalin and sliced ​​prior to step b). In one embodiment, the tissue sample is fixed in formalin, embedded in paraffin, and sectioned before step b). In one embodiment, the tissue sample is fixed on a glass slide before step b). In one embodiment, the glass slide contains gold. In one embodiment, the glass slide is a glass slide with a gold layer. In one embodiment, the tissue sample is fixed on a glass slide before step c). In one embodiment, the glass slide contains gold. In one embodiment, the glass slide is a glass slide with a gold layer. In one embodiment, the tissue sample is derived from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the five or more probes are in a mixture, and the tissue sample in step b) is contacted with the mixture to produce the probed tissue sample. In one embodiment, the probe conjugated to the mass tag has the following general structure, where X is a spacer base:

[0027]

[0028] In one embodiment, at least one of the five or more probes includes a fluorescent portion in addition to the mass tag. In one embodiment, the probes are selected from the set of proteins and nucleic acids. In one embodiment, at least one of the probes is an antibody. In one embodiment, the antibody is selected from the set of recombinant antibodies, nanobodies, single-strand fragment variable (scFv) antibodies, single-domain antibodies, and VHH single-domain antibodies. In one embodiment, at least one of the probes is selected from the set of affibodies, receptors, and ligands. In one embodiment, at least one of the probes is an aptamer. In one embodiment, at least one of the probes binds to an RNA target. In one embodiment, at least one of the probes binds to a miRNA target. In one embodiment, at least one of the probes binds to a DNA target. In one embodiment, the mass tag is a non-rare earth metal mass tag. In one embodiment, the mass tag contains multiple amino acids. In one embodiment, the mass tag is photocleavable. In one embodiment, the method further includes irradiating the mass tag with light prior to step c) to photocut at least a portion of the mass tag. In one embodiment, the method further includes mass spectrometry imaging of the tissue sample after step a) but before step b). In one embodiment, a matrix compound is applied to the tissue sample to be probed prior to step c). In one embodiment, the matrix compound is selected from the group consisting of α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tissue sample to be probed is treated after the step of applying the matrix compound to the tissue sample to be probed, the treatment comprising matrix recrystallization. In one embodiment, the tissue sample is treated after paraffin embedding and before step b), the treatment comprising deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further treated, the treatment comprising rehydration. In one embodiment, the rehydration is performed using a series of ethanol / water mixtures and aqueous saline buffers. In one embodiment, the tissue sample is further processed, including antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer at pH 6.In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the number of the different probes is 10 or more. In one embodiment, a subset of the different probes is capable of binding i) estrogen receptor (ER), ii) progesterone receptor (PR), iii) human epidermal growth factor receptor 2 (HER2), and iv) Ki67. In one embodiment, a subset of the different probes is capable of binding T-cell biomarkers i) CD3 (T-cells), ii) CD4 (T-helper cells), iii) CD8 (cytotoxic T-cells), and iv) CD45RO (memory T-cells). In one embodiment, one of the different probes is capable of binding the B-cell biomarker CD20. In one embodiment, one of the different probes is capable of binding the macrophage biomarker CD68. In one embodiment, one of the different probes is capable of binding an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the set of members: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0029] In one embodiment, the present invention provides a multiplexed method for co-detecting different hormone receptors in a tissue sample on a single slide, the method comprising: a) providing a tissue sample immobilized on a single slide; b) contacting the tissue sample with a first probe capable of binding a first hormone receptor, the probe being conjugated to a first non-rare earth metal mass tag comprising a plurality of amino acids; c) contacting the tissue sample with a second probe capable of binding a second hormone receptor, the probe being conjugated to a second non-rare earth metal mass tag comprising a second plurality of amino acids, wherein the second hormone receptor is different from the first hormone receptor, and the second plurality of amino acids have a different mass than the first plurality of amino acids, and wherein steps b) and c) together produce a tissue sample to be detected; and d) detecting at least one of the mass tags or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, the tissue sample is a thin tissue section. In one embodiment, the tissue sample is frozen and sliced ​​thinly before being immobilized on the slide. In one embodiment, the tissue sample is fixed and sliced ​​thinly with formalin before being immobilized on the slide. In one embodiment, the tissue sample is fixed in formalin, embedded in paraffin, and sliced ​​thinly before being immobilized on the slide. In one embodiment, the slide contains gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tissue sample is derived from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the probe is conjugated to a non-rare earth metal mass tag having the following general structure, where X is a spacer base:

[0030]

[0031] In one embodiment, at least one of the probes includes a fluorescent portion in addition to the non-rare earth metal mass tag. In one embodiment, the probe is selected from the set of proteins and nucleic acids. In one embodiment, at least one of the probes is an antibody. In one embodiment, the antibody is selected from the set of recombinant antibodies, nanobodies, single-strand fragment variable (scFv) antibodies, single-domain antibodies, and VHH single-domain antibodies. In one embodiment, at least one of the probes is selected from the set of affibodies, receptors, and ligands. In one embodiment, at least one of the probes is an aptamer. In one embodiment, at least one of the probes is capable of binding an RNA target. In one embodiment, at least one of the probes is capable of binding a miRNA target. In one embodiment, at least one of the probes is capable of binding a DNA target. In one embodiment, the non-rare earth metal mass tag is photocleavable. In one embodiment, the method further includes irradiating the non-rare earth metal mass tag with light prior to step d) to photocleave at least a portion of the non-rare earth metal mass tag. In one embodiment, steps b) and c) are performed simultaneously to generate the tissue sample to be probed. In one embodiment, the tissue sample is further subjected to mass spectrometry imaging after step a) but before step b). In one embodiment, a matrix compound is applied to the tissue sample to be probed before step d). In one embodiment, the matrix compound is selected from the group consisting of α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tissue sample to be probed is treated after the step of applying the matrix compound to the tissue sample to be probed, the treatment comprising matrix recrystallization. In one embodiment, the tissue sample is treated after paraffin embedding and before step b), the treatment comprising deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further treated, the treatment comprising rehydration. In one embodiment, the rehydration is performed using a series of ethanol / water mixtures and aqueous saline buffers. In one embodiment, the tissue sample is further processed, including antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer at pH 6.In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the first probe is capable of binding to the estrogen receptor (ER) and the second probe is capable of binding to the progesterone receptor (PR). In one embodiment, the process further includes contacting the tissue sample with additional different probes prior to step d), each of the additional different probes being conjugated to a different non-rare earth metal mass tag, the mass tag containing multiple amino acids and wherein each mass tag has a different mass. In one embodiment, the number of probes is 10 or more. In one embodiment, a subset of the additional different probes is capable of binding i) human epidermal growth factor receptor 2 (HER2) and ii) Ki67. In one embodiment, a subset of the additional different probes is capable of binding T-cell biomarkers i) CD3 (T-cells), ii) CD4 (T-helper cells), iii) CD8 (cytotoxic T-cells), and iv) CD45RO (memory T-cells). In one embodiment, one of the additional different probes is capable of binding the B-cell biomarker CD20. In one embodiment, one of the additional different probes is capable of binding the macrophage biomarker CD68. In one embodiment, one of the additional different probes is capable of binding to an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the set of members including PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0032] In one embodiment, the present invention provides a multiplexed method for co-detecting different types of tumor-infiltrating immune cells in tumor tissue on a single glass slide, the method comprising: a) providing a tumor tissue sample immobilized on a single glass slide; b) contacting the tumor tissue sample with a first probe capable of binding to a first tumor-infiltrating immune cell type, the probe being conjugated to a first non-rare-earth metal mass tag comprising a plurality of amino acids; c) contacting the tumor tissue sample with a second probe capable of binding to a second tumor-infiltrating immune cell type, the probe being conjugated to a second non-rare-earth metal mass tag comprising a second plurality of amino acids, wherein the second tumor-infiltrating immune cell is a different type from the first tumor-infiltrating immune cell, and the second plurality of amino acids have a different mass than the first plurality of amino acids, and wherein steps b) and c) together produce the tumor tissue sample to be detected; and d) detecting at least one of the mass tags or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, the tumor tissue sample is a thin tissue slice. In one embodiment, the tumor tissue sample is frozen and sliced ​​thinly before being immobilized on the glass slide. In one embodiment, the tumor tissue sample is formalin-fixed and sliced ​​thinly before being immobilized on the slide. In one embodiment, the tumor tissue sample is formalin-fixed, paraffin-embedded, and sliced ​​thinly before being immobilized on the slide. In one embodiment, the slide comprises gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tumor tissue sample is derived from a breast tumor. In one embodiment, the probe is conjugated to a non-rare earth metal mass tag having the following general structure, where X is a spacer base:

[0033]

[0034] In one embodiment, at least one of the probes includes a fluorescent portion in addition to the non-rare earth metal mass tag. In one embodiment, the probe is selected from the set of proteins and nucleic acids. In one embodiment, at least one of the probes is an antibody. In one embodiment, the antibody is selected from the set of recombinant antibodies, nanobodies, single-strand fragment variable (scFv) antibodies, single-domain antibodies, and VHH single-domain antibodies. In one embodiment, at least one of the probes is selected from the set of affibodies, receptors, and ligands. In one embodiment, at least one of the probes is an aptamer. In one embodiment, at least one of the probes is capable of binding an RNA target. In one embodiment, at least one of the probes is capable of binding a miRNA target. In one embodiment, at least one of the probes is capable of binding a DNA target. In one embodiment, the non-rare earth metal mass tag is photocleavable. In one embodiment, the method further includes irradiating the non-rare earth metal mass tag with light prior to step d) to photocleave at least a portion of the non-rare earth metal mass tag. In one embodiment, steps b) and c) are performed simultaneously to generate the tumor tissue sample to be probed. In one embodiment, the tumor tissue sample is further subjected to mass spectrometry imaging after step a) but before step b). In one embodiment, a matrix compound is applied to the tumor tissue sample to be probed before step d). In one embodiment, the matrix compound is selected from the group consisting of α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tumor tissue sample to be probed is treated after the step of applying the matrix compound to the tumor tissue sample, the treatment including matrix recrystallization. In one embodiment, the tumor tissue sample is treated after paraffin embedding and before step b), the treatment including deparaffinization. In one embodiment, the deparaffinization is performed with xylene. In one embodiment, the tumor tissue sample is further treated, the treatment including rehydration. In one embodiment, the rehydration is performed using a series of ethanol / water mixtures and aqueous saline buffers. In one embodiment, the tumor tissue sample is further processed, including antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer at pH 6.In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the first probe is capable of binding CD4 and the second probe is capable of binding CD8. In one embodiment, the process further includes contacting the tumor tissue sample with additional different probes prior to step d), each of the additional different probes being conjugated to a different non-rare earth metal mass tag, the mass tag containing multiple amino acids and wherein each mass tag has a different mass. In one embodiment, the number of probes is 10 or more. In one embodiment, one of the additional different probes is capable of binding the T-cell biomarker CD3. In one embodiment, one of the additional different probes is capable of binding the macrophage biomarker CD68. In one embodiment, one of the additional different probes is capable of binding an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the set of members: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0035] In one embodiment, the present invention provides a composition comprising a probe conjugated to i) a mass tag (via an optically cleavable connector) and ii) a fluorophore. In one embodiment, the probe is capable of binding a biomarker selected from the set of: i) estrogen receptor (ER), ii) progesterone receptor (PR), iii) human epidermal growth factor receptor 2 (HER2), and iv) Ki67. In one embodiment, the probe is capable of binding a T-cell biomarker selected from the set of: i) CD3 (T-cells), ii) CD4 (T-helper cells), iii) CD8 (cytotoxic T-cells), and iv) CD45RO (memory T-cells). In one embodiment, the probe is capable of binding an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the set of: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0036] In one embodiment, the present invention provides a multiplexed method for detecting different immune checkpoint molecules in tumor tissue on a single slide, the method comprising: a) providing a tumor tissue sample on a single slide; b) contacting the tumor tissue sample with a mixture comprising a plurality of different probes, each different probe capable of binding to a different immune checkpoint molecule, each of the probes being conjugated to a non-rare earth metal mass tag, the mass tag comprising a plurality of amino acids; and c) detecting at least one of the mass tags or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, wherein one of the plurality of different probes is capable of binding to an immune checkpoint molecule selected from a set of members including PD-1, PD-L1, PDL2, CTLA-4, OX40, CD27, and TIM3. In one embodiment, wherein the tumor tissue sample comprises a biopsy from a human patient. In one embodiment, wherein the tumor tissue biopsy is conjugated with a probe targeting a PD-L1 mass tag. In one embodiment, further comprising treating the human patient with an immune checkpoint inhibitor specific to PD-L1. In one embodiment, the PD-L1 specific checkpoint inhibitor is selected from the group consisting of atezolizumab, avelumab, and duvalibumab. In one embodiment, at least one of the probes is an antibody. In one embodiment, the 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.

[0037] In one embodiment, the present invention provides a multiplexed method for co-detecting three or more (more preferably five or more) different types of carbohydrates in a tissue sample, the method comprising: a) providing a tissue sample; b) contacting the tissue sample with three or more (more preferably five or more) different carbohydrate-binding proteins to achieve binding of the carbohydrate-binding proteins to the tissue sample to produce a tissue sample to be detected, each of the carbohydrate-binding proteins being capable of reacting with a different carbohydrate and each of the carbohydrate-binding proteins being conjugated to a unique mass tag; and c) using mass spectrometry imaging of the tissue sample to be detected to detect the mass tag or fragments thereof as molecular ions. In one embodiment, the tissue sample is a thin tissue section. In one embodiment, the tissue sample is frozen and sectioned prior to step b). In one embodiment, the tissue sample is fixed with formalin and sectioned prior to step b). In one embodiment, the tissue sample is fixed with formalin, embedded in paraffin, and sectioned prior to step b). In one embodiment, the tissue sample is fixed on a glass slide prior to step b). In one embodiment, the slide comprises gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tissue sample is fixed onto the slide prior to step c). In one embodiment, the slide comprises gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tissue sample is derived from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the carbohydrate-binding protein conjugated to the mass tag has the following general structure, wherein X is a spacer and CBP is a carbohydrate-binding protein:

[0038]

[0039] In one embodiment, the three or more (more preferably five or more) carbohydrate-binding proteins are in a mixture, and the tissue sample in step b) is contacted with the mixture to generate the probed tissue sample. In one embodiment, the mixture further contains one or more probes, each of which is reactive with a different target within the tissue, and each of the probes is conjugated to a unique mass tag. In one embodiment, at least one of the five or more carbohydrate-binding proteins contains a fluorescent portion in addition to the mass tag. In one embodiment, at least one of the five or more carbohydrate-binding proteins is a lectin. In one embodiment, the mass tag is a non-rare earth metal mass tag. In one embodiment, the mass tag contains multiple amino acids. In one embodiment, the mass tag is photocuttable. In one embodiment, the method further includes irradiating the mass tag with light prior to step c) to photocut at least a portion of the mass tag.

[0040] In one embodiment, the present invention provides a multi-omics method for detecting different biomolecules or fragments thereof in the same tissue sample, the method comprising: a) providing a tissue sample; b) applying a digesting agent to at least a portion of the tissue sample; c) performing mass spectrometry imaging of the tissue sample; d) contacting the tissue sample with three or more (more preferably five or more) different probes to achieve binding of the probes to the tissue sample to produce a probed tissue sample, each of the probes being reactive with a different target within the tissue sample, and each of the probes being conjugated to a unique mass tag; and e) using mass spectrometry imaging of the probed tissue sample to detect the mass tag or fragment thereof as a molecular ion. In one embodiment, the digesting agent is an enzyme. In one embodiment, the enzyme is selected from the group consisting of nucleases, proteases, kinases, phosphatases, and glycosidases. In one embodiment, the digesting agent is a chemical. In one embodiment, the chemical is selected from the group consisting of cyanogen bromide (CNBr) and hydroxylamine. In one embodiment, a mixture of different digesting agents is alternatively applied in step b). In one embodiment, steps b) and c) are alternatively performed after step e). In one embodiment, after step e) and before the step of applying the digestion reagent to the tissue sample, the tissue sample is further processed, the processing including detaching the bound probe from the tissue sample. In one embodiment, the detachment of the bound probe includes denaturing the tissue sample. In one embodiment, the denaturing treatment is selected from the group consisting of: dissociation agents, solutions with pH ≤ 5, solutions with pH ≥ 10, reducing agents, oxidizing agents, heat, organic solvents, and detergents. In one embodiment, the tissue sample is a thin tissue section. In one embodiment, the tissue sample is frozen and sectioned before step b). In one embodiment, the tissue sample is fixed with formalin and sectioned before step b). In one embodiment, the tissue sample is fixed with formalin, embedded in paraffin, and sectioned before step b). In one embodiment, the tissue sample is fixed on a glass slide before step b). In one embodiment, the glass slide contains gold. In one embodiment, the slide is a glass slide with a gold layer. In one embodiment, the tissue sample is derived from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the probe attached to the mass tag has the following general structure, where X is a spacer base:

[0041]

[0042] In one embodiment, the three or more (more preferably five or more) probes are in a mixture and the tissue sample in step d) is contacted with the mixture to generate the probed tissue sample. In one embodiment, at least one of the five or more probes includes a fluorescent portion in addition to the mass tag. In one embodiment, the probes are selected from the set of proteins and nucleic acids. In one embodiment, at least one of the protein probes is selected from the set of antibodies, recombinant antibodies, affibodies, nanobodies, single-strand fragment variable (scFv) antibodies, single-domain antibodies, VHH single-domain antibodies, receptors, ligands, and carbohydrate-binding proteins. In one embodiment, at least one of the nucleic acid probes is an aptamer. In one embodiment, at least one of the nucleic acid probes binds to an RNA target. In one embodiment, at least one of the nucleic acid probes binds to a miRNA target. In one embodiment, at least one of the nucleic acid probes binds to a DNA target. In one embodiment, the mass tag is a non-rare earth metal mass tag. In one embodiment, the mass tag contains multiple amino acids. In one embodiment, the quality label is photocuttable. In another embodiment, the method further includes illuminating the quality label with light prior to step e) to photocut at least a portion of the quality label.

[0043] In one embodiment, the present invention provides a composition comprising a general chemical structure:

[0044]

[0045] Wherein A is a first linker unit (also called a first spacer unit) comprising at least one amine, X is a protecting group covalently bonded to the nitrogen atom of the amine, and B is a second linker unit (also called a second spacer unit). In one embodiment, the protected amine is an Fmoc-protected amine. In one embodiment, the composition has the following chemical structure:

[0046]

[0047] In one embodiment, the first and / or second connector (or the first and / or second spacer unit) comprises polyethylene glycol. In another embodiment, the first and / or second connector (or the first and / or second spacer unit) comprises a 2,2'-(ethylenedioxy)-di-(ethylamine) chemical connector. Brief description of the attached diagram

[0049] The patent or application document contains at least one color drawing. A copy of the published text of the patent or patent application with the color drawing will be provided by the patent office upon request and payment of the necessary fees.

[0050] Figure 1 Bottom-up application of mass spectrometry imaging (MSI) for multipath direct label-free localization of analytes in tissue samples. Adapted from [Arentz, Mittal et al. (2017) Adv Cancer Res 134:27-66]. This existing approach is multipath-based but inherently untargeted because it does not use labeled probes such as optically cleavable mass-labeled probes (PC-MT probes) and therefore requires complex methods for identifying detected analytes, which in some cases can also reduce the spatial resolution of the images (e.g., due to the need for in situ tissue proteolysis for protein detection and identification).

[0051] Figure 2A -C. Basic design and application of preferred optically severable mass tags (PC-MT) and optically severable mass marker probes (PC-MT-probes). Figure 2A Preferred peptide-based PC-MTs are used. The curves represent peptide-based mass units, the ellipses are optically cleavable linkers (PC-linkers), and "NHS" represents the probe reactive portion, shown in this embodiment as the primary amine reactive NHS-ester leaving group. It should be noted that the peptide-based PC-MT is N-terminally blocked / protected (e.g., acetylated - not depicted) to prevent self-reaction / polymerization of the PC-MT in the presence of primary amine reactivity in the probe reactive portion (and to avoid the internal primary amine, such as the ε-amine on the lysine amino acid, being or protected / blocked). Mass encoding of the mass units is achieved using different amino acids (or their isotopes, analogs, derivatives, or modifications, including natural and non-natural amino acids). It should be noted that a fluorophore (a starburst shape with "F") is optionally included on the PC-MT, which can assist method development by allowing direct comparison of mass spectrometry imaging (MSI) results with conventional fluorescence imaging. Figure 2B PC-MT is linked to probes such as antibodies or amine-modified nucleic acids to generate PC-MT probes. This is typically achieved through a one-step chemical reaction between the probe-reactive portion of the PC-MT (e.g., the NHS-ester leaving group lost in this case) and the probe, followed by purification or unpurified PC-MT probe. Figure 2CThe PC-MT probe is then used to “stain” the tissue (i.e., the PC-MT probe binds to the target in the tissue), a procedure similar to conventional immunohistochemistry (IHC) or in situ hybridization (ISH), followed by MSI. Photodissection (releasing a quality reporter) can be achieved prior to MSI via exogenous ultraviolet treatment, or online using the instrument’s laser beam with matrix-assisted laser desorption / ionization MSI (MALDI-MSI). It should be noted that in some embodiments, the quality reporter may contain a portion of the photodissected PC connector.

[0052] Figure 3Detailed chemical structures and uses of preferred optically cleavable linkers (PC-linkers), optically cleavable mass tags (PC-MTs), and optically cleavable mass-tagged probes (PC-MT-probes). In conventional Fmoc-based solid-phase peptide synthesis (SPPS) processes, Fmoc-PC-linkers (step 1) are incorporated into peptide-based PC-MTs along with other amino acids (or their isotopes, analogs, derivatives, or modifications, including natural and non-natural amino acids). The Fmoc-PC-linker contains optional linker units, which can have various chemical compositions. The illustrated PC-MT is oriented with the N-terminus on the left and the C-terminus on the right. “APRLRFYSL” is an example amino acid sequence of the mass unit. The PC-MT contains an optional spacer unit, shown as a portion of the PC-linker plus the GSGGK amino acid sequence as an example. The PC-MT also contains optional mass unit linkers. The spacer unit and mass unit linkers can have various chemical compositions. The optically cleavable core (PC-core) of the PC-linker is a rapid and efficient 1-(2-nitrophenyl)-ethyl-based moiety. The probe reactive portion (e.g., shown as an NHS-ester leaving group) can be generated, for example, on the ε-amine of the lysine amino acid contained therein (or can be generated, for example, on a C-terminal carboxylic acid group – not depicted). “Ac” is the N-terminal acetylation of the α-amine used to prevent self-reaction / polymerization of the PC-MT shown. (Step 2) The PC-MT is reacted with a probe such as an antibody (shown) or an amine-modified nucleic acid (not shown) to form a PC-MT-probe. In the described embodiment, the NHS-ester leaving group of the PC-MT (the probe reactive portion) is reacted with a primary amine on the antibody (NHS ester is lost and an amide bond is formed). The core structure of the PC-MT-probe is defined as the portion of the PC-MT that connects the mass unit to the probe. This is generally the common structure of all PC-MT-probe materials, corresponding to the common structure of all PC-MT materials. (Step 3) Finally, the PC-MT-probe binds to a target in the tissue, followed by photodissection (to release the mass reporter) and mass spectrometry imaging (MSI) (MSI not depicted). It should be noted that, in this embodiment, a small residual portion of the PC-connector (mass unit connector) is retained as part of the photocut mass reporter, thereby generating primary amine groups in this embodiment, which may contribute to ionization in positive mode MSI.

[0053] Figure 4 A comparison of the structure of the example PC-connector of the present invention with that used in the work of Lemaire et al. For details of the work of Lemaire et al., see [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67] and U.S. Patent No. 8,221,972. The PC-connector is shown as being incorporated into the example peptide sequence.

[0054] Figure 5A -B. Example method for PC-MT signal amplification. Figure 5A Multiple PC-MTs, such as multiple PC-MTs with probe reactive moieties based on NHS-esters, can be conjugated to the surface of amine-terminated gold nanoparticles (“NP”; shown) or dendritic polymers (not shown). The dendritic polymer or NP can then be conjugated to a probe (depicted as an “antibody probe”). Figure 5B For nucleic acid probes, primary amine-modified nucleic acid sequences (which are preferably not part of the “target-binding” sequence (i.e., the target hybridization sequence)) can be used to facilitate the ligation of many PC-MTs for each probe molecule (e.g., where the probe reactive portion is primary amine reactive).

[0055] Figure 6 15-way MSI based on PC-MT-Ab using a bead-array as a model system. Fifteen different forms of streptavidin-resistant PC-MT-Ab were generated through direct one-step labeling using 15 novel amine-reactive NHS-activated PC-MT reagents. The PC-MT-Abs were used to detect 20 μm polymer streptavidin microbeads. The microbeads were then aggregated and used to form an array in the footprint of a microscope slide, followed by MALDI-MSI. The inset shows color-coded 15-way MALDI-MS “quality images” of representative regions of the bead array. The color-coded overlapping spectra are from representative individual microbeads within the quality images of each of the 15 different PC-MT-Ab-probe forms detected (black arrows indicate different PC-MT quality reporter peaks). It should be noted that although the 1-Da naturally occurring isotopes of the peptide PC-MT are readily resolveable by MALDI-MS, they are not visible in the provided spectra due to the compact X-axis scale depiction.

[0056] Figure 7A -E. 5-way MIHC using PC-MT-Ab on mouse brain FFPE tissue sections. FFPE tissue sections were simultaneously stained with PC-MT-Ab against five different protein targets and then subjected to MALDI-MSI. For MSI, matrix application was performed by sublimation followed by recrystallization (MSI in cation reflectance mode). Adjacent tissue sections were also subjected to standard immunofluorescence staining. Figure 7A Color-coded 5-way overlapping MALDI-MSI “mass images” of synaptic proteins (blue), MAP-2 (orange), NeuN (green), myelin (red), and Glut-1 (cyan). Different colors are associated with different m / z values ​​of the monoisotope PC-MT mass reporter spectral peaks. In other structures, the hippocampus (*) and cerebellum were observed. ( Figure 7B Color immunofluorescence overlay of cerebellum showing synaptic proteins (blue), NeuN (green), and myelin (red). Immunofluorescence was performed in "single-path" mode on adjacent tissue sections and overlay images. Figure 7C This shows a standalone MALDI-MS quality image (sub-region) of the less prominent Glut-1 biomarker (reddish-purple in this case), which primarily detects capillaries in the brain (cross-section). Figure 7D The corresponding immunofluorescence of Glut-1 (in this case, it is green). Figure 7E The color-coded overlapping MALDI-MS spectra show selected pixels from the 5-way quality image (pixels indicated by color-coded arrows in inset a). The black arrows in the spectra indicate the m / z peaks of the PC-MT quality reporter. It should be noted that although the 1-Da isolated natural isotopes of the peptide PC-MT are readily resolveable by MALDI-MS, they are not visible in the provided spectra due to the compact X-axis scale depiction.

[0057] Figure 8 MISH using PC-MT directly conjugated to oligonucleotide probes. In situ hybridization was performed on mouse sagittal brain tissue sections using a U6 snRNA probe (positive control) and a plant-specific miR-159 probe (negative control). To compare conventional “FISH” with “MISH”, fluorescence images (red) and mass images (green) corresponding to the m / z values ​​of the monoisotope PC-MT mass reporter material peaks are shown. Yellow arrows highlight cerebellar and hippocampal features detected using U6.

[0058] Figure 9A -D. Untargeted small molecule MALDI-MSI and large molecule MALDI-MSI based on PC-MT-Ab in the same tissue section. Figure 9A First, unfixed fresh frozen mouse sagittal brain tissue sections were subjected to direct, non-targeted MALDI-MSI. Color MALDI-MS images (red, green, and blue, respectively) show three well-known lipid substances (thioglycosides, m / z 888.7; phosphatidylinositol, m / z 885.4; and phosphatidylethanolamine, m / z 790.5). Figures 9B-9C Then, the same tissue sections were processed for a second round of MALDI-MSI. For this purpose, the matrix was flushed away, the tissue was fixed, and targeted multipath MALDI-MSI of macromolecular antigens was performed using PC-MT-Ab. For confirmatory purposes, images of selected biomolecules from the first and second rounds of MALDI-MSI were overlaid. Figure 9BImages of thioglycosides (red) from the first round of MALDI-MSI (direct small molecule detection) overlap with images of NeuN (green) from the second round of MALDI-MSI (multi-channel MIHC). Figure 9C Images of thioglycosides (red) from the first round of MALDI-MSI (direct small molecule detection) overlap with images of myelin basic protein (green) from the second round of MALDI-MSI (multi-channel MIHC). Figure 9D The image shows an example of overlapping spectra from the first round of MALDI-MSI (Direct Small Molecule Detection), color-coded with... Figure 9A The images in the image are matched (indicating three lipid qualities). Figure 9A The color-coded arrows in the image indicate the region from which the MALDI-MS spectrum originates.

[0059] Figure 10A -B. Preferred connection sites between mass units and the PC-core. Figure 10A This shows two configurations where the mass unit is ultimately attached to the benzene ring of the PC-nucleus (in this case, the case after photocutting is depicted; for an example of configuration 1 before photocutting, see...). Figure 3 Step 2). In this embodiment, a photocleavable peptide is used and ultimately ligated to the surface, as shown in the figure (similar to a PC-MT probe ultimately ligated to a tissue surface). In preferred configuration 1, the photocleaved benzene ring of the PC-core (indicated by *) does not remain ligated to the photocleaved mass reporter, as measured by mass spectrometry (MS), while in configuration 2, it is ligated. Figure 10B Overlapping mass spectra were obtained after optically cutting the surfaces of the two configurations of the mass reporter. For each configuration, the expected monoisotope peaks of the mass reporter are labeled with their respective m / z values.

[0060] Figure 11A -L. Comparison of different PC connectors and comparison of pre-optical cutting with in-line optical cutting using a MALDI-MS laser beam. Figure 11A Examples of microbead arrays include MALDI-MSI quality images, where the microbeads are double-marked with PC-MT (red) and PC-MT-L (green), each containing quality unit 1. (However, since only the PC-MT leaves a small remnant of the light-cut PC-connector attached to the quality report, the quality report can be distinguished by a difference of 43 m / z.) Figures 11B-11IFour different microbead materials were prepared, each double-labeled with PC-MT and PC-MT-L containing the same mass units (mass units 1-4 in Table 1). Four separate microbead arrays (one array per microbead material) were formed from each of these materials, and each array was pre-irradiated with UV for 5 minutes for photocutting. Another four microbead arrays were formed and pre-irradiated with UV for 25 minutes. MALDI-MSI was then performed on a total of eight separate microbead arrays. Individual pixels from each array showed representative spectra (each of the eight spectra contained peaks corresponding to mass reporters from PC-MT and PC-MT-L containing the same mass units). Figure 11J Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, each containing mass unit 1. This probe was used for immunostaining of mouse brain tissue sections, followed by MALDI-MSI. Color-coded quality images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is green. Pre-UV conditions of 0, 5, and 25 minutes were tested, where pre-UV was performed immediately before matrix application and before MALDI-MSI analysis. With 0-minute pre-UV, photocutting could only occur during MALDI-MSI analysis using the instrument's laser beam. Spectra of representative pixels are shown below. Figure 11K and Figure 11L As shown.

[0061] Figure 12 Immunohistochemistry based on mass spectrometry for fluorescent PC-MT. The "Fluorescence" inset shows fluorescence images (yellow) of three sagittal mouse brain tissue sections detected with fluorescent anti-NeuN Fluor-PC-MT1, non-fluorescent anti-NeuN PC-MT7, and negative control fluorescent anti-Cas9 Fluor-PC-MT1, respectively. The "MALDI-MSI" inset shows MALDI-MS images of the tissue sections with the same detection (red represents the quality reporter from Fluor-PC-MT1, and green represents the quality reporter from PC-MT7). Blue arrows indicate the hippocampus, and white arrows indicate the cerebellum. The inset spectra are from representative single pixels within the hippocampus, and the corresponding quality reporter peaks are indicated by black arrows when detected.

[0062] Figure 13A -E. Recombinant antibodies compared to conventional antibodies. 37 μm streptavidin PMMA microbeads were directly conjugated to PC-MT (microbead ID-tag), followed by loading of biotinylated protein G, and then binding-specific PC-MT-Abs (each PC-MT-Ab substance was loaded onto a specific microbead substance). Figure 13AMALDI-MSI mass image of microbead cell 1. The peak intensities of the mass spectra for various PC-MTs are colored as shown above the images. Microbeads with ID-tags 9, 10, and 15 were loaded with rAnti-MBP1, anti-MBP1, and rAnti-AB2, respectively (note that the numbers indicate mass cells from Table 1 in a specific PC-MT). Figure 13B )exist( Figure 13A The three overlapping spectra of three individual pixels within the three microbeads circled in () are shown. Based on ( Figure 13A The colors of the microbeads observed in the study were used to color-code the spectrum. Figure 13C MALDI-MSI mass images of microbead cell 2. The peak intensities of the mass spectra for various PC-MTs are colored as shown above the images. Microbeads with ID-tags 9, 10, and 15 were loaded with rAnti-NeuN7, anti-NeuN7, and rAnti-AB2, respectively (note that the numbers indicate mass cells from Table 1 in a specific PC-MT). Figure 13D )exist( Figure 13C The three overlapping spectra of three individual pixels within the three microbeads circled in () are shown. Based on ( Figure 13C The colors of the microbeads observed in the study were used to color-code the spectrum. Figure 13E Separately, mouse sagittal brain sections were stained with anti-NeuN7 and rAnti-NeuN7 and subjected to MALDI-MSI. PC-MT7 from the antibodies is stained green in the images. White arrows indicate the cerebellum, blue arrows indicate the hippocampus, and yellow arrows indicate dotted nucleus staining patterns. In the provided spectra, the strongest pixels from the hippocampus in both images were selected, with red traces indicating PC-MT7 from the “recombinant” rAnti-NeuN7 antibody probe and blue traces indicating PC-MT7 from the “conventional” non-recombinant anti-NeuN7 antibody probe.

[0063] Figure 14A -D. Multipath mass spectrometry-based immunohistochemistry (MIHC) of 12 biomarkers in human tonsil FFPE tissue sections. MIHC was performed as shown in Figure 7, except that 12 different biomarkers were used (see Table 1 for PC-MT assignment for each antibody). Furthermore, pan-cytokeratin antibody (CK) was labeled with PC-MT and fluorophores. Figure 14A CK immunofluorescence images of whole tonsil tissue sections taken at 5-micron resolution using a GenePix 4200A fluorescence microarray scanner. Figure 14B MALDI-MS images of the same tissue sections, showing intensity maps of the single isotopic m / z values ​​of PC-MT from the CK antibody (all MALDI-MS images are at 10 μm spatial resolution). Figure 14CMulticolor MALDI-MS images of selected biomarkers from whole tissue sections displaying differential structural patterns are overlaid. Color coding is indicated in the legend below the images. Figure 14D Individual MALDI-MS images of all 12 biomarkers are shown as gradient colors of representative subregions of tissue sections (biomarker identities indicated by markers). A “blank” is also shown, which is an adjacent tissue section stained with isotype control IgG with PC-MT (the same PC-MT as CD3). Gradient color bars are shown at the bottom. For comparison, the display bars for all biomarkers are set to a full intensity threshold of 25 (arbitrary peak intensity units) and a minimum display intensity of 2.5, except for CK, CD20, and Ki67, which produce particularly strong signals and are therefore set to 50 and 5, respectively.

[0064] Figure 15A -D. Multipath mass spectrometry-based immunohistochemistry (MIHC) of 12 biomarkers in human breast cancer FFPE tissue sections. MIHC was performed as shown in Figure 14 (see Table 1 for PC-MT allocation of each antibody). Figure 15A Multicolor MALDI-MS images of breast cancer tissue sections are overlaid, showing intensity maps of single isotope m / z values ​​of PC-MT from selected antibodies, revealing differential structural patterns (all MALDI-MS images are at 10 μm spatial resolution). Color coding is indicated in the legend above the images. The clinical annotations for this biosample, based on the pathology report provided by the biospecimen supplier (OriGene), are as follows: adenocarcinoma of the breast (ductal), TNM stage of pT1cpN3apMX, minimum stage IIIC, 75% tumor, and PR- / ER- / HER2+ according to conventional IHC. Figure 15B Individual MALDI-MS images of all 12 biomarkers are displayed as gradient colors across the entire tissue section (biomarker identity indicated by markers). A “blank” is also shown, which is an adjacent tissue section stained with isotype control IgG with PC-MT (the same PC-MT as CD3). Gradient color bars are also displayed. For comparison, the display bars for all biomarkers were set to a full intensity threshold of 20 (arbitrary peak intensity units) and a minimum display intensity of 2, except for CK, CD3, CD4, CD68, and Ki67, which produced particularly strong signals and were therefore set to 50 and 5, respectively. Figure 15C Multicolor MALDI-MS images of identical breast cancer tissue sections are overlaid. In this case, for simplicity, only CK, HER2, and ER (color-coded above the images) are displayed to allow for visual differentiation of the relative distribution of these three important biomarkers. Figure 15DMulticolor MALDI-MS images of selected biomarkers on different breast cancer tissue sections are overlaid (top; color coding below the images) and individual MALDI-MS images (gradient bar; small image at the bottom). In this case, the clinical annotation of the biosample, based on the pathology report provided by the biosample supplier (OriGene), is as follows: breast cancer, ductal, lobular, metastatic, TNM stage of T2N2aMX, minimum stage IIIA, 95% tumor, and PR+ / ER+ / HER2- according to conventional IHC (i.e., PR / ER / HER2 profile is the opposite of the previous tissue). For comparison, the display bar of the gradient color images is set to a full intensity threshold of 20 (arbitrary peak intensity units) and a minimum display intensity of 2, except for CK, which produces a particularly strong signal and is therefore set to 100 and 10, respectively.

[0065] Figure 16 The common elements of the schemes of the present invention (MIHC and MISH) are compared with those of conventional IHC (e.g., [Katikireddy and O'Sullivan (2011) Methods Mol Biol 784:155-67]) and conventional ISH (e.g., [Renwick, Cekan et al. (2014) Methods Mol Biol 1211:171-87]), as well as with conventional direct MSI (e.g., [Caprioli, Farmer et al. (1997) Anal Chem 69:4751-60]) and with MSI of microbead arrays (e.g., U.S. Patent No. 9,523,680, which is hereby incorporated by reference). It should be noted that many protocol variations are possible, for example, depending on whether FF or FFPE tissue is used, whether the protocol is based on IHC / MIHC or ISH / MISH, and / or what type of optical detection method is used for conventional IHC or ISH (such as directly labeled primary antibodies or secondary detection methods; and colorimetric measurements relative to fluorescence readout). Figure 16 Only the common basic elements of the proposed scheme are shown.

[0066] Figure 17 Examples of damage to mouse brain tissue sections on indium tin oxide (ITO)-coated glass slides subjected to the mass spectrometry-based immunohistochemistry (MIHC) procedure of this invention. The top two images are on ITO-coated glass slides, and the bottom image is on gold-coated glass slides. Tissue section loss or damage can be variable. Major sites of damage to the tissue sections are indicated by black arrows.

[0067] Figure 18Comparison of MIHC results detected by anti-NeuN antibody on mouse brain FFPE tissue sections with and without matrix recrystallization using DHB matrix sublimation.

[0068] Figure 19A -C. MALDI-MSI of sagittal mouse brain FFPE tissue sections stained with PC-MT-labeled lectin probes. Figure 19A & Figure 19B (This refers to color quality images of PC-MT corresponding to three lectins: PHA-E4 (red), PNA (green), and WGA (blue).) Figure 19B Competitive inhibition (blocking) confirmed the specificity of lectin binding (in this case, lectin binding to WGA). A mixture of PC-MT lectin probes was pre-incubated with N,N′,N″-triacetylchitotriose (TCT), a soluble sugar that specifically binds to WGA (and the soluble sugar remained intact during the tissue staining / probing step). TCT blocking was omitted as a control. Figure 19C Total average spectra were captured from each quality image in inset b. The monoisotopic PC-MT peaks (WGA, PNA, and PHA-E4) of the three lectins were labeled. Orange traces represent tissue treated with TCT blocking, and purple traces represent untreated tissue. TCT blocking showed a 70% reduction in the peak intensity of WGA PC-MT, while the other two lectins remained unchanged.

[0069] Figure 20A -C. Direct label-free MALDI-MSI on the same tissue sections, followed by MIHC. Using FF sagittal mouse brain tissue sections. Quality images of directly detected lipids and PC-MT derived from various antibodies are shown. Figure 20A MSI-1. Initial direct label-free MALDI-MSI of endogenous lipids (see the legend in the image for example lipids). Figure 20B MSI-2. Subsequent MIHC showed the selected antibody PC-MT (see the legend in the image, which shows the antibody PC-MT). Figure 20C Image merging of selected analytes from MSI-1 and MSI-2 (analytes are shown in the legend in the images). Example 4 explains the expected colocalization of lipid thioglycosides (ST) with myelin, but not between ST and NeuN.

[0070] Figure 21A -B. PC-MT probe in non-imaging mass spectrometry applications. PC-MT-Ab is bound to protein G agarose beads, the beads are washed, PC-MT is lightly released from the PC-MT-Ab bound to the beads, and the supernatant is analyzed by non-imaging standard MALDI-MS. Figure 21A Stacked 3D projections of MALDI-MS spectra from six samples corresponding to six different concentrations of PC-MT-Ab added to Protein G beads (the concentration range of PC-MT-Ab added to the beads is indicated by black arrows). For data normalization purposes, control peptides were included in the samples at fixed concentrations. Figure 21B The ratio of the intensity of the light-emitted PC-MT to the peak intensity of the control peptide monoisotope was obtained and plotted as a function of the concentration of PC-MT-Ab added to the protein G beads.

[0071] Figure 22 An exemplary schematic diagram is shown illustrating the parallel relationship between using undigested tissue and digested tissue. FFPE = Formalin Fixation and Paraffin Embedding (FFPE).

[0072] Table 1. Quality Unit Sequence for PC-MT and Quality Report Material Quality (For example, see PC-MT structure) Figure 3 ).

[0073] Table 1.1. Amino acid isotopes.

[0074] Invention Description

[0075] This invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for the targeted detection and localization of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for example, for research applications and for clinical applications such as those of pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies). Specifically, mass spectrometry imaging (MSI) is used, for example, as a mode for detecting and localizing biomolecules in tissues or cells. More specifically, the field of this invention relates to optically cleavable mass-tagged reagents attached to probes such as antibodies and nucleic acids and used to enable multiplexed immunohistochemistry and in situ hybridization with MSI as the detection / readout mode.

[0076] Current tissue imaging methods (such as fluorescence IHC) lack the multi-path and / or multi-omics levels required to reveal complex biological systems and human diseases. Mass spectrometry imaging (MSI) is typically limited to non-targeted analysis of small molecules and peptides and lacks the ability to target specific intact molecules (such as proteins), post-translational modifications (such as glycans), and nucleic acids (such as DNA and RNA). We have developed a method for readily labeling probes (including antibodies, lectins, and nucleic acids) using novel optically cleavable peptide mass tags (PC-MTs) for highly multi-pathway MSI targeting macromolecules in tissues. When combined with non-targeted MSI, highly multi-pathway and multi-omics tissue imaging can be achieved on a single sample. Combined multimodal fluorescence and MS IHC imaging can also be achieved on a single tissue sample by using dual-labeled antibody probes. What is needed is a novel optically cleavable mass tag (PC-MT) and MALDI-MSI procedure to overcome these limitations.

[0077] Here we report a novel optically cleavable mass tag (PC-MT) and MALDI-MSI procedure that overcomes these limitations. PC-MT is a modified peptide comprising: a mass unit, a highly efficient optically cleavable linker (PC-linker) incorporated into the peptide via solid-phase synthesis, a spacer group, and an NHS-ester probe reactive moiety near the C-terminus. A PC-MT antibody probe is generated in a one-step reaction. The rapid and efficient optical-nuclear method used in the novel PC-linker [Olejnik, Sonar et al. (1995) Proceedings of the National Academy of Science (USA) 92:7590-7594] provides robust sensitivity in practice, allowing high-path MSI of a variety of biomarkers in multiple tissues, including mouse brain (Example 2), human tonsils, and breast cancer (Example 9), as illustrated here. Furthermore, a novel dual-labeled antibody combining PC-MT and a fluorophore allows for direct association of MSI with conventional immunofluorescence (Example 9). Finally, the versatility of this approach was demonstrated by the ability to perform multi-pathway PC-MT-based targeting of MSIs, including label-free, untargeted small molecule MSIs (lipids) and macromolecular biomarkers, on the same tissue slices, which is impossible to achieve with standard IHC (Experimental Example 4).

[0078] This invention relates to compositions and methods for preparing and using novel optically cleavable connectors (PC-connectors), optically cleavable mass tags (PC-MTs), and optically cleavable mass-tagged probes (PC-MT-probes) that overcome the aforementioned limitations of earlier targeted mass spectrometry (MSI) methods, enabling highly multiplexed MSI targeting biomolecules in biological samples such as tissues and cells using probes such as antibodies and nucleic acids.

[0079] Figure 2 shows the basic design of peptide-based PC-MT, the obtained PC-MT probe, and its use.

[0080] As shown in Figure 2a, peptide-based PC-MT comprises: i) a probe-reactive portion, such as an NHS-ester leaving group (N-hydroxysuccinimide ester) exhibiting amine reactivity; ii) an internal PC-linker (ellipse in Figure 2a) introduced into the peptide chain during solid-phase peptide synthesis (SPPS); iii) a selectively detectable mass unit (curve in Figure 2a) containing an amino acid or its isotope or analog / derivative thereof, which can be chemically incorporated using SPPS; and iv) an optional fluorophore (starburst shape marked with "F" in Figure 2a).

[0081] PC-MT is covalently linked to the probe via a chemical reaction between the probe reactive portion of PC-MT and the probe to generate a PC-MT-probe (Figure 2b). The probe can be, for example, a protein such as an antibody or a nucleic acid such as an amine-modified oligonucleotide or aptamer.

[0082] Cells / tissues are "stained" with PC-MT probes (i.e., PC-MT probes bind to their targets in the cells / tissues), and after photocutting, a quality reporter region is released (photo-release) for detection by MSI (Figure 2c; note that the quality reporter contains mass units and, in some embodiments, also includes a portion of the photocut PC-connector). It should be noted that photocutting can be achieved by a MALDI-MS laser beam and / or by any light, such as UV or near-UV light with wavelengths in the 200 nm to 400 nm range.

[0083] The improvements in PC-MT, the resulting PC-MT probes, and their applications are summarized as follows: i) Fast and efficient optically cleavable cores (PC-cores; see [link]). Figure 3Step 1), which has previously been demonstrated in a range of other applications when incorporated into other compounds such as photo-cleavable biotin (PC-biotin) and photo-cleavable phosphoramide (PC-phosphoramide) [Olejnik, Sonar et al. (1995) Proceedings of the National Academy of Science (USA) 92:7590-7594; Olejnik (1996) Nucleic Acids Research 24:361-366; Olejnik, Krzymanska-Olejnik et al. (1998) Nucleic Acids Res 26:3572-6; Martinez, Patkaniowska et al. (2002) Cell 110:563-74; Pandori, Hobson et al. (2002) Chem Biol 9:567-73; Mitra, Shendure et al. (2003) Anal Biochem 320:55-65; Lim and Rothschild (2008) Anal Biochem 383:103-115; Lim, Liu et al. (2014) Rapid communications in mass spectrometry: RCM 28:49-62; Zhou, Liu et al. (2016) Sci Rep 6:26125 [See also U.S. Patent Nos. 5,643,722, 5,986,076, 6,218,530, 8,906,700 and 10,060,912, which are hereby incorporated by reference]; ii) Novel Fmoc-protected light-cuttable connector (Fmoc-PC connector; see Figure 3 ), which contains the PC-core and can be incorporated into the peptide chain using standard Fmoc-based SPPS chemistry (see Figure 3 ); iii) Using the probe reactivity portion of PC-MT (e.g., the NHS-ester probe reactivity portion; see Figure 3 Steps 1-2) allow for easy one-step probe labeling; iv) built-in optional fluorescent labeling on PC-MT (see Figure 2a) facilitates method development by allowing conventional fluorescence imaging in addition to MSI; and v) the ability to perform non-targeted label-free small molecule MSI and multi-path PC-MT-based targeted MSI of macromolecules on the same tissue slice (see Experimental Example 4).

[0084] This invention is not intended to be limited to tissues. For example, digested tissues can be used. Furthermore, the compositions and methods described in this invention can be applied to cells grown or deposited on a surface or to biofilms grown or deposited on a surface. For example, MALDI-MSI is used in clinical microbiology for the rapid identification of rapidly growing microorganisms [(2019) NatCommun 10:4029; Oviano and Bou (2019) Clin Microbiol Rev 32]. Bacterial cells are grown or deposited on a substrate and then MALDI-MSI is performed. In another example, using the compositions and methods described in this invention, cells derived from cancer biopsies and cells deposited on a surface can be analyzed. Another example is bacteria of a single species or multiple species grown on a surface to form complex heterogeneous patterns. Another example is MALDI-MSI of biofilms. Recent advances have been made in applying this method to Bacillus subtilis biofilms grown on agar by using a sprayer to deposit solutions of specific matrix compounds compatible with MALDI-MSI, such as 2,5-dihydroxybenzoin acid [Li, Comi et al. (2016) J Mass Spectrom 51:1030-1035]. The method can also be applied to complex, intact multicellular organisms deposited on surfaces. For example, *C. elegans*, a free-living, transparent nematode about 1 mm long, inhabits temperate soil environments. MSI has previously been applied to *C. elegans*, demonstrating the feasibility of using the compositions and methods described in this invention [Menger, Clendinen et al. (2015) Current Metabolomics 3:130-137]. The compositions and methods described in this invention can also be applied to subcellular or molecular assemblies grown or deposited on surfaces, including organic and inorganic nanostructures. Lanni et al. recently described examples of MSI analysis of single-cell and subcellular structures in a review [Lanni, Rubakhin et al. (2012) J Proteomics 75:5036-5051]. More recently, subcellular resolution has been achieved with MALDI-MSI using specialized techniques compatible with the compositions and methods described in this invention, such as transport mode geometry [Niehaus, Soltwisch et al. (2019) Nat Methods 16:925-931].

[0085] In another embodiment, the PC-MT and PC-MT probe can be used for encoding and / or detection in microarrays and microbead arrays (e.g., U.S. Patents 9,523,680, 9,513,285 and 10,060,912, which are hereby incorporated by reference). Invention Details

[0087] Optically cut quality label (PC-MT)

[0088] The preferred chemical structure of the Fmoc-protected, light-cuttable connector (Fmoc-PC connector) is as follows: Figure 3 As shown, this is a key structural unit in the synthesis of PC-MT. The compound contains at least one Fmoc-protected primary amine terminus, one free carboxyl terminus, and an intermediate, lightly cleavable core (PC-core) based on 1-(2-nitrophenyl)-ethyl. Optionally, the Fmoc-PC-connector also contains a connector unit, such as... Figure 3 As shown, it links a lightly cleavable core based on 1-(2-nitrophenyl)-ethyl to the fmoc-protected primary amine and carboxyl moieties. Overall, the minimal configuration of the Fmoc-PC-linker allows for the incorporation of PC-linkers into peptides to produce lightly cleavable mass tags (PC-MTs). Figure 3 Step 1). PC-linkers are incorporated using standard Fmoc-based solid-phase peptide synthesis (SPPS) in the same manner as amino acids, which is currently the preferred mode of chemical peptide synthesis [Behrendt, White et al. (2016) J Pept Sci 22:4-27]. However, it should be understood that other protecting groups and other peptide synthesis methods are possible, such as Boc protecting groups and related peptide synthesis chemistry [Stawikowski and Fields (2012) Curr Protoc Protein Sci Chapter 18: Unit 18 1].

[0089] As in Figure 3 As shown, the resulting PC-MT includes, but is not limited to, the following features:

[0090] i) PC-nucleus based on 1-(2-nitrophenyl)-ethyl disclosed in Figure 3 In step 1, the optical cutting point is located at... Figure 3 Step 1 is indicated by the black arrow, and the photocutting reaction is as follows: Figure 3Step 3 is shown. It should be noted that the PC-core used in the PC-connector of this invention provides rapid and efficient photocutting, and as shown in this specification (Experimental Example 6), it offers superior sensitivity in mass spectrometry compared to the PC-connector used in the aforementioned work of Lemaire et al., which contained an additional methoxy group on the nitrobenzene ring of the PC-core (for a comparison of PC-connector structures, see...). Figure 4 For the prior work of Lemaire et al., see [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67] and U.S. Patent No. 8,221,972.

[0091] ii) Probe-reactive moieties, such as the leaving group of primary amine-reactive N-hydroxysuccinimide esters (NHS-esters). For example, NHS-esters can be generated on the ε-amine of the lysine (K) side chain (see...). Figure 3 Step 1, NHS-ester), wherein conversion is performed using, for example, N,N'-disuccinimidyl carbonate (DSC) [Morpurgo, Bayer et al. (1999) J Biochem Biophys Methods 38:17-28] or DSS (disuccinimidyl octanoate). For example, probe reactive moieties can also be generated by chemically converting / modifying the carboxylic acid functional group at the C-terminus of peptide-based PC-MTs. Figure 3 (Not shown in the text). The probe reactive portion allows PC-MT to be easily linked to target probes such as antibodies or nucleic acids. It should be understood that a variety of probe reactive portions can be used, including but not limited to amine-reactive N-hydroxysuccinimide (NHS) esters, sulfonyl-N-hydroxysuccinimide (sulfonyl-NHS) esters, succinimide esters (SE), sulfonyl-succinimide esters (SSE), aldehydes or tetrafluorophenyl (TFP) esters; thiol-reactive maleimides or iodoacetamides; or, for example, multi-reactive epoxy portions. Azides and alkynes, such as those used in copper-containing or copper-free click chemistry, are also possible [McKay and Finn (2014) Chem Biol 21:1075-101].

[0092] iii) Attach the PC-core to an optional spacer base unit of the probe's reactive portion, for example in Figure 3Step 1 shows the addition of the GSGGK amino acid sequence to a portion of the PC-linker. It should be understood that this spacer unit is not necessary, as the reactive portion of the probe can be linked to the 1-(2-nitrophenyl)-ethyl-based PC-core at the same position without the spacer unit. It should also be understood that the spacer unit can have various chemical structures, such as a polyethylene glycol (PEG) spacer, to facilitate hydrophilic / water-soluble properties.

[0093] iv) Selectively detectable mass units, as an example, in Figure 3 The amino acid (peptide) sequence is shown as APRLRFYSL. It should be understood that any amino acid used can be, for example, natural and non-natural amino acids, as well as modified amino acids, isotopic amino acids, amino acid analogs / derivatives, and any combination thereof. While peptide-based mass units are preferred embodiments due to their ease of synthesis, robust performance in mass spectrometry, and the ability to obtain further specificity in mass spectrometry identification using established methods of fragmentation analysis based on tandem MS (e.g., MS / MS), selectively detectable mass units need not be peptides and can be, for example, any chemical entity detectable by mass spectrometry. Polymer mass units are preferred because they are generally easy to synthesize and the mass can be easily tuned by simply changing the number and type of monomer subunits. While peptides are considered a form of biopolymer (in addition to, for example, nucleic acids), polymers other than peptides, such as polyethylene glycol, can be used, which is easily synthesized and can be detected by, for example, MALDI-MS [Enjalbal, Ribiere et al. (2005) J Am Soc Mass Spectrom 16:670-8].

[0094] v) Optional mass unit connectors, such as Figure 3 As shown in step 1, the PC-core is connected to the mass unit. It should be understood that this mass unit connector is not required, as the mass unit can be connected to a 1-(2-nitrophenyl)-ethyl-based PC-core at the same location without a mass unit connector. It should also be understood that the mass unit connector can have various chemical structures, such as a polyethylene glycol (PEG) connector, to promote hydrophilic / water-soluble properties.

[0095] vi) A blocking group can be used on the N-terminal α-amine of the peptide-based PC-MT, for example, if the probe reactive moiety is primary amine reactive, acetylation can be used. Figure 3The "Ac" in the label is used to prevent the self-reaction or polymerization of PC-MT. In these cases, internal amino acids with free primary amines (e.g., lysine with ε-amine) can be avoided, or they can be blocked / protected. In cases where the reactive portion of the probe reacts with different functional groups (such as thiol groups), any such functional groups present on the peptide (e.g., from cysteine) can also be blocked / protected to prevent the self-reaction or polymerization of PC-MT.

[0096] vii) In addition to selectively detectable mass units, the PC-MT optionally includes a fluorophore or other detectable marker (e.g., a chromophore or an affinity ligand such as biotin) (the fluorophore is not on the surface of the fluorophore). Figure 3As depicted in Figure 2a, one embodiment can be shown. This can aid in method development, for example, allowing routine fluorescence imaging on the same cell / tissue sample in addition to MSI. A fluorophore can be attached to PC-MT, for example, an ε-amine containing lysine, using a range of commercially available amine-reactive dyes (e.g., Cy5-NHS or sulfonyl-Cy5-NHS). This attachment can be performed during or after SPPS. Fluorophore attachment can also be achieved using other chemical methods such as click chemistry [McKay and Finn (2014) Chem Biol 21:1075-101]. In a preferred embodiment, the fluorophore is attached to a spacer base unit. In other embodiments, the fluorophore can be attached to a mass unit or a mass unit adapter. However, the fluorophore can be attached to any part of the PC-MT structure, as long as it does not interfere with the reaction of the probe reactive portion with the probe, the photocutting reaction, or the MSI detection of the mass unit, and as long as it does not impair the solubility of the PC-MT labeling reagent in the solvent environment chosen for probe labeling. This approach offers the advantage that the quality reporter and the fluorophore are part of the same PC-MT labeling reagent, allowing, for example, evaluation of PC-MT probe labeling success by fluorescence methods (other than mass spectrometry). Alternatively, PC-MT lacking any fluorescent label (or other detectable label such as a chromophore or affinity ligand such as biotin) can be linked to the probe, and furthermore, the fluorescent label (or other detectable label such as a chromophore or affinity ligand such as biotin) can be alternatively linked to the same probe at a different location than PC-MT. This can be readily achieved, for example, by labeling the probe (e.g., an antibody) with both PC-MT having an NHS-ester probe reactive moiety and the aforementioned NHS-activated fluorophore. Probe labeling with PC-MT and the fluorophore can be performed simultaneously or sequentially (PC-MT first, then the fluorophore, or vice versa). In this embodiment using a primary amine reactive NHS group for probe labeling, labeling with both PC-MT and the fluorophore is readily possible because, for example, there are many primary amines in the antibody probe. In summary, this approach may have advantages over fluorescent PC-MT because it avoids any adverse effects on PC-MT solubility caused by the fluorophore's reaction to PC-MT labeling or during probe labeling (which is typically performed under aqueous conditions); it also avoids the adverse effects of the fluorophore on mass spectrometry readout (e.g., potentially poor ionization efficiency when the fluorophore is linked to a mass reporter). Furthermore, this alternative would allow for independent adjustment of the fluorescence and PC-MT labeling ratio.

[0097] Finally, it should be understood that, Figure 3The optional chemical connectors shown (i.e., connector units, mass unit connectors, and spacer base units) are not intended to limit the scope of the invention. These act as bridges between the PC core and essential components such as, for example, probe reactive portions and mass units. These chemical connectors can have a variety of chemical compositions. For example, the chemical connectors can simply be hydrocarbon chains, or alternatively, for example, to have better solubility in aqueous environments, 2,2'-(ethylenedioxy)-di-(ethylamine) chemical connectors [Pandori, Hobson et al. (2002) Chem Biol 9:567-73]. Polyethylene glycol (PEG) is another example and will be considered by those skilled in the art as a relatively stable, water-soluble, and biocompatible excellent chemical connector.

[0098] PC-MT probe

[0099] As in Figure 3 As shown in step 2, PC-MT is attached to the probe via the probe reactive portion. In the illustrated embodiment, the NHS-ester leaving group of PC-MT is lost upon reaction with the primary amine on the antibody, thereby forming an amide bond between PC-MT and the antibody. Although an antibody is depicted, the probe can be any kind, such as a protein, including but not limited to, antibodies, recombinant antibodies, affibodies, nanobodies, single-chain fragment variable (scFv) antibodies, single-domain antibodies, VHH single-domain antibodies (e.g., camel single-domain VHH antibodies), receptors, carbohydrate-binding proteins (e.g., lectins [Tsaneva and Van Damme (2020) Glycoconj J 37:533-551]) or ligands or fragments thereof. Probes can also be nucleic acids, such as DNA, RNA, or locked nucleic acids (LNAs) [Nielsen, Singh et al. (1999) J Biomol StructDyn 17:175-91], such as in oligonucleotide hybridization probes or DNA / RNA aptamers. Probes can also be other organic or biomolecules such as lipids, carbohydrates, steroids, or drugs. Labeling of probes with PC-MT can be at random sites (e.g., nonspecifically at any primary amine site of a protein, as would occur with PC-MT containing NHS-esters as the probe's reactive moiety) or the labeling can be site-specific (e.g., present at specific sites in carbohydrate regions on the heavy chain of certain antibodies). In some cases, modified probes may be needed to facilitate PC-MT labeling, such as primary amine-modified nucleic acid probes (e.g., in the case where the probe's reactive moiety is an NHS-ester).

[0100] Probes can have multiple tissue targets, meaning the part (molecular structure) the probe binds to. The following examples are not intended to limit the types of probe targets: different probes may target different biomolecules or biomarkers (e.g., different proteins), or they may target different binding sites within the same biomolecule or biomarker (e.g., different binding sites within the same protein). Probe targets include, but are not limited to, biomolecules or complexes or parts thereof, including proteins, post-translational modifications of proteins, glycoproteins, nucleic acids, lipids and their derivatives, drugs, metabolites, carbohydrates, glycans, proteoglycans, gangliosides, and organic compounds.

[0101] In PC-MT probes, the core structure is defined as the structure that connects the mass unit to the probe (see example...). Figure 3 The core structure (as described in Step 2, “core structure”) comprises, but is not limited to, a PC-core, optional spacer units, and optional mass unit linkers, and is typically a common structure in all PC-MT probe materials. In a preferred embodiment, the core structure is a non-neutral structure (e.g., containing ionizable groups such as, for example, sulfonates, phosphates, amines, or carboxylic acids). This improves the water solubility of the PC-MT labeling reagent and thus improves probe labeling reactions that are typically carried out in an aqueous environment. This also improves the water solubility of the PC-MT probe. In one embodiment, a sulfonated fluorophore is included on the core structure (e.g., sulfonyl-Cy5, as used in Example 7). Sulfonated fluorophores are used because fluorophores are typically polycyclic compounds and insoluble in water without such a sulfonate moiety. In another embodiment, aspartic acid and / or glutamic acid are included in the amino acid moiety of the spacer unit of the PC-MT to improve the water solubility of the PC-MT labeling reagent and / or the PC-MT probe.

[0102] In a preferred embodiment, multiple PC-MT molecules are linked to each probe molecule to enhance sensitivity in the mass spectrometry step. A carrier can be added to the probe to further increase the number of PC-MT labels and thus facilitate further signal amplification: in one embodiment, a polyamine-modified dendritic polymer or nanoparticle (NP) is labeled with PC-MT and then linked to a probe (e.g., an antibody) to increase the number of PC-MT labels (Figure 5a). Gold NPs are particularly attractive because they are as small as 13 nm and are routinely used with, for example, electron microscopy for the immunolabeling of target biomolecules [Ackerson, Powell et al. (2010) Methods Enzymol 481:195-230]. In one embodiment, the polyamine-terminated gold NP or branched dendritic polymer is commercially available (e.g., from Dendritech and Nanonovex Biotechnologies) and can be conjugated to PC-MT (via the amine-reactive NHS-ester probe reactive moiety). Alkyne groups can also be introduced into polyamine dendrimers or NPs using readily available NHS-activated reagents (e.g., NHS-DIBO-alkyne from Thermo Scientific [Waltham, MA]). Similarly, azido-modified antibodies or amine-modified nucleic acid probes can also be used with commercially available NHS-activated reagents (e.g., NHS-PEG4-azide from Thermo Scientific [Waltham, MA]). Finally, PC-MT / alkyne-modified dendrimers or NPs can be linked to azido-modified probes using highly deterministic, highly selective, mild, and bioorthogonal click chemistry [Kolb, Finn et al. (2001) Angew Chem Int Ed Engl 40:2004-2021] (azido and alkyne spontaneously and selectively form covalent bonds under physiological aqueous conditions and even in complex mixtures—in some cases, depending on the type of alkyne used), a copper catalyst is required.

[0103] In another implementation, multiple primary amine modifications are introduced into the nucleic acid probe to increase the number of PC-MT labeling sites. In some cases, it may be desirable to use nucleic acid "tails" that are not part of the target-binding (hybridization) sequence of the nucleic acid probe, whereby these "tails" carry multiple amine modifications to provide an increased number of PC-MT labeling sites (Figure 5b). This configuration reduces the potential interference of PC-MT with the target binding region of the nucleic acid probe. For example, amine modifications can be introduced into the nucleic acid probe using modified nucleotides. Examples include 5'-dimethoxytriphenylmethyl-5-[N-(trifluoroacetylaminohexyl)-3-propenylimino]-uridine, 2'-O-triisopropylsilyloxymethyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramide, commonly referred to as the amino-modifying agent C6-U phosphoramide, which is introduced during the chemical DNA synthesis process based on phosphoramide. Modified nucleotides can also be introduced into nucleic acid probes enzymatically (e.g., by DNA polymerase) using, for example, 5-[3-aminoallyl]-2'-deoxyuridine-5'-triphosphate (commonly known as aminoallyl-dUTP). Modifications other than amines used as PC-MT labeling sites can be introduced into nucleic acid probes in a similar manner.

[0104] In a preferred embodiment, PC-MT is attached to the probe in a one-step chemical reaction between the PC-MT and the probe (e.g., by mixing PC-MT containing NHS-ester as the reactive part of the probe with a nucleic acid such as an antibody or amine-modified probe). The labeling of probes with PC-MT in this invention is significantly different from the complex, multi-step process in the previous work of Lemaire et al. [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67] [See also U.S. Patent No. 8,221,972], wherein the probe (e.g., antibody) must first be conjugated to a non-photocleavable heterobifunctional crosslinker MBS (3-maleimide benzoic acid N-hydroxysuccinimide ester) by reaction with the NHS-ester moiety of MBS, the antibody is then purified by desalting column chromatography (to remove unreacted MBS linkers), and subsequently the thiol-reactive maleimide moiety generated on the antibody by the conjugated MBS linker is reacted with a peptide containing an internal photocleavage site and a cysteine ​​amino acid (thereby the cysteine ​​on the photocleavage peptide provides a free thiol moiety for the reaction of the maleimide on the MBS linker to the antibody). Similarly, the aforementioned imaging quality cytometry protocol [Giesen, Wang et al. (2014) Nat Methods 11:417-22] also uses a highly complex multi-step probe (i.e., antibody) labeling procedure, which includes preloading the polymer with metal ions, partially reducing the antibody and conjugating the two together, and purifying the polymer and antibody multiple times [Fluidigm, Quick Reference: “Maxpar X8 Antibody Labeling”, accessed September 2020, www.fluidigm.com / binaries / content / documents / fluidigm / resources / maxpar-x8-antibody-labeling-quick-reference-fldm-00015-rev01 / maxpar-x8-antibody-labeling-quick-reference-fldm-00015-rev01 / fluidigm%3Afile].

[0105] In one embodiment of the invention, for simplicity, further purification of the probe after the PC-MT labeling reaction may be avoided (thus not removing unreacted PC-MT), while in other embodiments, for example, unreacted PC-MT may need to be removed by size exclusion (gel filtration) chromatography or ultrafiltration (e.g., using an Amicon Ultra-0.5 centrifugal filter unit with an appropriate molecular weight cutoff to retain the labeled probe) to avoid background in downstream biomarker detection.

[0106] Tissue treated with PC-MT probe and mass spectrometry imaging (MSI)

[0107] The final step of this process involves treating (“staining”) cells or tissues with PC-MT probes (i.e., the PC-MT probes bind to a target in the cells / tissue), followed by imaging the photodissected quality reporter using MSI (see Figure 2c and...). Figure 3 Step 3). The two example embodiments referred to in this invention as mass spectrometry-based immunohistochemistry (MIHC) and mass spectrometry-based in situ hybridization (MISH) (where antibodies and nucleic acid probes are used, respectively) are similar to conventional immunohistochemistry (IHC) and in situ hybridization (ISH). Essentially, MIHC and MISH differ in that they use PC-MT-labeled probes instead of probes labeled with fluorophores or chromogenic agents, and use MSI instead of optical imaging (e.g., microscopy). The MIHC and MISH procedures (which are illustrated in detail later in the experimental examples) generally involve the basic steps described in the following paragraphs (although as with standard IHC and ISH, those skilled in the art will recognize that many protocol variations are possible). For a comparison of the fundamental common elements of the schemes of the present invention (MIHC and MISH) with those of conventional IHC (e.g., [Katikireddy and O'Sullivan (2011) Methods Mol Biol 784:155-67]) and conventional ISH (e.g., [Renwick, Cekan et al. (2014) Methods Mol Biol 1211:171-87]), as well as with conventional direct MSI (e.g., [Caprioli, Farmer et al. (1997) Anal Chem 69:4751-60]) and with MSI of microbead arrays (e.g., U.S. Patent No. 9,523,680, which is hereby incorporated by reference), see also the fundamental common elements of conventional direct ... Figure 16It should be noted that many protocol variations are possible, for example, depending on whether FF or FFPE tissue is used, whether the protocol is based on IHC / MIHC or ISH / MISH, and / or what type of optical detection method is used for conventional IHC or ISH (such as directly labeled primary antibodies or secondary detection methods; and colorimetric measurements relative to fluorescence readout). Figure 16 Only the common basic elements of the proposed scheme are shown.

[0108] The basic steps of MIHC are: i) fixing thin FFPE or fresh frozen tissue sections (e.g., sections 5-10 μm thick from FFPE or fresh frozen tissue blocks using a microtome or cryostat) onto conductive slides (e.g., metal-coated glass slides); it should be noted that although indium tin oxide (ITO) coated glass slides are almost universally used as conductive surfaces in MSI (e.g., see [Yalcin and de la Monte (2015) J Histochem Cytochem 63:762-71; Angel, Baldwin et al. (2017) Biochim Biophys Acta Proteins Proteom) [1865:927-935], but in this invention it has been found that, considering the extensive processing steps of MIHC and MISH described below, gold-coated glass slides are advantageous in preventing tissue from peeling off the slide and / or tissue damage during processing, while still providing the necessary conductive surface for MSI (e.g., glass slides with a 10 nm gold layer and a 2 nm titanium adhesion underlayer, from Platypus Technologies LLC, Madison, WI; or a 50 nm gold layer and a 5 nm chromium adhesion underlayer, from Substrata Thin Film Solutions / Angstrom Engineering Inc., ON, Canada); after tissue fixation are ii) deparaffinization (e.g., with xylene) (in the case of FFPE); iii) rehydration (if deparaffinization is performed), typically with a series of ethanol / water mixtures and aqueous saline buffers; iv) in the case of fresh frozen tissue, fixation in formalin or paraformaldehyde; v) antigen retrieval to reverse some of the adverse effects of formalin / paraformaldehyde fixation (e.g., at pH 6) Heating in citrate buffer or using formic acid); vi) Treating with blocking buffer to reduce background (typically a saline buffer containing nonionic detergents such as Tween-20 and protein blockers such as bovine serum albumin [BSA] and animal serum); vii) Simultaneous staining with a mixture of different PC-MT antibodies (PC-MT-Abs) for multiplexing (typically diluted in blocking buffer); viii) Washing in saline buffer with a nonionic detergent (such as Tween-20) to remove any unbound PC-MT-Abs, then washing in a volatile aqueous buffer (such as ammonium bicarbonate) to remove nonvolatile salts that may interfere with certain forms of mass spectrometry analysis; and ix) Drying the tissue slides before MSI. It has been found that using immunohistochemical mass spectrometry (MSI) protocols ( Figure 16The flowchart in the document shows that tissues fixed to gold slides (e.g., slides with a gold layer) yield better results. More specifically, fixing to gold slides helps avoid tissue damage or loss during the required slide processing steps (conductive slides for MSI). Previous use of gold slides in tissue MSI has been used... direct MSI, which lacks all the processing steps of the present invention (see...). Figure 16 (The flowchart in the document describes the process), and therefore avoids the problem of tissue loss / damage. This invention uses gold slides to improve tissue adhesion (more commonly, ITO conductive slides are used). direct MSI, but showed poor results in our protocol (see Example 10). Tissue damage and loss can occur during any liquid phase processing step of the slide.

[0109] The steps of MISH are as follows: i) tissue fixation, ii) deparaffinization, iii) rehydration, and iv) formalin / paraformaldehyde fixation as described with respect to MIHC; this is usually followed by v) partial protein digestion with protease K; vi) fixation of nucleic acids with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide); vii) tissue acetylation to cap free amines and reduce background (caused by nonspecific binding of probes); viiii) treatment with blocking buffer, which typically contains at least unrelated nucleic acids (e.g., yeast tRNA and / or salmon sperm DNA) to reduce background; ix) simultaneous staining (hybridization) with a mixture of different PC-MT nucleic acids (PC-MT-NA) for multiplexing (usually diluted in blocking buffer or similar buffer); x) washing in saline buffer to remove any unbound PC-MT-NA, followed by washing in a volatile aqueous buffer (such as ammonium bicarbonate) to remove nonvolatile salts that can interfere with certain forms of mass spectrometry analysis; and xi) drying the tissue slides before MSI.

[0110] However, as discussed previously, the probes of the present invention are not necessarily limited to antibodies (for MIHC) and nucleic acids (for MISH), and can be, for example, lectins, receptors, or ligands. Therefore, the present invention is not limited to MIHC and MISH methods, but to other embodiments in which tissues are treated (“stained”) using, for example, PC-MT conjugated lectins, receptors, or ligands, or any probe type or combination thereof.

[0111] Mass spectrometry imaging (MSI) (and implementations including MIHC and MISH): MSI is performed following the procedure described above, with MALDI-MSI used in one implementation:

[0112] For MALDI-MSI, the matrix compound is typically applied to the tissue in a thin and uniform layer. Examples of matrix compounds include α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), or 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In a preferred embodiment, matrix sublimation and subsequent recrystallization [Hankin, Barkley et al. (2007) J Am Soc Mass Spectrom 18:1646-52; Duenas, Carlucci et al. (2016) J Am Soc Mass Spectrom 27:1575-8] are used to achieve excellent spatial resolution (i.e., provided by limiting analyte delocalization during matrix application) and high sensitivity (i.e., provided by recrystallization, which allows the quality reporter to be fully co-crystallized with the matrix without significant analyte delocalization). Other matrix application methods can be used, such as by using commercially available sprayers (e.g., HTX™ sprayers, HTX Technologies, LLC, Chapel Hill, NC).

[0113] A variety of mass spectrometry techniques and instruments can be used for the MSI step, not just MALDI-MSI. Other MSI methods include, but are not limited to, desorption electro-electro-ionization mass spectrometry imaging (DESI-MSI) [Takats, Wiseman et al. (2004) Science 306:471-3], laser ablation electro-electro-ionization mass spectrometry imaging (LAESI-MSI) [Kulkarni, Wilschut et al. (2018) Planta 248:1515-1523], and atmospheric pressure (AP) matrix-assisted laser desorption / ionization (MALDI) mass spectrometry imaging (AP-MALDI-MSI) [Kompauer, Heiles et al. (2017) Nat Methods 14:90-96]. For example, DESI-MSI, which was initially best suited for small molecules such as metabolites and lipids, has also been adapted for protein / peptide detection [Takats, Wiseman et al. (2004) Science 306:471-3; Takats, Wiseman et al. (2008) CSH Protoc 2008: pdbprot 4994; Hsu, Chou et al. (2015) Anal Chem 87:11171-5; Towers, Karancsi et al. (2018) J Am Soc Mass Spectrom 29:2456-2466; Hale and Cooper (2021) Anal Chem 93:4619-4627]. In its basic form, DESI-MSI works by guiding electrosprayed charged solvent droplets onto a surface (e.g., tissue) to extract (desorb) and ionize the analyte, which is then carried into the inlet (e.g., transfer capillary) of a mass spectrometer [Takats, Wiseman et al. (2004) Science 306:471-3]. However, variants of DESI-MSI also exist, such as nano-DESI-MSI, which allows for the separation of desorption and ionization events [Roach, Laskin et al. (2010) Analyst 135:2233-6]. DESI-MSI can be readily performed under ambient conditions rather than in a vacuum, and therefore requires minimal sample preparation and is suitable for automation. Furthermore, as an ESI (electrospray ionization) based method, DESI-MSI does not require matrix application like MALDI-MSI. Matrix application in MALDI-MSI is not only an additional difficult and cumbersome step that can lead to reproducibility problems, but it also carries the risk of analyte delocalization.

[0114] Regardless of the type of MSI technology and instrument, the PC-MT undergoes photocutting to release the mass reporter from the PC-MT probe for MSI analysis (see Figure 2c and...). Figure 3 Step 3). It should be noted that, if Figure 3 As shown in step 3, the quality report contains quality units and, in some cases, also a portion of a light-cut PC connector (in...). Figure 3 In other cases not described herein, the quality report may not contain the photocut PC-MT portion, and therefore, the quality report and the quality unit are equivalent. The PC-MT can be pre-photocut by UV treatment prior to MSI, and in the case of MALDI-MSI, preferably also prior to matrix application to avoid matrix absorption of the incident UV light. Photocutting is also preferably performed on a dry slide to prevent the photocut quality report from delocalization due to diffusion. Pre-photocutting can be achieved, for example, by using a model XX-15 self-filtering 365nm peak lamp (UVP / Analytik Jena US LLC, Upland, CA), but many other light sources are also possible. In a preferred embodiment, pre-photocutting is performed for a short time (e.g., 5 minutes) with relatively low-intensity UV light (e.g., 3-10 mW / cm²). 2This can be achieved by using the instrument's laser beam (or the laser beam of any other laser-based mass spectrometer, such as in LAESI-MSI) to perform online optical cutting along with MALDI-MSI analysis. This alternative may promise improved spatial resolution for MSI because, in the case of MALDI-MSI, the mass reporter is not optically cut before matrix application (which could otherwise cause diffusion of the pre-optically cut mass reporter). However, the results in this specification indicate that this online optical cutting scheme provides poor sensitivity in the case of MALDI-MSI (Experimental Example 6). The advantages of pre-optical cutting before matrix application and MALDI-MSI can be explained as follows: i) pre-optical cutting avoids the light-blocking effect of the matrix compound (in fact, the function of the matrix is ​​to absorb the impacting UV laser during the MALDI-MSI process, converting it into heat, causing the mass reporter to evaporate and ionize for analysis); and ii) pre-optical cutting of the mass reporter will allow the released mass reporter to co-crystallize with the subsequently applied matrix (those skilled in the art will recognize that co-crystallization of the analyte with the matrix is ​​important for efficient desorption / ionization of the analyte in MALDI-MSI). Conversely, optical cutting consistent with MALDI-MSI analysis using the instrument's laser beam necessarily occurs after matrix application / crystallization, which may not allow for efficient co-crystallization of the matrix and the mass reporter (because during matrix application / crystallization, the mass reporter is still attached to the probe, which may still be attached to the tissue). However, other laser-based MSI methods and instruments (e.g., those that do not require a matrix compound) may promise to provide efficient in-line optical cutting and sufficient sensitivity, provided that the laser wavelength is adequately matched with the optical cutting wavelength.

[0115] It should be noted that for the preferred PC connector ( Figure 3 Photocutting leaves small remnants of the PC connector attached to the quality report, resulting in the formation of free primary amines on the quality report after photocutting (see...). Figure 3 Step 3) can help ionize the mass reporter in positive mode MALDI-MSI, and therefore this design can improve sensitivity.

[0116] It should also be pointed out that, Figure 3 The light-cutting point is depicted (for the light-cutting point represented by lightning, see...). Figure 3 Step 3) Connect the PC-MT quality reporter (before photocutting) to the benzene ring of the PC-core, and the probe is ultimately connected to the benzene ring of the PC-core at a location different from the photocutting site. This is a preferred embodiment of the invention because, importantly, the photocut 1-(2-nitrophenyl)-ethyl portion of the PC-connector does not remain connected to the quality reporter after photocutting, as in Figure 3As shown in step 3 (instead, it remains connected to the probe). Conversely, as taught by Olejnik et al. [Olejnik, Ludemann et al. (1999) Nucleic Acids Res27:4626-31] and Levy and Caprioli (US Patent No. 7,569,392), it is possible to completely reverse this orientation. However, in this configuration, the photocut 1-(2-nitrophenyl)-ethyl portion of the PC-connector remains connected to the quality reporter (in Figure 3 (Not depicted in the text), and thus, highly complex mass spectra with multiple peaks were observed, which are associated with mass reporters of various by-products of the photo-cut 1-(2-nitrophenyl)-ethyl moiety (see Experimental Example 5). This will reduce the sensitivity of MSI (by splitting the signal of a single mass reporter into many mass spectrum peaks, i.e., the by-products) and will confuse the differentiation of different mass reporters (e.g., by peak overlap).

[0117] It should be noted that when fluorophores are optionally used on PC-MT, they are preferably not placed on the mass reporter region. This avoids any potential interference from the fluorophore to the mass spectrometric detection of the mass reporter (e.g., the fluorophore may have its structure altered by photocutting light, thus obfuscating the analysis). However, in some cases, it may be useful to place the fluorophore on the mass reporter, such as to quantify the photocutting efficiency of PC-MT by fluorescence.

[0118] Finally, in general, as discussed in the preceding paragraphs, this invention is not limited to the probe type or mass spectrometry analysis type used for MSI. Therefore, more generally, the PC-MT probe-based MSI technique of this invention is referred to as PC-MT-MSI (wherein the aforementioned mass spectrometry-based immunohistochemistry [MIHC] and mass spectrometry-based in situ hybridization [MISH] methods are some of many possible methodological subtypes).

[0119] Multi-omics tissue imaging using PC-MT probes

[0120] A key advantage of this invention is its ability to perform multi-omics imaging of tissue samples, where "omics" refers to the measurement of a characteristic of a broad class of cellular molecules, including but not limited to genes (genomics), proteins (proteomics), small metabolites (metabolomics), glycans (glycomics), or RNA (transcriptomics) [(2012) Evolution of Translational Omics: Lessons Learned and the Path Forward]. In the case of tissue imaging, such a measurement of "characteristics" can be, for example, the spatial localization, morphological analysis, and co-localization analysis of these cellular molecules, and may also include quantification (e.g., quantifying biomarker levels or scoring the number of biomarker-positive cells). Thus, multi-omics is a combination of measurements and analyses of different omics groups (e.g., combined proteomics and glycomics). In this invention, in the context of tissue imaging, multi-omics can be achieved using, for example, different PC-MT probe types or classes (e.g., antibodies and lectins) and / or different MSI “modes” (e.g., non-targeted direct MSI of endogenous tissue biomolecules and MSI of biomolecules targeted by specific PC-MT probes). In a preferred embodiment, different omics measurements are performed on the same tissue slice. However, these measurements can also be performed individually, preferably from consecutive / adjacent tissue slices cut from the same tissue sample / block. Furthermore, different omics measurements can be performed simultaneously (e.g., by treating the tissue with a mixture of different PC-MT probe classes (such as antibodies and lectins)) or sequentially (one omics measurement followed by another) on tissue slices / samples. For example, for fluorescence imaging, this has been accomplished by using various methods of arranging lectins and antibodies [Zupancic, Kreft et al. (2020) Eur J Histochem 64]. In cases where different omics measurements are performed sequentially on tissue samples, the present invention is not intended to be limited to the order in which these measurements are performed, as many permutations can produce useful results, as will be apparent in the following paragraphs.

[0121] Finally, it should be noted that when carbohydrate-binding probes (such as lectin probes) are combined with antibody probes for simultaneous tissue treatment, it may be important to use antibody probes lacking glycosylation to avoid cross-reactions between lectin and antibody probes, which could confound tissue imaging results (e.g., producing artifacts that do not represent the true endogenous biomarker patterns of the tissue). This can be accomplished, for example, using antibodies lacking the glycosylated Fc domain, including but not limited to Fab fragment antibodies, F(ab')2 fragment antibodies, nanobodies, single-chain fragment variable (scFv) antibodies, and VHH single-domain antibodies (e.g., camel single-domain VHH antibodies). Intact antibody probes containing the Fc domain can still be used concurrently with carbohydrate-binding probes (such as lectins) as long as antibody glycosylation is absent, including but not limited to the use of recombinant antibodies (e.g., generated in prokaryotic expression systems) or antibodies that have been chemically or enzymatically deglycosylated (e.g., using the enzyme PNGase F). More broadly, when combining different probe classes for simultaneous tissue processing, it is important to take measures to avoid cross-reactions between probes to prevent artifacts in tissue imaging.

[0122] Multi-omics approaches in this invention are facilitated by using MSI methods that employ “soft” ionization for molecular analysis, as these methods generally do not cause molecular fragmentation or cause limited molecular fragmentation, and more specifically, do not atomize molecules for detection (allowing, for example, the detection of intact peptides or polymer-based mass tags in tissues, as well as intact endogenous biomolecules such as lipids, drugs, or metabolites). Soft ionization methods include, but are not limited to, laser desorption / ionization (LDI), matrix-assisted laser desorption / ionization (MALDI), desorption / ionization on silicon (DIOS), fast atom / ion bombardment (FAB), and electrojet ionization (ESI) [Siuzdak (2004) JALA 9:50-63], including derivatives of the previously discussed ESI, such as DESI and LAESI. The foregoing list is not intended to limit the invention to any particular type of “soft” ionization mass spectrometry. For the purposes of this invention, “soft” ionization-based methods are defined as methods for ionizing and detecting molecular ions (i.e., charged molecules consisting of two or more atoms held together by chemical bonds).

[0123] Dual Continuous MSI: In a simple multi-omics implementation, it is useful to first perform label-free direct MSI on endogenous small biomolecules (e.g., metabolomics), and then perform MSI on targeted biomolecules (e.g., protein targets such as in proteomics) using PC-MT-MSI, preferably on the same tissue slice. Targeted PC-MT-MSI is necessary for the detection of biomolecules that are typically not analyzable by direct MSI, such as macromolecular targets that cannot be well ionized on their own, may fragment in an undesirable manner even with soft ionization, and / or are not well resolved, for example, by mass spectrometry. Including PC-MT-MSI in a multi-omics workflow is also important when it is desired or necessary to target at least some known biomarkers. In general, this implementation of multi-omics tissue imaging can be important, for example, for co-localizing drug compounds (small molecule detection via direct label-free MSI) and drug targets (macromolecular detection via PC-MT-MSI, since drug targets are often proteins). In a preferred embodiment, label-free direct MALDI-MSI is first performed using freshly frozen (FF) tissue sections so that the tissue has not yet undergone tissue fixation (ideally for direct label-free MSI of endogenous molecules; however, formalin fixation and paraffin embedding [FFPE] are possible [Wisztorski, Franck et al. (2010) Methods Mol Biol 656:303-22]). In a preferred embodiment, the tissue sections are then washed, fixed if not previously fixed, and then subjected to PC-MT-MSI methods, such as MIHC and / or MISH, which include another MSI cycle, on the same tissue sections.

[0124] In some embodiments, it is useful to first perform label-free direct MSI on tissue sections, for example, for the direct detection of small biomolecules, followed by targeted MIHC and / or MISH on the same tissue sections for the detection of macromolecular targets and / or nucleic acid targets. This can be important, for example, for co-localization of drug compounds (small molecule detection via direct label-free MSI) and drug targets (macromolecular detection via targeted PCMT probe-based MSI). In a preferred embodiment, label-free direct MALDI-MSI is performed first using fresh frozen tissue sections so that the tissue has not yet undergone tissue fixation (ideally for direct label-free MSI of endogenous molecules; however, FFPE is possible [Wisztorski, Franck et al. (2010) Methods Mol Biol 656:303-22]). In a preferred embodiment, the tissue sections are then washed (e.g., in an organic solvent) to remove any remaining matrix compounds, and then MIHC and / or MISH and a subsequent round of MALDI-MSI are performed on the same tissue sections as described above. See Experimental Example 4 for more details.

[0125] It should be noted that this invention is facilitated by using mass spectrometry methods that employ “soft” ionization for molecular analysis, as these methods generally do not cause molecular fragmentation or cause limited molecular fragmentation, and more specifically, do not atomize molecules for detection (allowing, for example, the detection of peptides or polymer-based mass tags in tissues and endogenous biomolecules). Such methods include, but are not limited to, laser desorption / ionization (LDI), matrix-assisted laser desorption / ionization (MALDI), desorption / ionization on silicon (DIOS), fast atom / ion bombardment (FAB), and electrospray ionization (ESI) mass spectrometry [Siuzdak (2004) JALA 9:50-63]. The foregoing list is not intended to limit this invention to any particular type of “soft” ionization mass spectrometry. For the purposes of this invention, “soft” ionization-based methods are defined as methods capable of detecting molecular ions (i.e., charged molecules consisting of two or more atoms held together by chemical bonds).

[0126] In another preferred multi-omics implementation, the targeted PC-MT-MSI approach is combined with the non-targeted "bottom-up" omics approach.

[0127] Bottom-up omics refers to the fact that biomolecules are first broken down (digested) into smaller fragments that are easier to analyze by mass spectrometry. The identity and structure of the whole biomolecule can then be inferred from the mass spectrometry analysis. In other bottom-up cases, it is not necessary to know the identity of the biomolecules, because, for example, only the fingerprints of various mass substances (measured with high precision) may be associated with a disease state or stage. In the context of bottom-up tissue MSI, tissue is treated with digestion reagents to release biomolecule fragments that are easier to analyze by mass spectrometry. See, for example... Figure 22 Typically, digestion reagents are sprayed onto tissues in the form of a thin film to promote digestion without causing analyte delocalization, a process known as in situ digestion. However, achieving a balance between adequate digestion and minimal delocalization is challenging, and a perfect balance may not exist. These digestion reagents can be enzymes such as nucleases (e.g., restriction enzymes), proteases (e.g., chymotrypsin, trypsin, LysC, or AspN), kinases (e.g., PKC), phosphatases (e.g., alkaline phosphatase), or glycosidases (e.g., peptidyl N-glycosidase F [PNGase F]), or they may be chemical reagents such as cyanogen bromide (CNBr) or hydroxylamine [Gundry, White et al. (2009) Curr ProtocMol Biol Chapter 10: Unit 10 25; Barrett, Wither et al. (2017) J Proteome Res 16:4177-4184]. In situ tissue digestion and subsequent MSI have been previously reported. For example, Drake et al. [Drake, Powers et al. (2018) Curr Protoc Protein Sci 94:e68] sprayed the glycosidase PNGase F onto FF and FFPE tissues to proteolytically cleave N-linked glycans from the tissues, followed by MALDI-MSI of the released glycans. Other examples of using proteases as digestive agents in this context include using collagenases to digest specific protein types or using trypsin for general protein digestion [Angel, Schwamborn et al. (2019) Proteomics Clin Appl 13:e1700152; Lazova, Smoot et al. (2020) J Cutan Pathol 47:226-240].

[0128] When combining bottom-up MSI with PC-MT-MSI in a multi-omics tissue imaging protocol, this invention is not intended to limit to a specific sequence of operations. For example, PC-MT-MSI can be performed before or after bottom-up MSI (both options require two rounds of MSI). Conversely, tissue can be stained with a PC-MT probe, then digested in situ, followed by only one round of MSI. In the case of performing PC-MT-MSI first, it may be desirable to subsequently remove the PC-MT probe in case it might interfere with the subsequent bottom-up MSI. To do this, the probe can be detached from the tissue first by denaturation treatment, followed by washing away the probe. Such denaturation treatment can include, but is not limited to, dissociation agents, solutions with pH ≤ 5, solutions with pH ≥ 10, reducing agents, oxidizing agents, heat, organic solvents and / or detergents (e.g., ionic detergents such as SDS, nonionic detergents such as Triton X-100, or zwitterionic detergents such as CHAPS). See, for example Figure 22 .

[0129] It should be noted that although the above content covers a wide range of multi-omics methodologies, two specific examples of digestion using in situ proteases or glycosidases are provided in Experiment 15.

[0130] Table 1. Quality unit sequence and quality report quality for PC-MT (For example PC-MT structure, see...) Figure 3 ).

[0131]

[0132]

[0133] The shaded (bold) rows are mass unit 1 or variants of mass unit 1 used to minimize the variable MALDI-MS ionization efficiency. The letters in parentheses represent the stable isotopic amino acids shown in Table 1.1.

[0134] *The mass unit is the N-terminus acetylated on the α-amine; the mass report includes this acetylation plus the mass unit and a small portion of the photocut PC-connector (see [link]). Figure 3 Step 3).

[0135] Table 1.1. Amino acid isotopes.

[0136]

[0137] Experimental Section

[0138] Materials used in the experiment.

[0139] Water (LCMS grade) and xylene (semiconductor grade) were sourced from Acros Organics (Pittsburgh, PA). Methanol (LCMS grade) was sourced from JTBaker (Avantor, Radnor, PA). Ethanol (biological reagent for molecular biology), acetone (HPLC grade), N,N-dimethylformamide (anhydrous, ≥99.8%), 1,5-diaminonaphthalene (DAN, 97%), 2,5-dihydroxybenzoic acid (DHB, 98.0%), isopropanol (biological reagent for molecular biology), sodium chloride (BioXtra, ≥99.5%), sodium bicarbonate (99.7%–100.3%, for molecular biology experiments), ammonium bicarbonate (BioUltra, ≥99.5%), bovine serum albumin (heat shock fraction, protease-free, fatty acid-free, substantially globulin-free, pH 7, ≥98%), phosphate-buffered saline (PBS) (BioPerformance Certified, pH 7.4, P5368), glycine (ultrapure for molecular biology, ≥99%, Fluka Biochemika), paraformaldehyde (powder, 95%), citrate buffer (pH 7.4, P5368). 6.0, 10x, Antigen Retriever), 5-(ethylthio)-1H-tetrazole (5-ETT, 95%), 1-methylimidazolium (Reagent Plus, 99%), formamide (biological reagent for molecular biology, ≥99.5%), RNA, tRNA from baker's yeast, Denhardt solution (50 x 5 mL), Atto-647N-NHS ester and octyl β-D-glucopyranoside (OBG) (50% [w / v] stock solution) were all from Sigma Aldrich (St. Louis, MO). 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DyLight 650NHS ester, anti-streptavidin antibody clone S3E11, hydroxylamine, Slide-A-Lyzer TM0.1 mL MINIDialysis Devices (with a 20 kDa membrane) and biotinylated protein G were from Thermo Fisher Scientific (Waltham, MA). CHAPS (powder, ≥98%, MP Biomedicals), Invitrogen ultrapure salmon sperm DNA solution, and Electron Microscopy Sciences Secure-Seal Hybridization Chambers (1 well, 22 mm x 53 mm x 0.6 mm deep) were from Fisher Scientific (Hampton, NH). Tris HCl and Tris base (molecular biology grade), 5N sodium chloride (molecular biology grade), 0.5 M EDTA (pH 8; molecular biology grade), Tween-20 (molecular biology grade), SSC buffer 20X (molecular biology grade), and nuclease-free water were from Promega (Madison, WI). Antigen Retrieval Reagent-Basic (CTS013) was obtained from R&D Systems / BioTechne (Minneapolis, MN). Gold-coated microscope slides. All samples were obtained from Angstrom Engineering Inc. (Kitchener, ON, Canada). Mouse C57 brain sagittal FFPE sections (5 μm thick) and fresh frozen (FF) sections of mouse C57 brain sagittal sections (embedded in 2% w / v CMC, 10 μm thick) were obtained from Zyagen (San Diego, CA). FFPE human tonsil tissue blocks were obtained from amsbio LLC (Cambridge, MA), and FFPE human breast cancer tissue blocks were obtained from OriGene (Rockville, MD). FFPE tissue blocks were sent to Zyagen (San Diego, CA) for sectioning (5 μm thick) and mounting on slides.The antibodies used for multipath imaging of mouse brain slices were obtained from the following suppliers: anti-myelin basic protein antibody (MAB42282) from R&D Systems / BioTechne (Minneapolis, MN); anti-NeuN antibody (MAB377) and anti-synaptic-2 antibody (MABN1573) from Millipore Sigma (Burlington, MA); anti-GLUT1 antibody (PA146152) from Fisher Scientific (Hampton, NH); and anti-MAP2 antibody (ab11268), recombinant anti-NeuN antibody (ab209898), recombinant anti-Cas9 (ab218654; BSA and azide-free), recombinant anti-pan-cytokeratin antibody [C-11]-BSA and azide-free (ab264485), and recombinant anti-CD3rε antibody (anti-CD3repsilon). Antibody [CAL57] - BSA and azide-free (ab251607), Recombinant anti-CD4 antibody [EPR6855] - BSA and azide-free (ab181724), Recombinant anti-CD8α antibody [CAL66] - BSA and azide-free (ab251596), Recombinant anti-CD20 antibody [EP459Y] - BSA and azide-free (ab214282), Anti-CD45RO antibody [UCH-L1] (ab23), Recombinant anti-estrogen receptor α antibody [SP1] - BSA and azide-free (ab187260), Recombinant anti-progesterone receptor antibody [YR85] - BSA and azide-free (ab206926), Recombinant anti-ErbB 2. Antibodies [CAL27] - BSA and azide-free (ab251602), recombinant anti-histone H2A.X antibody [EPR22820-23] - ChIP grade - BSA and azide-free (ab256544), recombinant anti-CD68 antibody [EPR20545] - BSA and azide-free (ab227458), and recombinant anti-Ki67 antibody [EPR3610] - BSA and azide-free (ab209897) were all derived from Abcam (Cambridge, MA). Mouse IgG complete molecules (015-000-003), normal mouse serum (015-222-001), rabbit IgG complete molecules (011-000-003), and normal rabbit serum (011-000-001) were all derived from Jackson Immuno Research Laboratories, Inc. (West Grove, PA).For certain bead array experiments, recombinant anti-β-amyloid 1-42 antibody (ab224275) and recombinant anti-myelin basic antibody (ab230378) (both BSA- and azide-free) were from Abcam (Cambridge, MA). U6-amine-customized LNA oligonucleotides (339406YCO0191704), sense miR-159-amine-customized LNA oligonucleotides (339406YCO0191705), and miRCURY LNA miRNA ISH buffer sets for FFPE were from Qiagen (Germantown, MD). Streptavidin-coated 20 μm and 37 μm PMMA microbeads (microspheres) were from PolyAn GmbH (Berlin, Germany). 0.5 mL Ultrafree-MC centrifugal 0.45 μm filter devices were from Millipore Sigma (Burlington, MA). Both the NAP-5 Sephadex G-25 column and the PD SpinTrap G-25 column are from GE Healthcare Life Sciences (Pittsburgh, PA); 4-[4-[1-(9-fluorenylmethoxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy]butyric acid, referred to hereafter as 4-[4-[1-(9-Fmoc-amino)ethyl]-2-methoxy-5-nitrophenoxy]butyric acid, is from Santa Cruz Biotechnology (Dallas, TX). FlexWell. TM The 16-chamber self-adhesive gaskets (204916) are all from Grace Bio-Labs (Bend, Oregon).

[0140] Example 1. A 15-channel PC-MT-Ab based MSI model system using a microbead array as a model system.

[0141] PC-MT.

[0142] Peptide-based PC-MTs were prepared using standard Fmoc amino acid solid-phase peptide synthesis (SPPS) [Behrendt, White et al. (2016) J Pept Sci 22:4-27]. Fmoc-protected, light-cleavable amino acid linkers (see...) Figure 3 The Fmoc-PC linker is introduced into the peptide chain in the same manner as other amino acids, and the N-terminal α-amine of the peptide is acetylated with acetic anhydride using a standard procedure. An NHS-ester probe reactive moiety is generated on the ε-amine of the lysine (K) contained in the spacer unit (see [link to NHS-ester probe reactive moiety]). Figure 3Step 1, NHS-ester). The conversion of the ε-amine of lysine (K) to NHS-ester was achieved using dioctanoic acid disuccinimide ester (DSS). The use of bifunctional succinimide esters such as DSS or DSC (disuccinimide carbonate) has previously been reported for the conversion of primary amines to NHS-esters [Morpurgo, Bayer et al. (1999) J Biochem Biophys Methods 38:17-28]. PC-MT was purified by HPLC after synthesis using an Onyx Monolithic C18 column and a solvent system of 0.05% TFA in H2O / 0.05% TFA in acetonitrile. The exemplary chemical structure of PC-MT is shown below. Figure 3 As shown. Fifteen different PC-MTs were prepared, and... Figure 3 The differences shown are only in the amino acid sequence of the mass unit. Table 1 lists 15 different mass unit sequences (ID1, 2-3, and 5-16) and the monoisotopic masses of the mass reporter, as shown in... Figure 3 As shown in (Step 3), this includes a small portion of the N-terminal acetylated, peptide mass unit, and photocut PC connector.

[0143] In some cases, additional lysine residues are included in the spacer unit of PC-MT, and the spacer unit is modified with a fluorophore using a commercially available amine-reaction reagent (e.g., sulfonyl-Cy5-NHS, from Lumiprobe, Hunt Valley, Maryland). In this case, the amino acid sequence of the spacer unit is GS[K-sulfonyl-Cy5]GG[K-NHS] (SEQ ID NO:30), instead of GSGG[K-NHS] (SEQ ID NO:31) in the non-fluorescent PC-MT.

[0144] Preparation of PC-MT antibody (PC-MT-Ab)

[0145] Add 1 / 9 volume of 1M sodium bicarbonate to 100 μL of antibody solution (1 μg / μL in PBS), then add sufficient PC-MT from 1 mM stock solution in anhydrous DMF, in a 10-fold molar excess relative to the antibody. Incubate for 1 hour in the dark with gentle mixing. Quench the reaction with 1 / 9 volume of 1M glycine, then mix in the dark for 15 minutes. Finally, add 1 / 199 volume of 10% (w / v) BSA carrier stock solution in water to obtain 0.05% BSA (w / v). To remove unreacted PC-MT, treat the obtained PC-MT antibody (PC-MT-Ab) on a PD SpinTrap G-25 column using TBS (50 mM Tris, pH 7.5, 200 mM NaCl) as a pre-equilibration buffer according to the manufacturer's instructions. Further supplement the obtained PC-MT-Ab with 1 / 9 volume of 10X TBS. In some cases, this is done by using Slide-A-Lyzer. TM Extensive dialysis was performed in TBS using a 0.1 mL MINI dialysis apparatus (with a 20 kDa membrane) to purify PC-MT-Ab.

[0146] microbead array

[0147] Unless otherwise noted, streptavidin-coated 20 μm PMMA beads were processed in a 0.5 mL Ultrafree-MC centrifugal 0.45 μm filter apparatus (washed by 3 s vortexing of the bead suspension in the filter apparatus and filtered for 5 s at 15,000 rpm on a standard microcentrifuge to separate the beads from the solution). For each PC-MT-Ab form, 10,000 beads were used and processed separately unless otherwise noted. The beads were first washed 4 x 400 μL with bead blocking buffer (1% BSA [w / v] in TBS-T; it should be noted that TBS-T is TBS supplemented with 0.05% [v / v] Tween-20). The beads were then probed separately with 15 different forms of streptavidin-resistant PC-MT-Ab, each PC-MT-Ab form carrying a different PC-MT substance (i.e., different mass units - see Table 1 for mass units with ID1, 2-3, and 5-16). PC-MT-Ab was diluted to 1 μg / mL in bead blocking buffer and probed for 1 hour using 100 μL of the solution while gently mixing. The beads were then washed with TBS-T for 4 x 400 μL, and all 15 different bead forms were combined. The combined beads were then further washed with mass spectrometry grade water (MS-water) for 4 x 400 μL. As previously reported [Lim, Liu et al. (2014) Rapid communications in mass spectrometry: RCM 28:49-62; Zhou, Liu et al. (2016) Sci Rep 6:26125], bead arrays were formed on indium tin oxide (ITO) coated microporous substrates with a standard microscope slide footprint.

[0148] Light cutting

[0149] Finally, the substrate was dried in a vacuum drying chamber for 45 minutes and then irradiated with 365nm light at a distance of 5cm using an XX-15 lamp (UVP / Analytik JenaUS LLC, Upland, CA) at a distance of approximately 3mW / cm. 2 Alternatively, an LED Cube100IC (Honle UV Technology, Marlboro, MA) can be used to illuminate the substrate with 365nm light at approximately 30mW / cm². 2 Unless otherwise specified, use 5 minutes of light processing.

[0150] Matrix application

[0151] Next, according to publicly available reports [Hankin, Barkley et al. (2007) J Am Soc Mass Spectrom 18:1646-52; Duenas, Carlucci et al. (2016) J Am Soc Mass Spectrom 27:1575-8], the DHB or DAN matrix was applied to the dry substrate by sublimation followed by recrystallization.

[0152] MALDI-MS imaging (MALDI-MSI)

[0153] MALDI-MSI imaging was performed using a rapifleX MALDI-TOF-MS instrument (Bruker Daltonics, Billerica, MA) with the following parameters: reflector mode; laser spot size of 10 or 20 μm, using a 10 or 20 μm continuous grating scan; 300-500 laser emissions / pixel; and normalization to total ion count (TIC) in some cases. Image and spectral analysis were performed using flexImaging and flexAnalysis software (Bruker Daltonics, Billerica, MA).

[0154] result

[0155] Figure 6 The results in the inset show the MALDI-MS "mass image" of the microbead array. Different colors in the inset correspond to different m / z values ​​of the monospherical mass spectra of specific PC-MT mass reporters (see Table 1 for expected masses of mass reporters with ID1, 2-3, and 5-16). Fifteen distinct and mass-resolved mass reporters were observed, derived from PC-MT-Ab probes bound to different 20 μm microbeads in the array. Color-coded overlapping MALDI-MSI spectra from representative individual microbeads within the array are also shown. Figure 6 (Black arrow) does not show microbead crosstalk.

[0156] Example 2. Application of PC-MT-Ab 5-way MIHC on mouse sagittal FFPE brain tissue sections

[0157] Immunostaining with PC-MT-Ab

[0158] PC-MT and PC-MT-Ab were prepared as described in Example 1 and used for MIHC as follows: For deparaffinization and hydration, FFPE tissue sections were treated as follows (each treatment step was performed in a separate staining jar): treated with xylene 3 times for 5 minutes each time; treated with xylene:ethanol (1:1) once for 3 minutes; then hydrated with 100% ethanol twice for 2 minutes each time, hydrated with 95% ethanol twice for 3 minutes each time, hydrated with 70% ethanol once for 3 minutes, hydrated with 50% ethanol once for 3 minutes, and hydrated with TBS once for 10 minutes.

[0159] Antigen retrieval was achieved in a beaker preheated for 1 hour in a 95°C water bath with 200 mL of 1X citrate buffer (pH 6.0; see Materials), followed by cooling to room temperature for 30 minutes. The slides were then blocked for 1 hour in a staining jar with 50 mL of tissue blocking buffer (2% [v / v] normal serum [rabbit and mouse] and 5% (w / v) BSA in TBS-T; note that TBS-T is supplemented with 0.05% [v / v] Tween-20 TBS). For PC-MT-Ab staining, slides were treated overnight at 4°C with 200 μL / slide of a solution containing 2.5 μg / mL of each antibody, diluted in tissue blocking buffer (incubated in a humidified chamber to avoid evaporation, and each tissue slide was surrounded by a hydrophobic barrier to retain the liquid).

[0160] The slides were then washed as follows: three times with TBS for 5 minutes each, and then three times with 50 mM ammonium bicarbonate for 2 minutes each (Note that all solutions were in LCMS grade water, and all washes were performed with excess solution, and the slides were placed horizontally in the petri dish and gently shaken).

[0161] Finally, optical cutting, matrix application, and MALDI-MSI were performed as in Example 1.

[0162] It should be noted that in some cases, immunofluorescence is performed instead of PC-MT-Ab staining. In these cases, an antibody labeled with a 15-fold molar excess of DyLight 650NHS ester reagent (added from a 5 mM stock solution in DMF) is used instead of a PC-MT-labeled antibody (the same labeling procedure described above with respect to PC-MT-Ab in Example 1 is used in other respects). Furthermore, photocutting, matrix application, and MALDI-MSI are not performed; instead, the dried slide is imaged at 5 μm resolution on a GenePix 4200A fluorescence scanner (Molecular Devices, San Jose, CA). All other procedures are the same as described with respect to MIHC in this example.

[0163] result

[0164] 5-way MIHC was performed on sagittal sections of mouse brains via FFPE. For this purpose, different PC-MTs were directly conjugated to the antibody in one step. These five antibodies target myelin basic protein (a well-known axonal sheath marker, e.g., [van Tilborg, van Kammen et al. (2017) Sci Rep 7:16492]), NeuN (a neuronal nuclear marker, e.g., [Gusel'nikova and Korzhevskiy (2015) Acta Naturae 7:42-7]), synaptoprotein (a synaptic protein, e.g., [Mason (1986) Neuroscience 19:1319-33]), Glut-1 (rich in capillaries of brain tissue, e.g., [Tang, Gao et al. (2017) Nat Commun 8:14152]), and MAP-2 (a microtubule-associated protein present in neural tissue, e.g., [Wiche, Briones et al. (1983) EMBO J 2:1915-20]). MALDI-MSI was performed at 10 μm spatial resolution on an in-house Bruker rapifle XMSI instrument. Figure 7a shows a 5-color MALDI-MS "quality image" of the entire brain slice. Different colors correspond to different m / z values ​​of single isotope mass spectrometry peaks for a specific PC-MT. Myelin (red), NeuN (green), and synaptoprotein (blue) are the most prevalent and produce the most distinctive structural patterns. For example, NeuN produces the distinctive "vortex" pattern of the hippocampus (indicated by *). Furthermore, myelin, NeuN, and synaptoprotein highlight the three distinct layers of the cerebellum (using *). (Indicated). Less obvious biomarkers hidden in composite color images are best viewed as monochrome images. For example, Figure 7c shows a monochrome standalone MALDI-MS image of the Glut-1 biomarker (primarily showing cross-sections of brain capillaries). MAP-2 produces fairly uniform staining of brain slices (not shown as a standalone image). Furthermore, non-immunotype control immunoglobulins matched with substances carrying the same PC-MT do not show MALDI-MS signals (not shown). Color-coded overlapping mass spectra are shown for selected pixels of the MALDI-MS images (those marked with arrows in Figure 7a) (Figure 7e). Finally, MIHC results show a clear match with conventional immunofluorescence. For example, using antibodies to myelin, NeuN, and synaptic proteins labeled only with fluorophores produced the same pattern as MIHC in the cerebellum (Figure 7b). The Glut-1 MALDI-MS pattern also matches immunofluorescence (Figure 7d).

[0165] Example 3. MISH using PC-MT-NA miRNA hybridization probes on FFPE tissue sections

[0166] Preparation of PC-MT nucleic acid (PC-MT-NA)

[0167] The amine-modified locked nucleic acid (LNA) probe (see Materials) was labeled with PC-MT as follows: Sufficient PC-MT from a 10 mM stock solution in anhydrous DMF was added to 100 μL of LNA probe solution (10 μM in 200 mM sodium chloride and 200 mM sodium bicarbonate), resulting in a 200-fold molar excess relative to the LNA probe (2 μL of 10 mM stock solution was added every 30 minutes for a total of 5 additions). The reaction was carried out for a total of 2.5 hours under light protection and gentle mixing. To remove unreacted PC-MT, the obtained PC-MT-NA was treated on a NAP-5 Sephadex G-25 column using TE-150 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, and 150 mM NaCl) as a pre-equilibration buffer according to the manufacturer's instructions.

[0168] In situ hybridization with PC-MT-NA

[0169] For deparaffinization and hydration, FFPE tissue sections were treated in the same manner as in Example 2. The following procedure is adapted from Renwick et al. [Renwick, Cekan et al. (2013) J Clin Invest 123:2694-702]: Tissue sections were prepared for in situ hybridization by digestion with protease K, EDC fixation, and acetylation. For protease K digestion, 1.5 μL of a protease K stock solution from the miRCURY LNA miRNA ISH buffer set for FFPE (see Materials) was diluted to 2.0 mL in protease K buffer (5 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM NaCl), and 300 μL was incubated with each tissue section at 37 °C for 10 min. After protease K digestion, the tissue sections were washed twice for 10 min each with 0.2% (w / v) glycine in PBS. For EDC fixation, tissue sections were pretreated twice for 3 minutes each in 0.1M 1-methylimidazole solution, followed by treatment with 200 μL of EDC fixation solution for 1 hour (freshly prepared by adding 1.0 mL of 0.1M 5-ETT and 0.1M 1-methylimidazole to a 10 mg EDC HCl vial; pH adjusted to 8.0 with 10M NaOH). After EDC fixation, tissue sections were washed for 10 minutes with 0.2% (w / v) glycine in PBS. For acetylation, an acetylation solution was freshly prepared by adding 149 μL of triethanolamine, 2 μL of HCl (37%), and 5 μL of acetic anhydride to 846 μL of nuclease-free water, and 200 μL was incubated with each tissue section for 10 minutes. After acetylation, tissue sections were washed for 3 minutes in excess PBS. Next, the tissue sections were pre-hybridized for 1 hour at 25°C in 150 μL of hybridization buffer, which consisted of 50% formamide, 1.0 M NaCl, 75 mM Tris-HCl (pH 8.5), 1x Denhardt solution, 250 μg / mL baker's yeast tRNA, 500 μg / mL salmon sperm DNA, 5 mM CHAPS, and 0.1% Tween-20. After pre-hybridization, self-adhesive hybridization chambers were placed on glass slides (each chamber was 22 mm x 53 mm x 0.6 mm deep). For hybridization, the PC-MT U6 and 159LNA probes were diluted to 200 nM in the hybridization buffer, and the tissue sections were incubated overnight at 55°C (protected from light) in a humidity-controlled chamber. The tissue sections were then washed in 5X SSC buffer for 1 x 5 minutes, twice in 1X SSC buffer for 5 minutes each time, and in 0.2X SSC buffer at 55°C for 1 x 5 minutes, and then in 0.2X SSC buffer at room temperature for 1 x 5 minutes.

[0170] The slides were then washed as follows: three times with TBS for 5 minutes each, and then three times with 50 mM ammonium bicarbonate for 2 minutes each (Note that all solutions were in LCMS grade water, and all washes were performed with excess solution, and the slides were placed horizontally in the petri dish and gently shaken).

[0171] Finally, optical cutting, matrix application, and MALDI-MSI were performed as in Example 1.

[0172] It should be noted that in some cases, FISH is performed instead of PC-MT-NA staining. In these cases, nucleic acids labeled with Atto-647N-NHS ester reagent are used instead of PC-MT-labeled nucleic acids (the same labeling procedure described earlier in this embodiment regarding PC-MT-NA is used in other respects). Furthermore, photocutting, matrix application, and MALDI-MSI are not performed; instead, the dried slides are imaged at 5 μm resolution on a GenePix 4200A fluorescence scanner (Molecular Devices, San Jose, CA). All other procedures are the same as described in this embodiment regarding MISH.

[0173] result

[0174] To confirm MISH, an amine-reactive NHS-activated PC-MT labeling reagent was directly conjugated to a 5' amine-capped LNA hybridization probe (LNA = locked nucleic acid) to obtain better affinity [Vester and Wengel (2004) Biochemistry 43:13233-41; Sempere, Christensen et al. (2007) Cancer Res 67:11612-20; Robertson and Thach (2009) Anal Biochem 390:109-14; Nielsen (2012) Methods Mol Biol 822:67-84; Renwick, Cekan et al. (2013) J Clin Invest 123:2694-702; Kasai, Kakihara et al. (2016) Front Mol Neurosci 9:126; Lei, van Mil et al. (2018) Biotechnol Rep(Amst)18:e00255]. Then, mouse sagittal brain tissue sections were stained with probes and subjected to MALDI-MSI. The probes were U6, a snRNA commonly used as a positive control in miRNA FISH, and miR-159, a negative control plant-specific sequence [Lei, van Mil et al. (2018) Biotechnol Rep(Amst)18:e00255]. Figure 8 The results showed that MISH using the U6 probe produced tissue staining patterns similar to those of conventional FISH methods, while the negative control miR-159 probe did not produce any noticeable staining patterns.

[0175] Example 4. Multi-omics MSI: Non-targeted label-free MSI and targeted MIHC of lipids on the same FF tissue sections

[0176] Non-targeted label-free direct MSI on FF tissue sections

[0177] Untreated fresh frozen (FF) tissue sections were first analyzed directly in a non-targeted manner by MALDI-MSI (after thawing the tissue sections and only the matrix application as in Example 2 [in this case, DAN matrix]).

[0178] MIHC on the same FF tissue section

[0179] Next, the FF tissue sections do not require deparaffinization; however, fixation with polyoxymethylene (PFA) is performed as follows (each step in a separate staining vessel): pre-wash twice with cold acetone for 3 minutes each time (note that this is used to remove any remaining matrix compounds from the previous MALDI-MSI while providing solvent fixation) and air-dry for 10 minutes; fix with 1% PFA in PBS at pH 7.4 for 10 minutes (it should be noted that this solution is freshly prepared as follows: 1.0 g of PFA is dissolved in 60 mL of PBS containing 1.0 mL of 1 M NaOH with constant stirring on a heating block at approximately 60°C, followed by adjusting the pH to 7.4 with 1 M HCl [~1 mL], and adjusting the final volume to 100 mL with PBS); wash once with PBS for 10 minutes. The remaining steps of the PC-MT-Ab-based MIHC are performed as described in Example 2, from the antigen retrieval step to the end.

[0180] result

[0181] Highly advantageously, this allows for the detection of both untargeted, label-free small molecules and PC-MT-Ab-targeted macromolecules on the same tissue slice. For example, this would allow for the co-localization of small molecule drugs and drug-targets (such as their receptors), as well as related biomolecules involved in the cellular response to the drug. To demonstrate the fundamental feasibility of this capability, we first performed direct MALDI-MSI analysis on freshly frozen mouse sagittal brain tissue slices (fresh freezing is preferred over FFPE to facilitate small molecule detection without the need for tissue fixation or pre-washing). We used a negative-ion mode MALDI-MSI with a DAN matrix. Next, the tissue was washed / fixed with cold acetone to remove MALDI-MS matrix compounds and then further fixed with paraformaldehyde. MIHC was then performed, constituting a second round of MALDI-MSI (so-called dual MSI). The results of the first round of direct MALDI-MSI are shown in Figure 9a. The image colors are coded with the m / z values ​​of three well-known lipids identified from the METLIN database of the Scripps Center for Metabolomics [Smith, O'Maille et al. (2005) Ther Drug Monit 27:747-51], and are consistent with previous MALDI-MSI analyses of lipids from mouse brain tissue sections [Wang, Wang et al. (2018) Anal Chim Acta 1000:155-162] (thioglycosides [N24:1], red, observed m / z 888.7; phosphatidylethanolamine [40:6], blue, observed m / z 790.5; and phosphatidylinositol [38:4], green, observed m / z 885.4). These three lipids are significantly enriched in different brain structures; specifically, thioglycosides (red) show a different pattern from the other two lipids. However, phosphatidylethanolamine (blue) and phosphatidylinositol (green) also exhibit significant co-localization, as expected, they are two major structural lipids of the eukaryotic cell membrane [van Meer, Voelker et al. (2008) Nat Rev Mol Cell Biol 9:112-24] (the co-localized blue and green are shown as cyan in Figure 9a). For confirmatory purposes, Figures 9b and 9c show a two-color overlay of thioglycoside lipids detected in the first round of direct MALDI-MSI, and selected macromolecular biomarkers detected by MIHC in the second round of MALDI-MSI. Figure 9b shows thioglycosides (red) overlaid with the neuronal nuclear biomarker NeuN (green), detected by PC-MT-Ab as previously demonstrated in Example 2. These two biomolecules are not typically co-localized.Conversely, Figure 9c shows the same lipids, thioglycosides (red), covered with myelin basic protein (green), again detected by PC-MT-Ab in a multipath MIHC analysis. In this case, strong colocalization of thioglycosides and myelin is present (evidence of the yellow color resulting from the colocalization of green and red). This colocalization of thioglycosides and myelin is consistent with previous literature indicating that thioglycosides are primarily located in the myelin sheath of neuronal axons (Schwann cells / oligodendrocytes) [Eckhardt (2008) Mol Neurobiol 37:93-103; Hirahara, Wakabayashi et al. (2017) J Neurochem 140:435-450]. In contrast, thioglycosides are not expected to colocalize with the neuronal nuclear biomarker NeuN, as observed here (it should be noted that, as previously shown in Example 2, myelin and NeuN generally do not colocalize). Finally, an example spectrum from the first round of direct MALDI-MSI is shown in Figure 9d. These spectra are derived from selected image pixels chosen from three distinct colored “layers” observed in the olfactory bulb / neural layer of the mouse brain (see the colored arrows in Figure 9a for details on these layers). Future use of MS / MS or higher resolution FTICR mass spectrometry will provide more accurate identification of small molecules.

[0182] Example 5. Preferred connection sites between mass units and PC-cores

[0183] In this invention, the mass unit and the PC-core (see [link to PC-core] for more information) Figure 3 The preferred linkage mode of the benzene ring is ultimately through photo-cleavage sites (see Figure 10a, configuration 1; also see...). Figure 3 In this configuration, the photocleaved benzene ring of the PC-core does not remain attached to the photocleaved mass reporter detected by mass spectrometry (MS). While it is possible to alternatively attach the mass unit to the benzene ring of the PC-core via a site other than the photocleavage site, as taught by Olejnik et al. [Olejnik, Ludemann et al. (1999) Nucleic Acids Res 27:4626-31] and Levy and Caprioli (US Patent No. 7,569,392), this is not preferred because the photocleaved benzene ring of the PC-core remains attached to the photocleaved mass reporter detected by MS (see Figure 10a, configuration 2).

[0184] To demonstrate the benefits of preferred configuration 1, photocleavable peptides with the same mass unit peptide sequence were attached to the surface according to the two configurations shown in Figure 10a. (See also Figure 10a for the structure and sequence of the photocleavable peptides). The mass reporter was then photocleaved from the surface and measured by mass spectrometry. Preferred configuration 1 provides a clean single isotopic peak at the expected mass of the mass reporter (light gray trace in Figure 10b; 1,194.7 m / z; configuration 1), and only the accompanying peak clusters corresponding to the native isotopes of the mass reporter separated by 1 m / z (present, but not discernible in Figure 10b due to X-axis scaling). For configuration 2, the photocut mass reporter does indeed provide the expected monoisotopic mass reporter peak (black trace in Figure 10b; 1,399.9 m / z; configuration 2), but also produces a highly complex mass spectrum, which may be generated by byproducts of the photocut benzene ring still attached to the PC-nucleus of the mass reporter (see Figure 10b, “Byproducts of the PC-nucleus in configuration 2”). These byproducts may include, in part, oxygen adducts and losses resulting from the oxygen radical chemistry involved. Ultimately, this reduces sensitivity (by splitting the mass reporter signal into multiple mass spectral peaks) and confounds mass reporter identification in multipath analysis (through peak overlap).

[0185] Example 6. Comparison of different PC connectors and comparison of pre-cutting with MALDI-MS laser beam versus in-line optical cutting.

[0186] PC-MT

[0187] As described in Example 1, the preparation has Figure 3 The PC-MT with the structural configuration shown (in this embodiment, there are 4 PC-MTs, which are composed of mass units 1 and 2-4 listed in Table 1).

[0188] Furthermore, the so-called PC-MT-L is prepared in the same manner, the only difference being the use of the PC connector from Lemaire et al. ([Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67] [see also U.S. Patent No. 8,221,972], instead of Figure 3 The PC-linker described in [the document]. To achieve this, the PC-linker is introduced into the PC-MT-L during peptide synthesis using 4-[4-[1-(9-Fmoc-amino)ethyl]-2-methoxy-5-nitrophenoxy]butyric acid (see Materials). (For information on this PC-linker incorporated into common peptides, see [the document]). Figure 4 “Lemaire PC-Connector” - Note Figure 4The study described a common peptide to demonstrate the PC-linker itself; however, the common peptide lacked other features of PC-MT and PC-MT-L. Importantly, while the PC-linker of PC-MT-L is the same as that used by Lemaire et al., other features of PC-MT-L (its similarity to...) are missing. Figure 3 The PC-MT features described in [the text] are the same as those taught by Lemaire et al. These features ( Figure 3 This includes, but is not limited to, the probe reactive portion (in this case, NHS-ester), the N-terminal blocking of the peptide-based PC-MT (in this case, N-terminal acetylation), the peptide sequence of the mass unit (Table 1) and the peptide sequence in the spacer unit, and the specific connection of the mass unit to the benzene ring of the PC core via a photocleavage site.

[0189] microbead array

[0190] PC-MT-L was used in equimolar concentrations with PC-MT to simultaneously doubly label streptavidin-coated 37-micron PMMA beads. To achieve this, four bead materials were tested in four independent reactions by simultaneous doubling with PC-MT-L and PC-MT, each containing the same mass unit. This is possible because PC-MT leaves a small residual portion of the PC-connector attached to the photocut mass reporter (see [link to article]). Figure 3 Step 3), while PC-MT-L does not (therefore, even if the mass unit is the same for each microbead material, the mass of the photocut mass reporter is distinguishable in the mass spectrometer).

[0191] The quality labeling of the microbeads was performed as follows: Unless otherwise specified, streptavidin-coated 37-micron PMMA microbeads were treated in a 0.5 mL Ultrafree-MC centrifugal 0.45 μm filter apparatus (washed by 3 s vortexing of the microbead suspension in the filter apparatus and filtered for 5 s at 15,000 rpm on a standard microcentrifuge to separate the microbeads from the solution). 100,000 microbeads were used for each of the four microbead materials (treated individually unless otherwise specified). The microbeads were washed 4 x 400 μL with conjugation buffer (200 mM sodium bicarbonate and 200 mM NaCl). Each microbead precipitate was then resuspended in 100 μL of conjugation buffer, followed by the addition of 2 μL of each of PC-MT-L and PC-MT (in DMF) containing 500 μM. The reactants were mixed for 30 minutes, and then the microbeads were washed three times for 15 minutes each with 400 μL of glycine-hydroxylamine quencher (1 M glycine and 100 mM hydroxylamine in 10x concentrated TBS; freshly prepared). The microbeads were then washed four times for 15 minutes each with 400 μL of OBG saline (25 mM ammonium bicarbonate, 0.05% (w / v) octyl-β-D-pyranoside and 50 mM NaCl), and resuspended in the same buffer to 250 microbeads / μL.

[0192] Finally, the microbead array was formed as follows: 4 x 400 μL of microbeads were washed and resuspended in mass spectrometry-grade purified water (20,000 microbeads / 100 μL). Then, FlexWell microbeads were mounted on a gold-coated microscope slide (see Materials). TM 16-chamber self-adhesive gasket (see materials), microbeads were placed in a chemical fume hood at a density of 400 microbeads / mm. 2 Dry the slides overnight at the specified density. After drying, gently wash the slides in a tray with an excess of mass spectrometry-grade purified water (while the microbeads remain adhered to the slides), and then dry the slides again in a vacuum drying chamber for 45 minutes.

[0193] Photocutting of microbead arrays, matrix application and MALDI-MSI

[0194] The procedure was performed as in Example 1 (it should be noted that the microbeads, which were directly quality-labeled as detailed above, were not detected using PC-MT-Ab as in Example 1). In some cases, the UV irradiation was increased to 25 minutes.

[0195] C-MT-Ab

[0196] Prepared as described in Example 1, except that the anti-myelin antibody was simultaneously double-labeled with PC-MT-L and PC-MT at equimolar concentrations, each containing mass unit 1 listed in Table 1 (i.e., the PC-MT-L and PC-MT labeling reagents were premixed at equimolar concentrations [1 mM each in DMF], and the mixture was then added to the same antibody for double labeling [10-fold molar excess of each compared to the antibody used for labeling]).

[0197] Immunostaining was performed using PC-MT-Ab, light dissection, matrix application, and MALDI-MSI.

[0198] Perform as in Example 2. In some cases, UV irradiation is increased to 25 minutes. In other cases, UV irradiation is not performed, and only online optical cutting using the instrument's laser beam is permitted during MALDI-MSI.

[0199] result

[0200] First, using a microbead array, PC-MT was compared with PC-MT-L for mass units 1 and 2-4 listed in Table 1. Figure 11a shows an example mass image of the microbead array, with pixel plots representing the peak intensities of the mass report mass spectrum at 1,206.7 m / z (PC-MT with mass unit 1) and 1,163.7 m / z (PC-MT-L with mass unit 1) for mass unit 1. PC-MT is color-coded red in the image, and PC-MT-L is color-coded green. If the peaks had similar intensities, the two colors would be expected to produce a yellow color; however, the microbeads appear orange-red because PC-MT produces a much stronger signal. Representative mass spectra of individual pixels from the microbeads in the array for all four mass units under 5 and 25 minutes of pre-UV irradiation (i.e., UV photocutting before matrix application and before MALDI-MSI) are shown in Figures 11b-i. It should be noted that since each of the four microbead materials was double-labeled, each spectrum contains peaks from both PC-MT and PC-MT-L for a given mass unit (PC-MT produces a +43 m / z increase compared to the equivalent mass unit from PC-MT-L because PC-MT leaves a small residual portion of the photocut PC-connector attached to the mass reporter, while PC-MT-L does not). Then, for each mass unit and for each pre-UV time point, the PC-MT to PC-MT-L ratio of the mass spectrum peak intensities was calculated, averaging five pixels (five spectra) for each arrangement. For mass units 1 and 2–4, these ratios for the 5-minute pre-UV were 5:1, 8:1, 10:1, and 6:1, respectively, thus showing a 5–10 times higher PC-MT signal compared to PC-MT-L. Although the PC-MT-L signal shows a modest improvement over the PC-MT-L signal at 25 minutes UV, producing ratios of 2:1, 3:1, 7:1, and 5:1, the improvement is disproportionate to the increase in pre-UV time (which is 5 times longer), resulting in diminishing returns, and PC-MT consistently outperforms PC-MT-L.

[0201] Secondly, using MIHC (immunohistochemistry based on mass spectrometry), mouse brain tissue sections were stained with double-labeled antibody probes targeting myelin, and the mass units 1 of PC-MT and PC-MT-L were compared. In this case, in addition to 5-minute and 25-minute pre-UV treatments of the tissue (just before matrix application and before MALDI-MSI), a 0-minute pre-UV treatment was also tested, in which photodissection was consistent only with MALDI-MSI analysis using the instrument's laser beam. As with the microbead array, myelin imaging of the tissue in Figure 11j shows a two-color quality image, and again, PC-MT (red) provides a much higher signal than PC-MT-L (green), thus dominating the image. The spectra from representative pixels, shown in Figures 11k and 11l, confirm this result. In this case, the peak intensity ratio of PC-MT to PC-MT-L was 6:1 for both 5-minute and 25-minute pre-UV. Finally, the 0-minute pre-UV showed almost no detectable PC-MT or PC-MT-L signal (see Figure 11j). To quantify the effect of pre-UV, average spectra were generated from each of the entire region of interest in each tissue section (see the dashed outline in Figure 11j). These average spectra show that the single isotopic peak intensities of PC-MT were 2.5, 15.8, and 23.3 for 0, 5, and 25 minutes of pre-UV, respectively. Importantly, this confirms that photocutting, consistent with MALDI-MSI analysis using the instrument's laser beam (0-minute pre-UV), is far less sensitive to pre-UV treatment than photocutting (approximately 6-9 times lower in this example).

[0202] Example 7. Fluorescent PC-MT for Immunohistochemistry Based on Mass Spectrometry

[0203] Immunohistochemistry based on mass spectrometry (MIHC)

[0204] MIHC on mouse sagittal brain tissue sections was performed as in Example 2, with the following exception: recombinant anti-NeuN antibody (see Materials) was conjugated to PC-MT, with the conformation as shown. Figure 3 The sequence is shown (mass unit 1 from Table 1), but it additionally contains a sulfonyl-Cy5 fluorescent label linked to an ε-amine of the lysine amino acid added to the spacer unit. Therefore, the complete sequence of this fluorescent PC-MT (referred to as Fluor-PC-MT1) having mass unit 1 is as follows (from N-terminus to C-terminus): Acetyl-APRLRFYSL-[PC-linker]-GS[K-sulfonyl-Cy5]GG-[K-NHS]-COOH (SEQ ID NO:32). The resulting antibody probe (referred to as anti-NeuN Fluor-PC-MT1) was used for MIHC.

[0205] As a negative control, a recombinant anti-Cas9 antibody (see Materials) was conjugated to Fluor-PC-MT1 (note that Cas9 is a bacterial-specific protein that does not exist in mammals). The resulting antibody probe (called anti-Cas9Fluor-PC-MT1) was used for MIHC.

[0206] Finally, as a positive control, only the recombinant anti-NeuN antibody was conjugated to... Figure 3 The non-fluorescent PC-MT constructed in this manner (except for mass unit 7 from Table 1, referred to as PC-MT7) was used as an antibody probe (referred to as anti-NeuN PC-MT7) for MIHC.

[0207] Because Fluor-PC-MT1 contains fluorophores, fluorescence imaging was performed on the same tissue (on a gold-coated slide) in addition to MALDI-MSI (before pre-UV treatment for photocutting, before matrix application, and before MALDI-MSI analysis). Fluorescence imaging was performed as in Example 2.

[0208] result

[0209] Fluorescence images of mouse brain tissue in Figure 12 The fluorescence appears yellow (see “Fluorescence”). A strong and specific fluorescent signal was observed when tissue was probed with anti-NeuN Fluor-PC-MT1. In contrast, tissue probed with the negative control anti-Cas9 Fluor-PC-MT1 showed only a weak background (Cas9 is a bacterial protein absent in mammals). Tissue probed with the non-fluorescent anti-NeuN PC-MT7 showed only a very weak background fluorescence corresponding to the tissue's autofluorescence.

[0210] MALDI-MS images of mouse brain tissue in Figure 12 The markers are shown in red (from the quality reporter of Fluor-PC-MT1) and green (from the quality reporter of PC-MT7) (see “MALDI-MSI”). Tissue detected with anti-NeuN Fluor-PC-MT1 showed a strong and specific MALDI-MSI signal compared to tissue detected using the negative control anti-Cas9 Fluor-PC-MT1 (which also produced only a weak background) (see [link to relevant documentation]). Figure 12 (The red text in the "MALDI-MSI" thumbnail).

[0211] Anti-NeuN Fluor-PC-MT1 “staining pattern” and Figure 12 The fluorescence and MALDI-MS images are similar. Specifically, as in Example 2, anti-NeuN detects unique features such as the hippocampus ( Figure 12(blue arrow in the image) and cerebellum ( Figure 12 (White arrow in the image). However, in MALDI-MSI, the non-fluorescent anti-NeuN PC-MT7 probe produces a more unique pattern than the anti-NeuN Fluor-PC-MT1 probe (see white arrow in the image). Figure 12 (Green in the "MALDI-MSI" thumbnail). Based on Figure 12 The illustration in the image shows the spectrum from the hippocampus ( Figure 12 The blue arrows in the image represent the mass reporter peaks of Fluor-PC-MT1 and PC-MT7, which are individual pixels (black arrows in the spectrum). The superiority of the anti-NeuN PC-MT7 probe does not appear to be due to higher peak intensity (scaled to the same y-axis ratio in the spectrum). The superior results can be explained by the less nonspecific binding (and therefore less diffuse background) of the anti-NeuN PC-MT7 probe compared to the anti-NeuN Fluor-PC-MT1 probe. This may be addressed by reducing nonspecific binding through the use of a different fluorophore. Furthermore, increasing the length of the spacer unit allows for a greater distance between the fluorophore and the reactive portion of the probe (the NHS-ester on PC-MT in this example), which could improve probe labeling efficiency.

[0212] Finally, it should be noted that the spectrum displayed for the negative control anti-Cas9Fluor-PC-MT1 probe did not show any identifiable mass reporter peaks.

[0213] Example 8. Recombinant antibody versus conventional antibody

[0214] PC-MT-Ab

[0215] The following PC-MT-Abs were prepared as described in Example 1 (unless otherwise indicated - see Antibody Material) (it should be noted that the following numbers indicate the mass units from Table 1 used in a particular PC-MT): recombinant anti-amyloid β (referred to as rAnti-AB2); recombinant anti-myelin basic protein (referred to as rAnti-MBP1); anti-myelin basic protein (referred to as anti-MBP1); recombinant anti-NeuN (referred to as rAnti-NeuN7); and anti-NeuN (referred to as anti-NeuN7).

[0216] microbead array

[0217] The procedure was performed as described in Example 6, with the following exception: 37 μm streptavidin-coated PMMA microbeads were used for conjugation with PC-MT (these PC-MTs were referred to as microbead ID tags). The microbeads were then used to capture the aforementioned PC-MT-Ab as follows: the microbeads were processed in a filter apparatus as described in Example 6. Unless otherwise indicated, each PC-MT microbead material was processed separately. The microbeads were washed with 4 x 400 μL of OBG saline (formulation see Example 6). 200 μL of 100 μg / mL biotinylated protein G in OBG saline was added to each microbead precipitate and mixed for 30 minutes. The microbeads were washed with 8 x 400 μL of OBG saline. 200 μL of 1 μg / mL PC-MT-Ab solution in OBG saline was added to the microbead precipitate and mixed for 30 minutes. The microbeads were washed with 2 x 400 μL of OBG saline and then with 2 x 400 μL of OBG buffer (same as OBG saline, but without NaCl). At this stage, different microbead materials are combined. Next, microbead array formation, photocutting, matrix application, and MALDI-MSI are performed as in Example 6.

[0218] As a result of the above procedure, two bead cells were generated and analyzed separately in the bead arrays by MALDI-MSI. Each bead material in the cell had a unique directly linked bead ID tag and a bound PC-MT-Ab. All figures below represent the mass units used in a specific PC-MT as listed in Table 1:

[0219] Microbead Pool 1:

[0220] Microbead ID-Tag 9 / rAnti-MBP1

[0221] Microbead ID-Tag 10 / Anti-MBP1

[0222] Microbead ID-Label 15 / rAnti-AB2

[0223] Microbead Pool 2:

[0224] Microbead ID-tag 9 / rAnti-NeuN7

[0225] Microbead ID-Tag 10 / Anti-NeuN7

[0226] Microbead ID-Label 15 / rAnti-AB2

[0227] Immunohistochemistry based on mass spectrometry (MIHC)

[0228] MIHC of mouse sagittal brain tissue sections was performed using the aforementioned PC-MT-Ab:rAnti-NeuN7 and anti-NeuN7 as described in Example 2.

[0229] result

[0230] Figure 13a shows the MALDI-MS quality images of the microbead array in microbead cell 1. For the mass spectrum peak intensities of microbead ID-tags 9, 10, and 15, corresponding to the microbeads loaded with rAnti-MBP1, anti-MBP1, and rAnti-AB2, respectively, the microbeads are color-coded as blue, green, and yellow in the images. The PC-MT from PC-MT-Ab is color-coded as red. Therefore, the colocalization (overlay) of the blue microbead ID-tag 9 and the bound red rAnti-MBP1 appears as pink in the quality image. Conversely, when the green microbead ID-tag 10 is detected, very little or no signal of the corresponding red anti-MBP1 is observed, so these microbeads mainly appear green. Finally, the colocalization (overlay) of the yellow microbead ID-tag 15 and the bound red rAnti-AB2 appears as orange in the quality image. In summary, while both recombinant antibodies exhibited strong PC-MT-Ab signals on their respective microbeads (observed as pink and orange microbeads), the non-recombinant anti-myelin basic protein PC-MT-Ab (anti-MBP1) was difficult to detect. This is confirmed by the overlapping spectra shown in Figure 13b. The three spectra correspond to three individual pixels selected from the center of the three microbeads circled in Figure 13a and are color-coded according to the color of the microbeads observed in Figure 13a. The pink trace in Figure 13b shows a strong peak for microbead ID-tag 9 and a strong peak for the corresponding antibody PC-MT1 bound to rAnti-MBP1. Conversely, the green trace in Figure 13b shows a strong peak for microbead ID-tag 10, but only a weak peak (black arrow) for the corresponding antibody PC-MT1 bound to anti-MBP1. It should be noted that the intensity of the rAnti-MBP1 antibody PC-MT1 peak is 10 times that of the (non-recombinant) anti-MBP1. Finally, the orange trace in Figure 13b shows the strong peak of the microbead ID-tag 15 and the corresponding antibody PC-MT2 bound to rAnti-AB2.

[0231] Microbead cell 2 showed similar results. Figure 13c shows the MALDI-MS quality images of the microbead array in microbead cell 2. For the mass spectrum peak intensities corresponding to microbead ID-tags 9, 10, and 15 loaded with rAnti-NeuN7, anti-NeuN7, and rAnti-AB2, respectively, the microbead colors were encoded as blue, green, and yellow in the images. PC-MT from PC-MT-Ab was all color-coded as red. Therefore, the colocalization (overlay) of blue microbead ID-tag 9 and the bound red rAnti-NeuN7 appears as pink in the quality image. Conversely, when green microbead ID-tag 10 was detected, very little or no signal of the corresponding red anti-NeuN7 was observed, so these microbeads mainly appeared green. Finally, the colocalization (overlay) of yellow microbead ID-tag 15 and the bound red rAnti-AB2 appears as orange in the quality image. In summary, while both recombinant antibodies exhibited strong PC-MT-Ab signals on their respective microbeads (observed as pink and orange microbeads), the non-recombinant anti-NeuN PC-MT-Ab (anti-NeuN7) was difficult to detect. This is confirmed by the overlapping spectra shown in Figure 13d. The three spectra correspond to three individual pixels selected from the center of the three microbeads circled in Figure 13c, and are color-coded according to the color of the microbeads observed in Figure 13c. The pink trace in Figure 13d shows a strong peak for microbead ID-tag 9 and a strong peak for the corresponding antibody PC-MT7 bound to rAnti-NeuN7. Conversely, the green trace in Figure 13d shows a strong peak for microbead ID-tag 10, but only a very weak peak (black arrow) for the corresponding antibody PC-MT7 bound to anti-NeuN7. It should be noted that the intensity of the rAnti-NeuN7 antibody PC-MT7 peak is 60 times that of the (non-recombinant) anti-NeuN7. Finally, the orange trace in Figure 13d shows the strong peak of the microbead ID-tag 15 and the corresponding antibody PC-MT2 bound to rAnti-AB2.

[0232] The superior performance of recombinant PC-MT-Ab in the microbead array was summarized in mass spectrometry-based immunohistochemistry (MIHC) on mouse sagittal tissue sections. Figure 13e shows MALDI-MSI quality images of the tissue stained with anti-NeuN7 and rAnti-NeuN7. NeuN immunostaining showed the typical pattern of this biomarker, similar to that observed in Example 2. Notably, the hippocampus (blue arrow) and cerebellum (white arrow) were strongly stained, and a dotted pattern of nuclear staining (NeuN is a neuronal nuclear biomarker) was observed (yellow arrow). However, recombinant rAnti-NeuN7 showed a much greater sensitivity than non-recombinant anti-NeuN7, which is clearly seen in the mass spectrometry images in Figure 13e. The spectra shown in Figure 13e confirm this. The spectra were taken from the strongest pixel in the hippocampus of each image, where the intensity of the peak from the “recombinant” antibody (red trace) is approximately 7 times greater than that from the peak from the “conventional” non-recombinant antibody (blue trace).

[0233] In general, the superior performance of recombinant antibodies is believed to stem from their typically higher purity, absence of contaminating proteins, and the presence of buffers containing azides and amines, which enhances PC-MT labeling efficiency.

[0234] Example 9. Demonstration of 12-way MIHC in FFPE tonsil and breast cancer tissues

[0235] method

[0236] Except for the following, the procedure was performed as in Example 2: FFPE human tonsil and breast cancer tissues were used. The antigen retrieval procedure was performed as follows: basic antigen retrieval was carried out for 30 minutes in 60 mL of 95°C 1X antigen retrieval basal reagent (see Materials), followed by cooling at room temperature for 30 minutes in the same Coplin staining jar. Furthermore, 12 different PC-MT antibodies were used in this case (see Table 1 for PC-MT antibody allocation), and tissue detection was performed using 0.5 μg / mL of antibody. Finally, for PC-MT and fluorescently labeled pancytokeratin antibody (CK), PC-MT antibody labeling was performed as in Example 1, except that after adding the PC-MT labeling reagent and reacting for 1 hour, DyLight 650NHS ester reagent (added in 15-fold molar excess from 5 mM stock solution in DMF) was added and reacted for another 1 hour, followed by the remaining antibody labeling procedure detailed in Example 1.

[0237] result

[0238] We constructed a 12-way biomarker set suitable for assessing the tumor microenvironment in breast cancer, specifically: breast cancer-related biomarkers [Mueller, Haymond et al. (2018) Expert Rev Proteomics 15:131-152] estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 (proliferation biomarkers); biomarkers for tumor-infiltrating lymphocytes (TILs) and other immune-related cells [Blom, Paavolainen et al. (2017) Sci Rep 7:15580; Chistiakov, Killingsworth et al. (2017) Lab Invest 97:4-13; Halse, Colebatch et al. (2018) Sci Rep 8:11158; Poh and Ernst (2018) Front Oncol [8:49], which includes T-cell subset biomarkers CD3 (T-cells), CD4 (T-helper cells), CD8 (cytotoxic T-cells), and CD45RO (memory T-cells), B-cell biomarkers CD20 and CD68, biomarkers for macrophages and other mononuclear phagocytes; and finally, pancytokeratin (CK) antibodies as general epithelial cell biomarkers [Karantza (2011) Oncogene 30:127-38] and histone H2A.X antibodies as nuclear biomarkers [Rogakou, Pilch et al. (1998) J Biol Chem 273:5858-68]. It is important to clarify that the purpose of these studies is not to validate any given biomarker for the detection of any specific cell or cancer type, nor to determine the effectiveness of biomarkers in identifying any specific disease state. The aim is to demonstrate the functionality and applicability of the novel MIHC approach presented herein.

[0239] Each antibody was directly labeled with a unique PC-MT (see Table 1 for PC-MT allocation and reporter quality for each antibody). Notably, to eliminate the bias in MALDI-MS ionization efficiency that can occur with different PC-MT amino acid sequences, eight PC-MTs contained mass unit 1 (all mass units are shown in Table 1) or the same sequence composed of various stable isotopes (mass units Iso-1.1 to Iso-1.5, Iso-1.7, and Iso-1.8); the remaining four PC-MTs also contained the core sequence of mass unit 1, but extended 1–3 glycine and / or serine amino acids at the end (mass units 1.2 to 1.5), which was not expected to significantly alter the MALDI-MS ionization efficiency.

[0240] Furthermore, to enhance the MIHC procedure, the CK antibody was double-labeled with PC-MT and a fluorophore. The ability to combine conventional immunofluorescence with PC-MT-based MSI on the same tissue section would be extremely useful, even if immunofluorescence multiplexing is limited to less than 5 channels. Firstly, such a combination would facilitate method development and antibody probe validation. Secondly, since the spatial resolution of MALDI-MSI has not yet matched that of optical imaging, even high-resolution non-multiplexed co-registered fluorescence images can be used to aid in deconvolution of structures observed in highly multiplexed MALDI-MS images. While this could be achieved by staining adjacent tissues (one for fluorescence, one for MALDI-MSI) or by performing a sequential procedure on the same tissue section, both methods are cumbersome because they introduce numerous additional steps and variables that can lead to inaccurate co-registration between MALDI-MS and fluorescence images. To overcome this limitation, we sequentially labeled the CK antibody with two different amine-reactive NHS-ester-activated reagents, first with PC-MT and then with a fluorescent reagent. On a molar basis, a 10-fold excess of PC-MT labeling reagent was used relative to the antibody, and a 15-fold excess of fluorescent reagent was used (note that the antibody contains up to 80 available lysine amino acids for labeling with NHS-ester activated reagent [Mueller, Wrasidlo et al. (1988) Hybridoma 7:453-6]). Using this dual-labeled antibody as part of the group, tissue sections were stained once with a multipath antibody mixture, and then subjected to fluorescence imaging, PC-MT photocutting, MALDI-MS matrix application, and MALDI-MSI on the same tissue sections.

[0241] First, we used human tonsil tissue to validate most of the antibodies in the 12-way group. Tonsils are frequently used as positive controls for immune cell CD markers [Kap, van Meurs et al. (2009) J Histochem Cytochem 57:1159-67; Kalina, Fiser et al. (2019) Front Immunol 10:2434], including B cells [Kalina, Fiser et al. (2019) Front Immunol 10:2434] and T cells [Sada-Ovalle, Talayero et al. (2012) ClinExp Immunol 168:200-6; Geissler, Markwart et al. (2017) PLoS One 12:e0183214], and are also known to be strongly positive for Ki67 [Hsu, Yang et al. (2013) Histopathology 63:810-6]. Figure 14a shows an immunofluorescence image of the entire tissue section using a GenePix 4200A microarray scanner at a resolution of 5 μm. As expected, the CK antibody selectively stains the squamous epithelial layer that covers the tonsils and lines their many intussusceptions and crypts. Figure 14b shows the corresponding MALDI-MS image of CK PC-MT on the same tissue section, producing the same pattern (10 μm resolution was used here, which is the limit of the Bruker rapifleX instrument used for this work, but the Caprioli and Spengler group has developed techniques that can achieve a resolution of about 1-2 μm [Zavalin, Todd et al. (2012) J MassSpectrom 47:i; Kompauer, Heiles et al. (2017) Nat Methods 14:90-96]).

[0242] Figure 14c shows multicolor MALDI-MS images of PC-MT corresponding to selected biomarkers (selected biomarkers that produce differential patterns are used here because the human eye cannot distinguish a large number of overlapping colors; see Figure 14d for all 12 biomarkers). Notably, germinal centers within lymphoid follicles (e.g., white arrows) are strongly positive for Ki67 (a proliferation marker; blue in Figure 14c) and CD20 (a B-cell marker; cyan in Figure 14c; see also CD20 in Figure 14d for better observation of this biomarker in germinal centers). This is expected, as germinal centers are known sites containing proliferating B cells [MacLennan (1994) Annu Rev Immunol 12:117-39]. The strong Ki67 staining in germinal centers is also consistent with results previously reported using standard IHC [Hsu, Yang et al. (2013) Histopathology 63:810-6]. In contrast, T cells (e.g., CD3 and CD45RO, red and orange respectively in Figure 14c) were prevalent in the extrafollicular region (and some within the follicles), consistent with previous reports [Nave, Gebert et al. (2001) Anat Embryol (Berl) 204:367-73; Sada-Ovalle, Talayero et al. (2012) Clin Exp Immunol 168:200-6]. Interestingly, CD8+ cytotoxic T- cells were not widely distributed throughout the tissue, but were found in high concentrations in discrete areas within the tonsillar crypts, in the periepithelial and intraepithelial regions (green in Figure 14c). This is logical, as tonsillar crypts are known to harbor or trap microorganisms and pathogens [Jensen, Fago-Olsen et al. (2013) PLoS One 8:e56418; Rieth, Gill et al. (2018) JAMAOtolaryngol Head Neck Surg 144:231-237]. Figure 14d shows all 12 antibodies on representative subregions of tissue sections using a gradient color bar. “Blank” corresponds to PC-MT-labeled isotype control IgG, which was used to stain individual but adjacent tissue sections (the same PC-MT as on CD3 antibody) and provides no detectable signal. Conversely, all CD antibodies are positive to varying degrees, as expected, as are CK, Ki67, and histone antibodies, many of which show different patterns. HER2 and PR are negative.Although ER showed a slight positive result, this was not unexpected based on previous reports using standard IHC [Shirasaki, Watanabe et al. (2003) International Congress Series 1257:115-118], as it showed that ER was detected in all four evaluated tonsil tissues, while PR was not found (it is also worth noting that the tonsils presented in Figure 14 are of female origin).

[0243] Next, we applied the 12-way antibody group to breast cancer FFPE tissue sections to demonstrate utility and further validate PR, ER, and HER2 antibodies, which are typically negative on tonsillar tissue (except for mild ER staining discussed above). For the tissues presented in Figures 15a-5c, the clinical annotations based on the pathology report provided by the biospecimen supplier (OriGene) are as follows: breast cancer (ductal), TNM stage of pT1cpN3apMX, minimum stage IIIC, 75% tumor, and PR- / ER- / HER2+ according to conventional IHC. Figure 15a again shows the multicolor MALDI-MS image overlay of the selected biomarkers, displaying differential patterns. Tumors were identified by areas stained positive for both CK (red; epithelial marker) and HER2 (light green), with co-localization of the two colors typically appearing as yellow-orange. Discrete patches of CD20B- cells (dark green) were observed outside and around the tumor. CD8+ cytotoxic T-cells (cyan) are not widely distributed in the tissue, as in the tonsils, but bright staining is observed in discrete areas, including infiltration into the tumor (e.g., cyan arrows). The prevalence of CD8+ T-cells and their infiltration into tumors has been reported as a positive prognostic indicator for certain forms of breast cancer [Vihervuori, Autre et al. (2019) J Cancer Res Clin Oncol 145:3105-3114; Gao, Wang et al. (2020) BMC Cancer 20:179; Jin and Hu (2020) Cancers (Basel) 12]. Highly abundant CD68 staining (purple) is also present in extratumoral and peritumoral areas, indicating macrophages (and other mononuclear phagocytes) [Chistiakov, Killingsworth et al. (2017) Lab Invest 97:4-13]. Abundant CD68 staining is consistent with reports that macrophages can typically comprise up to 50% of tumor masses [Poh and Ernst (2018) Front Oncol 8:49]. The presence of tumor-associated macrophages (TAMs) can indicate a positive or negative prognosis for a variety of solid tumors, but is generally negative, as they possess tumor-promoting activities such as immunosuppression and promotion of angiogenesis and inflammation (see reviews by Poh et al. [Poh and Ernst (2018) Front Oncol 8:49] and Goswami et al. [Goswami, Ghosh et al. (2017) Cell Immunol 316:1-10]).

[0244] Figure 15b again shows MALDI-MS images of all 12 antibodies, as well as the “blank” (in this case, on an adjacent breast cancer tissue section) as previously described. While HER2 is positive as previously stated, PR is negative, both consistent with the biopsy pathology report. ER shows positive staining; however, this is in an extratumoral region, and the tumor itself is not ER+, again consistent with the pathology report. To illustrate this, for simplicity, Figure 15c shows a multicolor overlay of the three biomarkers (CK, HER2, and ER) represented only in primary colors. The tumor is indicated by colocalized CK (blue in this case) and HER2 (green) staining; however, ER staining (red) is almost exclusively confined to the extratumoral region of the tissue section.

[0245] Finally, to further validate the PR, ER, and HER2 antibodies, a second breast cancer tissue sample was analyzed. In this case, the clinical annotations from the pathology report provided by the biospecimen supplier (OriGene) were as follows: breast cancer, ductal, lobular, metastatic, TNM stage of T2N2aMX, minimum stage IIIA, 95% tumor, and PR+ / ER+ / HER2- according to conventional IHC (i.e., the PR / ER / HER2 profile was the opposite of the previous tissue). The MALDI-MS image in the top small plot of Figure 15d again shows a three-color overlay using primary colors for simple visualization of PR, ER, and CK in this case. PR (green) and ER (red) are both strongly positive, and colocalization with the CK epithelial biomarker (blue) produces white in many areas (occurring when all three colors have similar intensities). Figure 15d (below) also shows CK, PR, ER, and HER2 separately, again in gradient colors, indicating that PR+ / ER+ / HER2- is completely consistent with the pathology report.

[0246] Example 10. Gold-coated microscope slides for improving tissue adhesion in mass spectrometry imaging.

[0247] background

[0248] Most forms of MSI require conductive slides, including most forms of MALDI-MSI. Indium tin oxide (ITO) coated glass slides are commonly used, and gold-coated slides are also used due to their conductivity, and in some cases, because gold is suitable for chemical modification [Chaurand, Cornett et al. (2011) Mol Cell Proteomics 10: O110004259; Yang and Caprioli (2014) J Mass Spectrom 49: 417-22]. However, these are used for direct MSI applications, where tissue is fixed, and in some cases, a matrix compound (for MALDI-MS) is applied and MSI is performed. Therefore, liquid-phase treatment with little or no slide is required. In contrast, the methods of this invention (mass spectrometry-based immunohistochemistry and mass spectrometry-based in situ hybridization, MIHC and MISH) are similar to conventional IHC or ISH, but use PC-MT probes instead of fluorescent or chromogenic probes, and use MSI (and related procedures) instead of optical microscopy (for a comparison of IHC / ISH, direct MSI and MIHC / MISH protocols, see...). Figure 16 Therefore, MIHC and MISH require slides that are both conductive (unlike IHC and ISH) and provide stronger tissue adhesion than direct MSI requires. We have found that this is best achieved with gold surfaces, and to our knowledge, the use of gold surfaces with IHC-MT probes and MSI procedures has not been previously reported.

[0249] method

[0250] As in Example 2, except that the tissue was fixed on a glass slide (standard microscope slide size) with a different conductive coating. These include: indium tin oxide (ITO) slides (Bruker Daltonics, Billerica, MA); ITO slides silanized using (3-aminopropyl)triethoxysilane as the silanizing agent according to a published protocol [Qin, Hou et al. (2007) Colloids Surf B Biointerfaces 60:243-9]; ITO slides coated with poly-L-lysine solution (0.01%, sterile filtered, biological reagent, suitable for cell culture, Millipore-Sigma, St. Louis, MO) according to the manufacturer's instructions; ITO slides coated with chromium alum adhesive according to the manufacturer's instructions (American MasterTech Scientific Inc., Lodi, CA); ITO slides coated with BIOBOND tissue section adhesive according to the manufacturer's instructions (Ted Pella, Inc., Redding, CA); and commercially available silver-coated slides (Platypus Technologies). (Limited LLC, Madison, WI) and two types of gold-coated slides (glass slides with a 10 nm gold layer and a 2 nm titanium adhesion underlayer, such as those from Platypus Technologies LLC, Madison, WI; or glass slides with a 50 nm gold layer and a 5 nm chromium adhesion underlayer, such as those from Substrata ThinFilm Solutions / Angstrom Engineering Inc., ON, Canada). Visually assess tissue damage or loss until the antigen retrieval step is completed.

[0251] result

[0252] Except for two types of gold-coated slides where little to no tissue damage was observed, severe tissue damage or loss of varying degrees was observed in all cases. Damage could occur during any liquid-phase processing step, but was more likely to occur during or at the initiation of a heat-mediated antigen retrieval step. Figure 17 Visible light images of damaged mouse brain tissue sections on ITO slides after processing are shown, as well as examples of intact tissue sections on gold slides.

[0253] Example 11. Requirements for matrix sublimation and subsequent matrix recrystallization in mass spectrometry-based immunohistochemistry (MIHC)

[0254] background

[0255] Those skilled in the art will recognize that, in most forms of laser-based mass spectrometry (MALDI-MS), co-crystallization of the analyte with an excess of exogenously added matrix compound is often necessary for efficient analyte evaporation / ionization and detection, as it facilitates the absorption of laser energy by the mass spectrometer and its transfer to the analyte [Yao, Scott et al. (1998) J Am Soc Mass Spectrom 9:805-13; Duenas, Carlucci et al. (2016) J Am Soc Mass Spectrom 27:1575-8]. This is known as matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). Matrix-free LDI methods do exist, such as TAMSIM [Thiery, Shchepinov et al. (2007) Rapid Commun Mass Spectrom 21:823-9] and DIOS [Trauger, Go et al. (2004) Anal Chem 76:4484-9], but their applications are generally limited to niche applications (e.g., drug detection using DIOS, but non-laser-based methods are generally preferred for this). Furthermore, these matrix-free methods lack sensitivity. In MALDI mass spectrometry imaging (MALDI-MSI), numerous matrix application methods exist, including air brushes, automated commercial nebulizers, and sublimation as some examples [Gemperline, Rawson et al. (2014) Anal Chem 86:10030-5]. The goal is to maximize sensitivity while minimizing analyte diffusion / delocalization. While the present invention is not intended to be limited to any particular matrix application method, or to require no matrix at all, preferred embodiments utilize matrix sublimation followed by recrystallization [Hankin, Barkley et al. (2007) J Am Soc Mass Spectrom 18:1646-52; Duenas, Carlucci et al. (2016) J Am Soc Mass Spectrom 27:1575-8]. This provides excellent spatial resolution (i.e., provided by sublimation, which limits analyte delocalization during matrix application due to the lack of any liquid phase) and high sensitivity (i.e., provided by vapor-based recrystallization, which allows the analyte [PC-MT mass reporter] to be fully co-crystallized with the matrix without significant analyte delocalization).

[0256] method

[0257] Except for using only PC-MT anti-NeuN antibody (1 μg / mL detection concentration), the procedure was performed as described in Example 2, and a comparison was made between recrystallization and no recrystallization.

[0258] result

[0259] Sublimation using matrix (DHB), with and without recrystallization (see methods in Examples 1-2), MIHC using MALDI-MSI on mouse brain tissue sections, and PC-MT-labeled anti-NeuN antibody were performed. Figure 18 The image shown is a color MALDI-MS image. Figure 18 The results showed that when recrystallization was applied, a strong signal and a typical anti-NeuN pattern were observed in mouse brain tissue sections (see Example 2). Without recrystallization, no significant signal was detected in the images. To quantify the effect of matrix recrystallization after sublimation, the entire region of interest was analyzed from each tissue section (see Example 2). Figure 18 Each generated average spectrum is shown in the dashed outline (in the diagram). These average spectra reveal that the single isotopic peak intensities of PC-MT are 55.4 and 2.5 with and without recrystallization, respectively. Importantly, this demonstrates that matrix recrystallization after sublimation is necessary for a robust PC-MT signal in the MALDI-MSI step of MIHC.

[0260] Example 12. Mass spectrometry-based immunohistochemistry (MIHC) for multichannel imaging of biomarkers associated with anticancer immune checkpoint inhibitor drugs.

[0261] background

[0262] The normal biological function of the immune checkpoint pathway is to maintain peripheral immune tolerance, for example, by suppressing T-cell responses. To demonstrate this, mice deficient in key proteins of the immune checkpoint pathway (CTLA-4 or PD-1) exhibit autoimmune disorders [Haanen and Robert (2015) Prog Tumor Res 42:55-66]. It is now well known that T-cell activation induces the expression of immune checkpoints (such as PD-1 and CTLA-4) on activated T cells, which in turn acts as a form of negative feedback to suppress T-cell signaling and activation [Sharma and Allison (2015) Science 348:56-61; Darvin, Toor et al. (2018) Exp Mol Med 50:1-11; Wei, Duffy et al. (2018) Cancer Discov 8:1069-1086]. However, this immunosuppression can also block beneficial anti-tumor immune responses, and in some cases, the tumor itself can hijack immune checkpoints to its advantage. For example, tumor cells can express PD-L1 ligands, which bind to PD-1 on activated T cells to suppress anti-tumor immune responses [Sharma and Allison (2015) Science 348:56-61; Darvin, Toor et al. (2018) Exp Mol Med 50:1-11; Wei, Duffy et al. (2018) Cancer Discov 8:1069-1086]. Several antibody therapeutics are now available, such as pembrolizumab (as...). Sales, Merck, Kenilworth, NJ), which binds to PD-1 and blocks its interaction with PD-L1 / 2, thereby preventing immunosuppression (similarly, therapeutic antibodies against PD-L1 also block this interaction) [Kwok, Yau et al. (2016) Hum Vaccin Immunother 12:2777-2789; Wei, Duffy et al. (2018) Cancer Discov 8:1069-1086]. Currently FDA-approved immune checkpoint inhibitors include: ipilimumab, which is specific to CTLA-4; nivolumab, pembrolizumab, and cimipril, which are specific to PD-1; and atezolizumab, avelumab, and duvastatin, which are specific to PD-L1 [Vaddepally, Kharelel et al. (2020) Cancer (Basel) 12]. Although these drugs have made groundbreaking progress [Darvin, Toor et al. (2018) Exp Mol Med 50:1-11] and are effective against a variety of cancers [Sharma and Allison (2015) Science 348:56-61; Gorris, Halilovic et al. (2018) J Immunol 200:347-354], durable responses are achieved in only 20-40% of patients [Gorris, Halilovic et al. (2018) J Immunol 200:347-354]. Therefore, problems remain in understanding and predicting which patients will respond and which of the available treatments will be effective. Thus, it is crucial to understand the expression of a range of known immune checkpoints and related molecules on infiltrating immune cells within the tumor itself and the tumor microenvironment [Gorris, Halilovic et al. (2018) J Immunol 200:347-354]. Given the large number of known immune checkpoint molecules, including PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3, a method for highly multi-path IHC, such as the MIHC technique of this invention, will become an important clinical tool.

[0263] method

[0264] MIHC will be performed as described in Examples 2 and 9, but the 12-way antibody group for breast cancer biomarkers and infiltrating immune cell biomarkers detailed in Example 9 will be further expanded to include PC-MT antibodies against the following biomarkers associated with immune checkpoints and checkpoint inhibitor drugs: PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, CD28, and TIM3. To avoid overlap with other antibodies in this group, these new antibodies will be labeled with PC-MT containing mass units 7-14 (see Table 1) using the methods described in Examples 1, 2, and 9, for a total of 20-way antibody groups.

[0265] The antibodies used for PC-MT labeling will be commercially available from ABCAM (Cambridge, MA) and are as follows: recombinant anti-PD1 antibody [CAL20] - BSA and azide-free (ab251613); recombinant anti-PD-L1 antibody [73-10] - BSA and azide-free (ab226766); recombinant anti-PD-L2 antibody [EPR1163(2)] - BSA and azide-free (ab215254); recombinant anti-CTLA4 antibody [CAL49] - BSA and azide-free Recombinant anti-CD27 antibody [EPR8569] - without BSA and azide (ab256583); Recombinant anti-CD28 antibody [EPR22076] - without BSA and azide (ab243557); Recombinant anti-TIM3 antibody [EPR22241] - without BSA and azide (ab242080).

[0266] As described in Example 9, in addition to using the complete 20-way antibody group described above, MIHC will be performed on breast cancer tissue, which is an example of FFPE. However, the present invention is applicable to any tumor type, including but not limited to tissues prepared as FFPE (formalin-fixed paraffin-embedded) or FF (freshly frozen).

[0267] result

[0268] This approach is expected to allow for simultaneous multi-channel imaging of cancer biomarkers, biomarkers of infiltrating immune cells, and biomarkers of immune checkpoints (similar to Example 9, but with the added biomarkers described above), thereby providing pathologists with important information on cancer diagnosis, prognosis, and guiding therapies such as immune checkpoint inhibitors.

[0269] Example 13. PC-MT lectin for targeted multipath mass spectrometry imaging of glycans.

[0270] background

[0271] This embodiment describes tissue MSI using PC-MT-labeled lectin probes. Lectins are a class of carbohydrate-binding proteins (discussed previously) typically derived from plants. PC-MT-labeled lectin probes can be used for MSI of N-glycans and O-glycans in tissues, but are particularly important for O-glycans. Therefore, unlike N-glycans, there are no suitable glycosidases for in situ tissue digestion followed by direct label-free MSI. Instead, O-glycans must be chemically degraded sequentially until only the core Gal-β(1→3)-GalNAc carbohydrate remains. At this point, O-glycosidases can be used for core removal, but these procedures are not suitable for in situ tissue digestion and direct label-free MSI [Poiroux, Barre et al. (2017) Int J Mol Sci 18; Wilkinson and Saldova (2020) J Proteome Res 19:3890-3905]. PC-MT-labeled lectin probes avoid this problem and thus facilitate highly multi-path MSI targeting of N-glycans and O-glycans in tissues. Importantly, lectins not only exist to bind N-glycans and O-glycans [Tsaneva and Van Damme (2020) Glycoconj J 37:533-551], but also, for example, peanut (Arachis hypogaea) lectin (PNA) is selective for the Gal-β(1→3)-GalNAc core of O-glycans [Chacko and Appukuttan (2001) Int J Biol Macromol 28:365-71; Cummings, Darvill et al. (2015) Essentials of Glycobiology:611-625], and can be used to distinguish between O-glycans and N-glycans. Jacalin lectin from *Artocarpus integrifola* and lectin from *Agaricus bisporus* also exhibit specificity for O-glycan T / Tn antigens [Poiroux, Barre et al. (2017) Int J Mol Sci 18]. O-glycans are particularly important because alterations to O-glycans have been repeatedly associated with cancer [Chacko and Appukuttan (2001) Int J Biol Macromol 28:365-71; Poiroux, Barre et al. (2017) Int J Mol Sci 18]. Note that, as discussed in the detailed description of the invention, various types of probes, such as lectins and antibodies, can be combined, and all types of PC-MT probes can be combined with other “omics” MSI protocols (e.g., direct label-free metabolomics MSI).

[0272] method

[0273] The lectins were labeled with PC-MT in the same manner as the antibodies in Example 1, but with the following changes: the molar ratio of PC-MT labeling reagent to probe was maintained at 10:10; therefore, the amount of PC-MT labeling reagent was adjusted accordingly because the lectins have a different molecular weight than the antibodies. In this case, instead of using a PD SpinTrap G-25 column, an Amicon Ultra-0.5 centrifugal filter unit containing an Ultracel regenerated cellulose membrane (Millipore-Sigma) was used, according to the manufacturer's instructions, to remove unreacted PC-MT labeling reagent using six wash cycles with 450 μL of TBS. The nominal molecular weight limit (NMWL) of the filter unit was 10 kDa for wheat germ lectin (WGA; from Millipore-Sigma) and bean lectin (PHA-E4; from amsbio), and 50 kDa for peanut lectin (PNA; from Millipore-Sigma) and dolichos biflorus lectin (DBA; from Millipore-Sigma). Each lectin is labeled with a unique PC-MT as follows: For WGA, PNA, PHA-E4 and DBA lectins, they are labeled with PC-MT mass units ID 1, Iso-1.2, Iso-1.3 and 1.2, respectively (see Table 1 for details).

[0274] Subsequent tissue processing of sagittal mouse brain FFPE sections was performed as in Example 2, but the antigen retrieval step was omitted because it was not necessary in this case. Therefore, the processing steps were deparaffinization, hydration, blocking, detection / staining (using PC-MT lectin in this case), washing, drying, optical cutting, DHB matrix sublimation / recrystallization, and MALDI-MSI (without immunofluorescence). It should be noted that antigen retrieval can still be used, for example, when combining PC-MT lectin probes with PC-MT antibody probes. All four of the above-described PC-MT-labeled lectins were mixed together for multiplex detection / staining of tissue sections at concentrations ranging from 1 to 20 μg / mL. In some cases, the PC-MT lectin probe / stain mixture is supplemented with 1 mM of the soluble sugar N,N′,N″-triacetylchitotriose (TCT; bound to WGA lectin) and pre-incubated for 30 minutes before tissue probe / staining (and the soluble sugar remains present during the tissue probe / staining process). This is to confirm the specificity of PC-MT lectin (WGA) staining through competitive inhibition.

[0275] result

[0276] Figure 19a is a color quality image of the single isotope mass spectrometry peaks corresponding to the three lectins PHA-E4, PNA, and WGA. The results are consistent with previous reports by Kitada et al., who stained mouse brain tissue sections with fluorescently labeled lectins [Kitada, Kuroda et al. (2011) Anat Rec (Hoboken) 294:305-21]. Notably, in Kitada et al., PHA-E4 lectin staining was prevalent in the choroid plexus along with some WGA staining, as observed here using PC-MT lectins and MSI (choroid plexus is labeled in Figure 19a; see also Figure 19b). In contrast, Kitada et al.'s PNA lectin preferentially stained white matter (i.e., myelinated axons). Therefore, as expected, the PC-MT PNA lectin staining here (green in Fig. 19a) matches the previously observed PC-MT myelin antibody staining (see Example 2, Fig. 7a, red for the previous PC-MT myelin antibody staining).

[0277] Finally, to demonstrate the specificity of PC-MT lectin binding to tissue, the PC-MT lectin probe / stain mixture was pre-incubated with 1 mM of the soluble sugar N,N′,N″-triacetylchitotriose (TCT), which remained in the tissue probe / stain step that competitively inhibited (blocked) WGA binding (because TCT specifically binds to WGA [Damm, Mikkat et al. (2004) Pancreas 28:31-7]). As observed in the PC-MT quality image in Figure 19b, major WGA staining (blue in Figure 19b) was observed when TCT blocking was not used. When TCT blocking was used, WGA staining was selectively inhibited, while staining of other lectins PHA-E4 (red) and PNA (green) was still observed. To quantify this result, the overall average spectrum from each tissue section was obtained with and without TCT blocking (Figure 19c). When TCT blocking was used (orange trace), WGA... The PC-MT signal intensity was reduced by 70% compared to without TCT blocking (purple trace), while the peak intensities of the other two lectins, PNA and PHA-E4, were almost the same with and without TCT blocking, thus demonstrating specificity.

[0278] Example 14. Improved multi-omics MSI: Non-targeted label-free MSI and targeted MIHC of lipids on the same FF tissue sections

[0279] method

[0280] This example was performed using FF sagittal mouse brain tissue sections in the same manner as in Example 4, except that some enhancements were performed after the initial direct unlabeled MALDI-MSI (in this case, for endogenous lipids) and before the subsequent MIHC step using a PC-MT-labeled antibody (PC-MT-Ab) to better fix the tissue and better remove unfixed small endogenous organic compounds. Therefore, after the initial direct unlabeled MALDI-MSI of endogenous lipids, the tissue sections were treated as follows (each treatment step in a separate staining jar): pre-washed twice with acetone at -80°C for 3 minutes each (note: used to remove any remaining matrix compounds from the previous MALDI-MSI while providing solvent fixation), and vacuum dried for 10 minutes; fixed for 30 minutes with PBS containing 1% PFA at pH 7.4 (note: this solution was freshly prepared as follows: 1.0 g of PFA was dissolved in 60 mL of PBS and 1.0 mL of 1M acetone on a heating block at approximately 60°C with constant stirring). The solution was prepared in NaOH, then the pH was adjusted to 7.4 with 1M HCl (~1 mL), and the final volume was adjusted to 100 mL with PBS. The solution was washed once with PBS for 10 minutes, then twice with acetone at room temperature for 3 minutes each time. Finally, the solution was washed once with Carnoy's solution (6:3:1 EtOH / chloroform / acetic acid) for 3 minutes to further fix and remove unfixed endogenous organic compounds. The remaining steps of the PC-MT-Ab-based MIHC were performed as described in Example 2, from the antigen retrieval step to the end.

[0281] result

[0282] The quality images are shown in Figure 20. Figure 20a is the initial direct label-free MALDI-MSI of endogenous lipids (see Figure 20a for which example lipids are shown); Figure 20b is the subsequent MIHC showing the selected antibody PC-MT (see Figure 20b for which PC-MT antibodies are shown); and Figure 20c is a merged image of the selected analytes from the initial direct label-free MALDI-MSI and the subsequent MIHC (see Figure 20c for which analytes are shown). The expected co-localization of lipid thioglycosides (ST) with myelin was explained in Example 4, but not between ST and NeuN.

[0283] Example 15. Multi-omics tissue imaging: MIHC combined with bottom-up proteomics MSI.

[0284] In the detailed description of the invention, under the section entitled "Multi-omics Tissue Imaging Using PC-MT Probes," a wide variety of multi-omics imaging schemes incorporating PC-MT probe technology are described. The following examples illustrate some of these schemes but are not intended to limit the scope of the invention.

[0285] method

[0286] FFPE tissue sections of breast cancer will be used as in Example 9.

[0287] In one embodiment, in situ tissue glycan digestion with glycosidase PNGase F is performed according to Drake et al. [Drake, Powers et al. (2018) Curr ProtocProtein Sci 94:e68], followed by direct label-free MALDI-MSI of the released glycans. Next, after MALDI-MSI, the MALDI-MS matrix is ​​removed (in a staining jar) by washing twice with acetone at room temperature for 3 minutes each time, and the tissue is then completely dried under vacuum for 10 minutes. Next, 12-way MIHC is performed according to Example 9 to detect macromolecular protein biomarkers of breast cancer and tumor-infiltrating lymphocytes / immune cells (with the exception that the deparaffinization step in Example 9 is omitted, as it has already been performed according to the procedure of Drake et al.).

[0288] In another implementation, MIHC (targeted) will be performed first, followed by in situ protease digestion of the tissue sections, and direct, non-targeted, label-free MALDI-MSI of the released proteolytic fragments (from endogenous tissue proteins). See, for example... Figure 22In this case, MIHC will be performed again as described in Example 9, which includes MALDI-MSI as a final step. The MALDI-MS matrix (in the staining vessel) will then be removed by washing twice with acetone at room temperature for 3 minutes each time, and the tissue will then be completely dried under vacuum for 10 minutes. Subsequent in situ protease (trypsin) digestion and direct label-free MALDI-MSI of the released proteolytic fragments will be performed according to Lazova et al. [Lazova, Smoot et al. (2020) J Cutan Pathol 47:226-240], but the deparaffinization and antigen retrieval steps will be omitted as they have already been performed according to the MIHC procedure. Finally, in another embodiment, it may be desirable to remove the antibody (and protein blocking agent) from the initial MIHC procedure prior to in situ proteolytic digestion for subsequent direct label-free MALDI-MSI (so that only proteolytic fragments from endogenous tissue proteins are detected). In this case, the MALDI-MS matrix from the final step of the MIHC process is removed again by washing twice for 3 minutes each with room temperature acetone (all steps are performed in a staining jar). However, the tissue is then denatured for 5 minutes at 65°C in a 1% (w / v) SDS detergent solution in 50 mM Tris at pH 7.4, containing 1 mM dithiothreitol (DTT). This denaturation treatment is used to separate antibody probes (and protein blocking agents) from the tissue. This is followed by washing twice for 3 minutes each with standard 50 mM Tris at pH 7.4, and then twice for 3 minutes each with room temperature acetone (this will further help to completely remove the SDS detergent from the tissue). The tissue is then completely dried under vacuum for 10 minutes. Similarly, according to Lazova et al. [Lazova, Smoot et al. (2020) J Cutan Pathol 47:226-240], subsequent in situ protease (trypsin) digestion of the proteolytic fragments and direct label-free MALDI-MSI were performed (but again, the deparaffinization and antigen retrieval steps of Lazova et al. will be omitted, as they have already been performed according to the MIHC process).

[0289] result

[0290] By combining the results of MIHC with a bottom-up MSI protocol, a much larger spatial information content from the tissue can be obtained compared to either protocol alone. This is expected to yield better biomarker “features,” such as those determined by the machine learning statistical protocol of Lazova et al., which could, for example, be used in cancer on tumor tissue for diagnosis, staging, prognosis, subtyping, and prediction of optimal treatment pathways to improve patient outcomes.

[0291] Example 16. PC-MT probe in non-imaging mass spectrometry applications

[0292] background

[0293] PC-MT probes are not limited to mass spectrometry imaging applications, but can be used in conjunction with non-imaging mass spectrometry (MS) applications. For example, a set of PC-MT probes can be applied to heterogeneous biological samples, including but not limited to tissue or excised portions of tissue, tumors, cells derived from tumors (or derived from any tissue), liquid biopsies, cell cultures, blood and other body fluids, bacterial cells, and cultured infectious pathogens, to identify the presence of components in the heterogeneous sample. For example, one or more PC-MT antibody (PC-MT-Ab) probes can be used to identify pathogens present in a biological sample, said probes targeting the characteristic antigens of one or more pathogens. Readout will be achieved by detecting photocutted (photoemitted) PC-MT using non-imaging mass spectrometry methods (including but not limited to MALDI-MS or ESI-MS) with or without liquid chromatography prior to mass spectrometry analysis (e.g., LC-MS). Those skilled in the art will recognize that further purification of the analyte (e.g., desalting) is sometimes necessary prior to mass spectrometry analysis. In one implementation, using PC-MT-Ab probes targeting these antigens / epitopes (such as Spike protein antigens and / or their epitopes) can identify specific antigens / epitopes of specific variants of the SARS-CoV-2 virus that caused the widespread COVID-19 pandemic. In a second implementation, specific antigens / epitopes present on tumor-infiltrating lymphocytes in tumor biopsies can be targeted with specific PC-MT-Ab probes and detected using non-imaging mass spectrometry to determine the presence of various immune cell types in the tumor, which can provide information useful, for example, in terms of prognosis and treatment. While spatial information is lost due to this non-imaging protocol, those skilled in the art of immunodiagnostics (including ELISA and flow cytometry) know that the positive binding of specific combinations of antibodies (or other probe types) to biological samples can be used to identify the presence of specific components of the sample (such as bacterial cells or viruses in the case of infectious diseases). This targeting protocol for detecting the presence of specific biomarkers in a sample can be combined with other mass spectrometry methods (such as non-targeted detection of specific combinations of small molecules, lipids, metabolites, and proteins) to identify components of the sample (e.g., components of biological mixtures). The experimental example presented below confirms the binding of the PC-MT probe (PC-MT-Ab) to the surface, followed by non-imaging mass spectrometry analysis of PC-MT photoemission and photoemission of PC-MT. Although the surface to which the PC-MT-Ab is bound is microbeads in this case, ultimately, any type of PC-MT probe can bind to any sample, as illustrated in the example above.

[0294] method

[0295] PC-MT (mass unit Iso-1.1 in Table 1) and PC-MT-Ab were prepared as described in Example 1. It should be noted that, in this case, purified rabbit IgG was used as the model PC-MT-Ab (see the “Materials for Experimental Examples” section).

[0296] The obtained PC-MT-Ab was bound to protein G agarose beads (Thermo Fisher Scientific, Waltham, MA). The beads were then washed to remove unbound PC-MT-Ab, and PC-MT was photo-released from the PC-MT-Ab bound to the beads. The photo-released PC-MT present in the supernatant was analyzed by non-imaging MALDI-MS. The complete procedure is as follows: Unless otherwise indicated, the protein G agarose beads were processed in a 0.5 mL Ultrafree-MC centrifugal 0.45 μm filter device (see the “Materials for Experimental Examples” section). (It should be noted that washing was performed as follows: the beads were briefly vortexed in the washing solution, followed by a brief rotation at 15,000 rpm in a standard microcentrifuge, and the filtrate was discarded in the bottom chamber of the filter device; the washed beads were retained in the top chamber of the filter device). 1 μL of bead precipitate was used for each sample (each sample was processed in parallel in a separate filter device). Microbeads of each sample were first briefly pre-washed with 4 x 400 μL of OBG-saline (formulation see Example 6). The washed microbead precipitate was then resuspended in 100 μL of PC-MT-Ab solution (prepared in OBG-saline at concentrations of 0.625, 1.25, 2.5, 5, 7.5, and 10 μg / mL). The microbeads were gently mixed for 30 minutes (protected from light) to allow PT-MT-Ab to bind to protein G on the microbeads. The microbeads were then briefly washed with 4 x 400 μL of OBG-saline, followed by a brief wash with 4 x 400 μL of MS-water to remove unbound PC-MT-Ab.

[0297] The microbeads were then resuspended and transferred to a clear, thin-walled polypropylene PCR microcentrifuge tube containing 100 μL of MS-water. The microbeads were then briefly centrifuged at 15,000 rpm in a standard microcentrifuge, and approximately 80 μL of the supernatant was discarded, leaving approximately 20 μL of MS-water with the microbead precipitate in the tube. The microbeads were resuspended in the tube by a brief mixing and used an Honle LED Cube 100IC (Honle UV Technology, Marlboro, MA) at 25% power (30 mW / cm²). 2The test tubes were exposed to UV light for 5 minutes (at 360 nm) through the side walls (with the tubes placed on their sides). While the microbeads were still in the test tubes, 20 μL of the following solution was added to each sample: 10 mg / mL α-cyano-4-hydroxycinnamic acid (CHCA; Sigma-Aldrich, St. Louis, MO), 80% acetonitrile, 0.2% (v / v) trifluoroacetic acid (TFA), and 25 femtomol / μL of unmodified control peptide with the sequence APRLRFYSL (SEQ ID NO: 33) (custom-synthesized by GenScript, Piscataway, NJ). The microbeads were then mixed for 15 minutes to fully extract the light-released PC-MT. The microbeads were briefly spun at 15,000 rpm in a standard microcentrifuge, and then 2 μL of each sample supernatant (excluding any microbeads) was spotted onto a standard steel MALDI-MS target for standard non-imaging MALDI-MS analysis on a rapifleX MALDI-TOF-MS instrument (Bruker Daltonics, Billerica, MA).

[0298] result

[0299] Figure 21a shows the spectra of photoemitted PC-MT from six samples corresponding to six different concentrations of PC-MT-Ab added to protein G beads, obtained without imaging MALDI-MS analysis. As expected, monoisotope peaks of photoemitted PC-MT (reporter) and control peptide were observed at m / z 1,210.6 and 1,122.6, respectively. To quantify the results, the ratio of the monoisotope peak intensity of photoemitted PC-MT to that of the control peptide (at a fixed concentration) was calculated and plotted in Figure 21b. A linear response (R0) as a function of PC-MT-Ab concentration was observed. 2 =0.9779).

[0300] All publications and patents mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the described embodiments of the invention that will be apparent to those skilled in the art and related fields fall within the scope of the appended claims.

Claims

1. A multiplexed method for co-detecting a plurality of different types of biomarkers in a tissue sample on a single slide, the method comprising: a) providing a tissue sample on a single slide; b) contacting the tissue sample with a plurality of different antibodies to effect binding of the antibodies to the tissue sample, each of the antibodies being reactive with a different biomarker, and each of the antibodies being conjugated to a unique photo-cleavable mass tag, wherein the antibody conjugated to the photo-cleavable mass tag has the following general structure: c) illuminating the photo-cleavable mass tag with light prior to step d), thereby photo-cleaving at least a portion of the mass tag; and d) detecting the mass tag or fragments thereof as molecular ions using mass spectrometric imaging.

2. The method of claim 1, further comprising, after step a) but prior to step b), performing direct mass spectrometric imaging on the tissue sample.

3. The method of claim 1, wherein the plurality of different antibodies are in a mixture, and the tissue sample in step b) is contacted with the mixture.

4. The method of claim 1, wherein the mass tag is a non-rare earth metal mass tag.

5. The method of claim 1, wherein the mass tag comprises a plurality of amino acids.

6. The method of claim 1, wherein the tissue sample is fresh frozen and sectioned prior to being mounted on the single slide.

7. The method of claim 1, wherein the slide comprises gold.

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

9. The method of claim 1, wherein the tissue sample is formalin fixed and paraffin embedded and sectioned prior to being mounted on the single slide.

10. The method of claim 9, wherein the sample is treated prior to the step of contacting the tissue sample with antibodies, the treatment comprising deparaffinization.

11. The method of claim 10, wherein the deparaffinization is performed with xylene.

12. The method of claim 10, wherein the tissue sample is further treated, the treatment comprising rehydration.

13. The method of claim 12, wherein the rehydration is performed with a series of ethanol / water mixtures and aqueous saline buffer.

14. The method of claim 12, wherein the tissue sample is further treated, the treatment comprising antigen retrieval.

15. The method of claim 14, wherein the antigen retrieval is performed by heating in citrate buffer at pH 6.

16. The method of claim 14, wherein the antigen retrieval is performed by using formic acid.

17. The method of claim 1, wherein a matrix compound is applied to the mass tag prior to step d).

18. The method of claim 17, wherein the matrix compound is selected from the group consisting of a-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid).

19. The method of claim 17, wherein the matrix compound is applied by sublimation.

20. The method of claim 17, wherein the tissue sample is treated after the step of contacting the sample with a matrix compound, the treatment comprising matrix recrystallization.

21. The method of claim 1, wherein the tissue is from a tumor.

22. The method of claim 21, wherein the tumor is a breast tumor.

23. The method of claim 1, wherein at least one of the plurality of different antibodies comprises a fluorescent moiety in addition to the mass tag.

24. The method of claim 1, wherein the number of different antibodies is 8 or more.

25. The method of claim 24, wherein a subset of the different antibodies reacts with i) estrogen receptor (ER), ii) progesterone receptor (PR), iii) human epidermal growth factor receptor 2 (HER2), and iv) Ki67.

26. The method of claim 24, wherein a subset of the different antibodies reacts with T-cell biomarkers i) CD3 (T-cells), ii) CD4 (T-helper cells), iii) CD8 (cytotoxic T-cells), and iv) CD45RO (memory T-cells).

27. The method of claim 24, wherein one of the different antibodies reacts with the B-cell biomarker CD20.

28. The method of claim 24, wherein one of the different antibodies reacts with the macrophage biomarker CD68.

29. The method of claim 24, wherein one of the plurality of different antibodies reacts with an immune checkpoint molecule.

30. The method of claim 29, wherein the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

31. A conjugate having the following structure:

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