Novel photocleavable mass tags for multiplexed mass spectrometric imaging of tissues using biomolecular probes

Abstract

Immunohistochemistry (INC) and in situ hybridization (ISH) methods for targeted detection and mapping of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for example, for research applications and clinical use by pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies), include mass spectrometry imaging (MSI) as a mode for detecting and mapping biomolecules within tissues or cells. The methods utilize photocleavable mass tag reagents coupled to probes such as antibodies and nucleic acids and used to achieve multiplexed immunohistochemistry and in situ hybridization, with MSI as the detection / readout modality. The methods also encompass multi-omic MSI procedures, in which MSI of photocleavable mass tag probes is combined with other modes of MSI, such as direct, label-free MSI of endogenous biomolecules from biological samples (e.g., tissues).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. CA236097 awarded by the National Cancer Institute. The government has certain rights in this invention.

[0005] FIELD OF THE INVENTION

[0006] The field of the present invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for targeted detection and mapping of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for example, for research applications and clinical applications by pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies). In particular, the use of mass spectrometry imaging (MSI) as a mode for detecting and mapping biomolecules, for example, within tissues or cells. More specifically, the field of the present invention relates to photocleavable mass tag reagents attached to probes such as antibodies and nucleic acids and used to achieve multiplexed immunohistochemistry and in situ hybridization, using MSI as a detection / readout modality. Probe types other than antibodies and nucleic acids are also included in the field of the present invention, including, but not limited to, carbohydrate-binding proteins (e.g., lectins), receptors, and ligands. Finally, the field of the invention also encompasses multi-omic MSI procedures, in which MSI of photocleavable mass tag probes is combined with other modes of MSI, such as direct, unlabeled MSI of endogenous biomolecules from biological samples (e.g., tissues), whereby the biomolecules can be intact or digested (e.g., chemically digested or enzymatically). [Background technology]

[0007] Background of the Invention

[0008] Immunohistochemistry (IHC) and in situ hybridization (ISH) are widely used to determine the structural organization 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 the preferred method for studying extracellular amyloid plaques and intracellular tau-based neurofibrillary tangles in neurodegenerative disorders [Deng, Bigio et al. (2011) Methods Mol Biol 793:259-72; Dugger and Dickson (2017) Cold Spring Harb Perspect Biol 9]. In oncology, IHC and ISH can be used to diagnose, subtype, and determine optimal treatment for various cancers, including the evaluation of tumor-infiltrating lymphocytes (TILs), which have prognostic value [Halse, Colebatch et al. (2018) Sci Rep 8:11158] [Renwick, Cekan et al. (2013) J Clin Invest 123:2694-702; Zaha (2014) World J Clin Oncol 5:382-92]. IHC and ISH analysis is generally performed on tissue samples collected, for example, by tumor biopsy or surgical resection. Typically, tissue samples are fresh-frozen (FF) or formalin-fixed, paraffin-embedded (FFPE), then sectioned (e.g., at 10 μm) and mounted on glass microscope slides. Fluorophores or chromogenic agents conjugated to antibodies or nucleic acid probes are the most common method for visualizing the spatial distribution of targeted biomolecules (e.g., protein antigens or genetic material such as miRNA) using microscopy [Katikireddy and O'Sullivan (2011) Methods Mol Biol 784:155-67].

[0009] It is often important to simultaneously determine the localization and potential colocalization of multiple biomarkers. This is important, for example, for mapping the locations of hundreds of possible proteins and / or miRNAs involved in cellular regulation and dysregulation 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, resulting in spectral overlap [Stack, Wang et al. (2014) Methods 70:46-58]. The multiplexing limit for standard fluorescence microscopy is generally 3–5, while hyperspectral / multispectral methods are limited to 8 [Tsurui, Nishimura et al. (2000) J Histochem Cytochem 48:653-62; Stack, Wang et al. (2014) Methods 70:46-58; Parra, Uraoka et al. (2017) Sci Rep 7:13380; Gorris, Halilovic et al. (2018) J Immunol 200:347-354]. Furthermore, these multiplexing methods often require repeated staining followed by cycling strategies such as photobleaching or probe removal / denaturation (e.g., PerkinElmer's OPAL multispectral platform) [Wahlby, Erlandsson et al. (2002) Cytometry 47:32-41; Schubert, Bonnekoh et al. (2006) Nat Biotechnol 24:1270-8; Gerdes, Sevinsky et al. (2013) Proc Natl Acad Sci USA 110:11982-7; Blom, Paavolainen et al. (2017) Sci Rep 7:15580]. Such methods are complex, tedious, and involve incomplete cycling, potentially confounding results [Giesen, Wang et al. (2014) Nat Methods 11:417-22; Blom, Paavolainen et al. (2017) Sci Rep 7:15580].

[0010] In contrast, mass spectrometry imaging (MSI) facilitates high levels of multiplexing without the limitations of optical methods mentioned above (limited only by mass resolution typically below 1 Da). Briefly (see Figure 1 for details), these methods scan a tissue specimen with a mass spectrometer, generating a complete mass spectrum at each "pixel," thereby enabling simultaneous imaging of any given mass species within the spectrum [Arentz, Mittal et al. (2017) Adv Cancer Res 134:27-66]. The Caprioli group first introduced this technique based on matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) [Caprioli, Farmer et al. (1997) Anal Chem 69:4751-60], and the technique has since been widely adopted 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) methods, such as ESL-based DESI-MS imaging [Takats, Wiseman et al. (2004) Science 306:471-3]. While MALDI and DESI MSI techniques currently cannot match the spatial resolution of optical methods (e.g., a 10 μm laser focus with the newer Bruker rapifleX MALDI-MS instrument), improved resolution is possible using innovative designs such as atmospheric pressure MALDI-MSI with transmission geometry (2 μm) [Zavalin, Todd et al. (2012) J Mass Spectrom 47:i] or laser focusing objectives (1.4 μm) [Kompauer, Heiles et al. (2017) Nat Methods 14:90-96].

[0011] However, MSI of intact macromolecules such as proteins is typically impossible due to insufficient mass resolution and low sensitivity [Buchberger, DeLaney, et al. (2018) Anal Chem 90:240-265]. Identification of specific biomolecules requires tandem MS / MS fragmentation, ultrahigh mass resolution instruments, and / or bottom-up proteomic approaches (e.g., in situ proteolysis of tissues). To overcome this limitation, several targeted MSI approaches have been introduced that enable multiplex workflows similar to traditional IHC and ISH using labeled antibodies and nucleic acid probes. TAMSIM (Targeted Multiplex Mass Spectrometry Imaging) is a matrix-free laser desorption / ionization (LDI) method that uses antibodies conjugated to 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, mass tags are not easily synthesized, and only 3-plex imaging has been demonstrated [Thiery, Anselmi et al. (2008) Proteomics 8:3725-34]. Furthermore, those skilled in the art will recognize that efficient analyte vaporization / ionization and detection, called matrix-assisted laser desorption / ionization (MALDI) mass spectrometry, requires analyte co-crystallization with an excess of exogenously added matrix compounds to facilitate absorption and transfer of the mass spectrometer's laser energy 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], and therefore the TAMSIM method lacks sensitivity.

[0012] In contrast, peptide mass tags are easily generated using standard solid-phase synthesis, their masses are easily adjusted by altering the sequence, and peptides generally ionize with high efficiency. Lemaire et al. first introduced a photocleavable peptide-based MSI method for tissue-targeted imaging called Tag-Mass [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67]. However, mass tagging of probes (e.g., antibodies) is a complex, multi-step process involving intermediate chemical linkers. Furthermore, the photocleavable cores used in peptides provide suboptimal sensitivity. These drawbacks have so far limited the general use of Tag-Mass, and as a result, only two-plex MSI has been achieved to date [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67; Franck, Arafah et al. (2009) Mol Cell Proteomics 8:2023-33; El Ayed, Bonnel et al. (2010) Med Sci Monit 16:BR 233-45].

[0013] Imaging mass spectrometry uses antibodies tagged with rare earth metals in combination with inductively coupled plasma mass spectrometry (ICP-MS) [Giesen, Wang et al. (2014) Nat Methods 11:417-22]. This approach has achieved the highest multiplexing level to date, with at least 32-plex tissue staining. However, this method requires specialized MS equipment and is a destructive approach that reduces molecules to elements (atomization) for detection and analysis. Therefore, it is not compatible with performing non-targeted direct MSI analysis of biomolecules (in conjunction with targeted MSI using mass-tagged probes, as in Example 4 of the present invention). For an example review of ICP-MS, see Wilschefski et al. [Wilschefski and Baxter (2019) Clin Biochem Rev 40:115-133]. Further drawbacks of the imaging mass cytometry approach include that the probe labeling process is also highly complex, involving preloading the polymer with metal ions, partially reducing the antibody, coupling 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 / fhiidigm%3Afile]. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Katikireddy and O'Sullivan (2011) Methods Mol Biol 784:155-67 [Non-patent document 2] Howat and Warford (2014) Methods 70:1-2 [Non-patent document 3] Stack, Wang et al. (2014) Methods 70:46-58 [Non-patent document 4] Deng, Bigio et al. (2011) Methods Mol Biol 793:259-72 [Non-patent document 5] Dugger and Dickson (2017) Cold Spring Harb Perspect Biol 9 [Non-patent document 6] Halse, Colebatch et al. (2018) Sci Rep 8:11158 [Non-Patent Document 7] Renwick,Cekanら(2013)J Clin Invest 123:2694-702 [Non-licensed document 8] Zaha(2014)World J Clin Oncol 5:382-92

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[0015] Summary of the Invention

[0016] The present invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for targeted detection and mapping of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for example, for research applications and clinical applications by pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies). In particular, the use of mass spectrometry imaging (MSI) as a mode for detecting and mapping biomolecules, for example, within tissues or cells. More specifically, the field of the present invention relates to photocleavable mass tag reagents that are conjugated to probes such as antibodies and nucleic acids and used to achieve multiplexed immunohistochemistry and in situ hybridization, using MSI as a detection / readout modality.

[0017] In one embodiment, the present invention provides a multiplex method for simultaneously 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 effect binding of the antibodies to the tissue sample, each of the antibodies reactive with a different biomarker and each of the antibodies conjugated to a unique mass tag; and c) detecting the mass tags or fragments thereof as molecular ions using mass spectrometry imaging. In one embodiment, the method further comprises performing direct mass spectrometry imaging on the tissue sample after step a) but before step b) (i.e., tissue imaging is achieved in a single specimen). In one embodiment, the five or more antibodies are in a mixture, and the tissue sample is contacted with the mixture in step b). In one embodiment, the mass tags are non-rare earth metal mass tags. In one embodiment, the mass tags comprise multiple amino acids. In one embodiment, the tissue sample is fresh frozen and sectioned prior to mounting on 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 formalin-fixed, paraffin-embedded, and sectioned before mounting on the single slide. In one embodiment, the tissue sample is processed prior to contacting the sample with the antibody, the processing comprising deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further processed, the processing 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, the processing comprising antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer (pH 6). In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the antibody conjugated to a mass tag has the following general structure: [ka] and X is a spacer. In one embodiment, a matrix compound is applied to the mass tags prior to step c). In one embodiment, the matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tissue sample is subjected to a treatment after contacting the sample with the matrix compound, the treatment comprising matrix recrystallization. In one embodiment, the mass tags are photocleavable. In one embodiment, the method further comprises irradiating the mass tags with light prior to step c) to photocleave at least a portion of the mass tags. In one embodiment, the tissue is 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 moiety in addition to the mass tag. In one embodiment, the number of different antibodies is eight or more. In one embodiment, the subset of distinct 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, the subset of distinct antibodies reacts with the T cell biomarkers i) CD3 (T cells), ii) CD4 (T helper), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, one of the distinct antibodies reacts with the B cell biomarker CD20. In one embodiment, one of the distinct antibodies reacts with the macrophage biomarker CD68. In one embodiment, one of the distinct antibodies reacts with an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0018] In one embodiment, the present invention provides a multiplex method for simultaneously detecting human epidermal growth factor receptor 2 (HER2) and estrogen receptor (ER) proteins 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, wherein the antibody is conjugated to a first non-rare earth metal mass tag, the mass tag comprising a plurality of amino acids; c) contacting the sample with an ER-specific antibody, wherein the antibody is conjugated to a second non-rare earth metal mass tag, the mass tag comprising a second plurality of amino acids, the second plurality of amino acids having a different mass from the first plurality of amino acids; and d) detecting the mass tag or a fragment thereof as a molecular ion using mass spectrometry imaging. In one embodiment, steps b) and c) are performed simultaneously. In one embodiment, the method further comprises performing direct mass spectrometry imaging on the tissue sample after step a) but before step b). In one embodiment, the tissue sample is fresh-frozen and sectioned before mounting on 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 formalin-fixed, paraffin-embedded, and sectioned before mounting on the single slide. In one embodiment, the tissue sample is processed prior to contacting the sample with the antibody, the processing comprising deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further processed, the processing 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, the processing comprising antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer (pH 6). In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the antibody conjugated to a mass tag has the following general structure: [ka] and X is a spacer. In one embodiment, a matrix compound is applied to the mass tags prior to step d). In one embodiment, the matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the tissue sample is subjected to a process after contacting the sample with the matrix compound, the process comprising matrix recrystallization. In one embodiment, the mass tags are photocleavable. In one embodiment, the method further comprises irradiating the mass tags with light prior to step d) to photocleave at least a portion of the mass tags. 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 comprises 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, and the mass tags comprising a plurality of amino acids. In one embodiment, the number of different antibodies is eight or more. In one embodiment, one of the distinct antibodies reacts with the progesterone receptor (PR). In one embodiment, a subset of the distinct antibodies reacts with the following T cell biomarkers: i) CD3 (T cells), ii) CD4 (T helper), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, one of the distinct antibodies reacts with the B cell biomarker CD20. In one embodiment, one of the distinct antibodies reacts with the macrophage biomarker CD68. In one embodiment, one of the distinct antibodies reacts with an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0019] In one embodiment, the present invention provides a multiplex method for simultaneously detecting different types of tumor-infiltrating immune cells in tumors 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 effect binding of the antibody to the tissue sample, the antibody being conjugated to a first non-rare earth metal mass tag, the mass tag comprising multiple amino acids; c) contacting the tissue sample with a second antibody specific for a second type of tumor-infiltrating immune cell to effect binding of the antibody to the tissue sample, the antibody being conjugated to a second non-rare earth metal mass tag, the mass tag comprising multiple amino acids; and d) detecting the mass tag or a fragment thereof as a molecular ion using mass spectrometry imaging. In one embodiment, steps b) and c) are performed simultaneously. In one embodiment, the method further comprises performing direct mass spectrometry imaging on the tissue sample after step a) but before step b). In one embodiment, the tissue sample is fresh frozen and sectioned before mounting on 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 formalin-fixed, paraffin-embedded, and sectioned before mounting on the single slide. In one embodiment, the tissue sample is processed prior to contacting the sample with the antibody, and the processing comprises deparaffinization. In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tissue sample is further processed, and the processing comprises 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, and the processing comprises antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer (pH 6). In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the antibody conjugated to a mass tag has the following general structure: [ka] and X is a spacer. 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 alpha-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 subjected to a treatment after contacting the sample with the matrix compound, the treatment comprising matrix recrystallization. In one embodiment, the mass tag is photocleavable. In one embodiment, the method further comprises irradiating the mass tag with light prior to step d) to photocleave 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, prior to step d), the method further comprises contacting the tissue with additional, distinct antibodies, each of the additional, distinct antibodies conjugated to a non-rare earth metal mass tag, wherein the mass tag comprises multiple amino acids. In one embodiment, one of the additional, distinct antibodies reacts with CD4. In one embodiment, one of the additional, distinct antibodies reacts with the macrophage biomarker CD68. In one embodiment, one of the additional, distinct antibodies reacts with an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3. In one embodiment, the invention provides a composition comprising i) a mass tag via a photocleavable linker, and ii) an antibody conjugated to a fluorophore. In one embodiment, the antibody reacts with a biomarker selected from the group consisting of 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 group consisting of i) CD3 (T cells), ii) CD4 (T helper), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells).In one embodiment, the antibody reacts with an immune checkpoint molecule, hi one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0020] In one embodiment, the present invention provides a multiplex 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 reactive with a different immune checkpoint molecule, each of the antibodies 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 a fragment thereof as a molecular ion using mass spectrometry imaging. In one embodiment, one of the plurality of different antibodies reacts with an immune checkpoint molecule selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3. In one embodiment, the tumor tissue comprises a biopsy from a human patient. In one embodiment, the biopsied tumor tissue reacts with an antibody against PD-L1. In one embodiment, the method further comprises treating the human patient with an immune checkpoint inhibitor specific for PD-L1. In one embodiment, the checkpoint inhibitor specific for PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0021] In one embodiment, the present invention provides a multiplex method for simultaneously detecting three or more (and 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 (and more preferably five or more) different probes to produce a probed tissue sample, each of the probes conjugated to a unique mass tag, wherein at least three or more (and more preferably five or more) of the probes each bind to a different biomarker in the tissue sample; and c) detecting the unique mass tags or fragments thereof from at least three or more (and preferably five or more) of the bound probes using mass spectrometry imaging of the probed tissue sample (e.g., tissue imaging is accomplished on a single specimen), wherein the mass tags are detected 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 formalin-fixed and sectioned prior to step b). In one embodiment, the tissue sample is formalin-fixed, paraffin-embedded, and sectioned prior to step b). In one embodiment, the tissue sample is mounted on a 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 mounted on a 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 five or more probes are in a mixture, and in step b), the tissue sample is contacted with the mixture to produce the probed tissue sample. In one embodiment, the probes conjugated to mass tags have the following general structure: [ka] and X is a spacer. In one embodiment, at least one of the five or more probes comprises a fluorescent moiety in addition to the mass tag. In one embodiment, the probe is selected from the group consisting of a protein and a nucleic acid. In one embodiment, at least one of the probes is an antibody. In one embodiment, the antibody is selected from the group consisting of a recombinant antibody, a nanobody, a single-chain fragment variable (scFv) antibody, a single-domain antibody, and a VHH single-domain antibody. In one embodiment, at least one of the probes is selected from the group consisting of an affibody, a receptor, and a ligand. 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 an 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 comprises a plurality of amino acids. In one embodiment, the mass tag is photocleavable. In one embodiment, the method further comprises irradiating the mass tag with light prior to step c) to photocleave at least a portion of the mass tag. In one embodiment, the method further comprises performing mass spectrometry imaging on the tissue sample after step a) but before step b). In one embodiment, a matrix compound is applied to the probed tissue sample before step c). In one embodiment, the matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the probed tissue sample is subjected to a treatment after the step of applying the matrix compound to the probed tissue sample, the treatment comprising matrix recrystallization. In one embodiment, the tissue sample is subjected to a treatment after the 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 processed, the processing 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, the processing comprising antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer (pH 6). In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the number of distinct probes is 10 or more. In one embodiment, the subset of distinct probes can bind to i) estrogen receptor (ER), ii) progesterone receptor (PR), iii) human epidermal growth factor receptor 2 (HER2), and iv) Ki67. In one embodiment, the subset of distinct probes can bind to T cell biomarkers: i) CD3 (T cells), ii) CD4 (T helper), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, one of the different probes is capable of binding to the B cell biomarker CD20. In one embodiment, one of the different probes is capable of binding to the macrophage biomarker CD68. In one embodiment, one of the different probes is capable of binding to an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0022] In one embodiment, the present invention provides a multiplex method for simultaneously detecting different hormone receptors in a tissue sample on a single slide, the method comprising: a) providing a tissue sample mounted on a single slide; b) contacting the tissue sample with a first probe capable of binding to a first hormone receptor, the probe being conjugated to a first non-rare earth metal mass tag, the mass tag comprising a plurality of amino acids; c) contacting the tissue sample with a second probe capable of binding to a second hormone receptor, the probe being conjugated to a second non-rare earth metal mass tag, the mass tag comprising a second plurality of amino acids, the second hormone receptor being different from the first hormone receptor and the second plurality of amino acids having a different mass from the first plurality of amino acids, steps b) and c) together producing a probed tissue sample; 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 sectioned before mounting on the slide. In one embodiment, the tissue sample is formalin fixed and sectioned before mounting on the slide. In one embodiment, the tissue sample is formalin fixed, paraffin embedded and sectioned before mounting 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 tissue sample is from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the probe conjugated to a non-rare earth metal mass tag has the following general structure: [ka] and X is a spacer. In one embodiment, at least one of the probes comprises a fluorescent moiety in addition to the non-rare earth metal mass tag. In one embodiment, the probe is selected from the group consisting of a protein and a nucleic acid. In one embodiment, at least one of the probes is an antibody. In one embodiment, the antibody is selected from the group consisting of a recombinant antibody, a nanobody, a single-chain fragment variable (scFv) antibody, a single-domain antibody, and a VHH single-domain antibody. In one embodiment, at least one of the probes is selected from the group consisting of an affibody, a receptor, and a ligand. 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 to an RNA target. In one embodiment, at least one of the probes is capable of binding to an miRNA target. In one embodiment, at least one of the probes is capable of binding to a DNA target. In one embodiment, the non-rare earth metal mass tag is photocleavable. In one embodiment, the method further comprises, prior to step d), irradiating the non-rare earth metal mass tag with light 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 produce the probed tissue sample. In one embodiment, the method further includes performing mass spectrometry imaging on the tissue sample after step a) but before step b). In one embodiment, a matrix compound is applied to the probed tissue sample before step d). In one embodiment, the matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the probed tissue sample is subjected to processing after the step of applying the matrix compound to the probed tissue sample, the processing comprising matrix recrystallization. In one embodiment, the tissue sample is subjected to processing after the paraffin embedding and before step b), the processing comprising deparaffinization. In one embodiment, the deparaffinization is performed using xylene.In one embodiment, the tissue sample is further processed, the processing 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, the processing comprising antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer (pH 6). In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the first probe is capable of binding to an estrogen receptor (ER) and the second probe is capable of binding to a progesterone receptor (PR). In one embodiment, prior to step d), the method further comprises contacting the tissue sample with additional, different probes, each of the additional, different probes conjugated to a different non-rare earth metal mass tag, the mass tags comprising a plurality of amino acids, and each mass tag having 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 to i) human epidermal growth factor receptor 2 (HER2) and ii) Ki67. In one embodiment, the subset of additional different probes can bind to the following T cell biomarkers: i) CD3 (T cells), ii) CD4 (T helper), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, one of the additional different probes can bind to the B cell biomarker CD20. In one embodiment, one of the additional different probes can bind to the macrophage biomarker CD68. In one embodiment, one of the additional different probes can bind to an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0023] In one embodiment, the present invention provides a multiplex method for simultaneously detecting different types of tumor-infiltrating immune cells in tumor tissue on a single slide, the method comprising: a) providing a tumor tissue sample mounted on a single slide; b) contacting the tumor tissue sample with a first probe capable of binding to a first tumor-infiltrating immune cell type, wherein the probe is conjugated to a first non-rare earth metal mass tag, the 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, wherein the probe is conjugated to a second non-rare earth metal mass tag, the mass tag comprising a second plurality of amino acids, the second tumor-infiltrating immune cells being of a different type from the first tumor-infiltrating immune cells, the second plurality of amino acids having a different mass from the first plurality of amino acids, steps b) and c) together producing a probed tumor tissue sample; and d) detecting at least one of the mass tags or fragments thereof as a molecular ion using mass spectrometry imaging. In one embodiment, the tumor tissue sample is a thin tissue section. In one embodiment, the tumor tissue sample is frozen and sectioned before mounting on the slide. In one embodiment, the tumor tissue sample is formalin fixed and sectioned before mounting on the slide. In one embodiment, the tumor tissue sample is formalin fixed, paraffin embedded and sectioned before mounting 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 from a breast tumor. In one embodiment, the probe conjugated to a non-rare earth metal mass tag has the following general structure: [ka] and X is a spacer. In one embodiment, at least one of the probes comprises a fluorescent moiety in addition to the non-rare earth metal mass tag. In one embodiment, the probe is selected from the group consisting of a protein and a nucleic acid. In one embodiment, at least one of the probes is an antibody. In one embodiment, the antibody is selected from the group consisting of a recombinant antibody, a nanobody, a single-chain fragment variable (scFv) antibody, a single-domain antibody, and a VHH single-domain antibody. In one embodiment, at least one of the probes is selected from the group consisting of an affibody, a receptor, and a ligand. 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 to an RNA target. In one embodiment, at least one of the probes is capable of binding to an miRNA target. In one embodiment, at least one of the probes is capable of binding to a DNA target. In one embodiment, the non-rare earth metal mass tag is photocleavable. In one embodiment, the method further comprises, prior to step d), irradiating the non-rare earth metal mass tag with light 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 produce the probed tumor tissue sample. In one embodiment, the method further includes performing mass spectrometry imaging on the tumor tissue sample after step a) but before step b). In one embodiment, a matrix compound is applied to the probed tumor tissue sample before step d). In one embodiment, the matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid). In one embodiment, the matrix compound is applied by sublimation. In one embodiment, the probed tumor tissue sample is subjected to a treatment after the step of applying the matrix compound to the probed tumor tissue sample, the treatment comprising matrix recrystallization. In one embodiment, the tumor tissue sample is subjected to a treatment after the paraffin embedding and before step b), the treatment comprising deparaffinization.In one embodiment, the deparaffinization is performed using xylene. In one embodiment, the tumor tissue sample is further processed, the processing comprising 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, the processing comprising antigen retrieval. In one embodiment, the antigen retrieval is performed by heating in a citrate buffer (pH 6). In one embodiment, the antigen retrieval is performed using formic acid. In one embodiment, the first probe is capable of binding to CD4 and the second probe is capable of binding to CD8. In one embodiment, prior to step d), the method further comprises contacting the tumor tissue sample with additional, different probes, each of the additional, different probes conjugated to a different non-rare earth metal mass tag, the mass tags comprising a plurality of amino acids, and each mass tag having 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 to the T cell biomarker CD3. In one embodiment, one of the additional different probes is capable of binding to 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 group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0024] In one embodiment, the present invention provides a composition comprising a probe conjugated to i) a mass tag via a photocleavable linker and ii) a fluorophore. In one embodiment, the probe is capable of binding to a biomarker selected from the group consisting 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 to a T cell biomarker selected from the group consisting of i) CD3 (T cells), ii) CD4 (T helper), iii) CD8 (cytotoxic T cells), and iv) CD45RO (memory T cells). In one embodiment, the probe is capable of binding to an immune checkpoint molecule. In one embodiment, the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, OX40, CD27, and TIM3.

[0025] In one embodiment, the present invention provides a multiplex method for detecting different 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 tumor tissue sample with a mixture comprising a plurality of different probes, each capable of binding to a different immune checkpoint molecule, each of the probes 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 a molecular ion using mass spectrometry imaging. In one embodiment, one of the plurality of different probes is capable of binding to an immune checkpoint molecule selected from the group consisting of PD-1, PD-L1, PDL2, CTLA-4, OX40, CD27, and TIM3. In one embodiment, the tumor tissue sample comprises a biopsy from a human patient. In one embodiment, the tumor tissue biopsy binds to a mass-tagged probe for PD-L1. In one embodiment, the method further comprises treating the human patient with an immune checkpoint inhibitor specific for PD-L1. In one embodiment, the checkpoint inhibitor specific for PD-L1 is selected from the group consisting of atezolizumab, avelumab, and durvalurnab. In one embodiment, at least one of the probes is an antibody. In one embodiment, the antibody is selected from the group consisting of a recombinant antibody, a nanobody, a single-chain fragment variable (scFv) antibody, a single-domain antibody, and a VHH single-domain antibody.

[0026] In one embodiment, the present invention provides a multiplex method for simultaneously detecting three or more (and 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 (and more preferably five or more) different carbohydrate-binding proteins to effect binding of the carbohydrate-binding proteins to the tissue sample to produce a probed tissue sample, wherein each carbohydrate-binding protein is reactive with a different carbohydrate and each carbohydrate-binding protein is conjugated to a unique mass tag; and c) detecting the mass tag or a fragment thereof as a molecular ion using mass spectrometry imaging of the probed tissue sample. 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 formalin-fixed and sectioned before step b). In one embodiment, the tissue sample is formalin-fixed, paraffin-embedded, and sectioned before step b). In one embodiment, the tissue sample is mounted on a slide before 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 mounted on a 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 from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the carbohydrate binding protein conjugated to a mass tag has the following general structure: [ka] wherein X is a spacer and CBP is a carbohydrate binding protein. In one embodiment, the three or more (and more preferably five or more) carbohydrate-binding proteins are in a mixture, and in step b), the tissue sample is contacted with the mixture to produce the probed tissue sample. In one embodiment, the mixture further comprises one or more probes, each reactive with a different target in the tissue, and each probe is conjugated to a unique mass tag. In one embodiment, at least one of the five or more carbohydrate-binding proteins comprises a fluorescent moiety 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 comprises a plurality of amino acids. In one embodiment, the mass tag is photocleavable. In one embodiment, the method further comprises, prior to step c), irradiating the mass tags with light to photocleave at least a portion of the mass tags.

[0027] In one embodiment, the present invention provides a multi-omic method for detecting different biomolecules or fragments thereof in the same tissue sample, the method comprising: a) providing a tissue sample; b) applying a digestion agent to at least a portion of the tissue sample; c) performing mass spectrometry imaging on the tissue sample; d) contacting the tissue sample with three or more (and more preferably five or more) different probes to cause binding of the probes to the tissue sample to produce a probed tissue sample, wherein each of the probes is reactive with a different target within the tissue sample and each of the probes is conjugated to a unique mass tag; and e) detecting the mass tags or fragments thereof as molecular ions using mass spectrometry imaging of the probed tissue sample. In one embodiment, the digestion 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 digestion 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 digestion agents is applied instead in step b). In one embodiment, steps b) and c) are instead performed after step e), respectively. In one embodiment, the tissue sample is further subjected to a treatment after step e) prior to the step of applying the digestion agent to the tissue sample, the treatment comprising separating the bound probes from the tissue sample. In one embodiment, the separation of the bound probes comprises subjecting the tissue sample to a denaturing treatment. In one embodiment, the denaturing treatment is selected from the group consisting of a chaotropic agent, a solution of pH≦5, a solution of pH≧10, a reducing agent, an oxidizing agent, heat, an organic solvent, and a detergent. 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 formalin-fixed and sectioned before step b). In one embodiment, the tissue sample is formalin-fixed, paraffin-embedded, and sectioned before step b).In one embodiment, prior to step b), the tissue sample is mounted on a 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 from a tumor. In one embodiment, the tumor is a breast tumor. In one embodiment, the probe conjugated to a mass tag has the following general structure: [ka] and X is a spacer. In one embodiment, the three or more (and more preferably five or more) probes are in a mixture, and in step d) the tissue sample is contacted with the mixture to produce the probed tissue sample. In one embodiment, at least one of the five or more probes comprises a fluorescent moiety in addition to the mass tag. In one embodiment, the probes are selected from the group consisting of proteins and nucleic acids. In one embodiment, at least one of the protein probes is selected from the group consisting of antibodies, recombinant antibodies, affibodies, nanobodies, single-chain 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 an 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 comprises a plurality of amino acids. In one embodiment, the mass tag is photocleavable. In one embodiment, the method further comprises, prior to step e), irradiating the mass tag with light so as to photocleave at least a portion of the mass tag.

[0028] In one embodiment, the present invention provides compounds having the following general chemical structure: [ka] a composition comprising: wherein A is a first linker unit (also referred to as 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 referred to as 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: [ka] It has. In one embodiment, the first and / or second linker (or first and / or second spacer unit) comprises polyethylene glycol. In one embodiment, the first and / or second linker (or first and / or second spacer unit) comprises a 2,2'-(ethylenedioxy)-bis-(ethylamine) chemical linker. [Brief explanation of the drawings]

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color will be provided by the Office upon request and payment of the necessary fee.

[0031] [Figure 1]Bottom-up application of mass spectrometry imaging (MSI) for multiplexed, direct, label-free mapping of analytes in tissue samples. Adapted from [Arentz, Mittal, et al. (2017) Adv Cancer Res 134:27-66]. This existing approach, while multiplexed, is inherently untargeted because it does not use labeled probes such as photocleavable mass-tagged probes (PC-MT-probes) and therefore requires complex identification methods for the detected analytes, potentially reducing the spatial resolution of the images (e.g., because in situ tissue proteolysis is required for protein detection and identification).

[0032] [Figure 2A]Figures 2A-2C. Basic design and use of preferred photocleavable mass tags (PC-MTs) and photocleavable mass-tagged probes (PC-MT-probes). (Figure 2A) Preferred peptide-based PC-MTs. The curved lines represent peptide-based mass units, the ovals represent photocleavable linkers (PC-linkers), and "NHS" represents the probe-reactive moiety, shown in this example as a primary amine-reactive NHS ester leaving group. Note that peptide-based PC-MTs are N-terminally blocked / protected (e.g., acetylated—not shown) to prevent self-reaction / polymerization of the PC-MTs if the probe-reactive moiety is primary amine-reactive (and internal primary amines, such as the s-amine on lysine amino acids, are avoided or protected / blocked). Mass coding of the mass units is achieved using different amino acids (or their isotopes, analogs, derivatives, or modifications, including natural and unnatural amino acids). Note that PC-MTs optionally contain a fluorophore (starburst with an "F"), which can aid in method development by enabling direct comparison of mass spectrometry imaging (MSI) results with conventional fluorescence imaging. (Figure 2B) PC-MTs are conjugated to a probe, such as an antibody or amine-modified nucleic acid, to create a PC-MT-probe. This is typically accomplished in a one-step chemical reaction between the probe-reactive moiety of the PC-MT (e.g., the NHS-ester leaving group, which is lost in this case) and the probe, with or without subsequent purification of the PC-MT-probe. (Figure 2C) The PC-MT probe is then used to "stain" tissue (i.e., the PC-MT probe is bound to the target in the tissue) in a procedure similar to traditional immunohistochemistry (IHC) or in situ hybridization (ISH), followed by MSI. Photocleavage to liberate the mass reporter can be achieved by exogenous UV treatment prior to MSI, or in parallel with matrix-assisted laser desorption / ionization MSI (MALDI-MSI) using the instrument's laser beam. Note that the mass reporter in some embodiments can include an attached photocleavable PC-linker moiety. [Figure 2B]Figures 2A-2C. Basic design and use of preferred photocleavable mass tags (PC-MTs) and photocleavable mass-tagged probes (PC-MT-probes). (Figure 2A) Preferred peptide-based PC-MTs. The curved lines represent peptide-based mass units, the ovals represent photocleavable linkers (PC-linkers), and "NHS" represents the probe-reactive moiety, shown in this example as a primary amine-reactive NHS ester leaving group. Note that peptide-based PC-MTs are N-terminally blocked / protected (e.g., acetylated—not shown) to prevent self-reaction / polymerization of the PC-MTs if the probe-reactive moiety is primary amine-reactive (and internal primary amines, such as the s-amine on lysine amino acids, are avoided or protected / blocked). Mass coding of the mass units is achieved using different amino acids (or their isotopes, analogs, derivatives, or modifications, including natural and unnatural amino acids). Note that PC-MTs optionally contain a fluorophore (starburst with an "F"), which can aid in method development by enabling direct comparison of mass spectrometry imaging (MSI) results with conventional fluorescence imaging. (Figure 2B) PC-MTs are conjugated to a probe, such as an antibody or amine-modified nucleic acid, to create a PC-MT-probe. This is typically accomplished in a one-step chemical reaction between the probe-reactive moiety of the PC-MT (e.g., the NHS-ester leaving group, which is lost in this case) and the probe, with or without subsequent purification of the PC-MT-probe. (Figure 2C) The PC-MT probe is then used to "stain" tissue (i.e., the PC-MT probe is bound to the target in the tissue) in a procedure similar to traditional immunohistochemistry (IHC) or in situ hybridization (ISH), followed by MSI. Photocleavage to liberate the mass reporter can be achieved by exogenous UV treatment prior to MSI, or in parallel with matrix-assisted laser desorption / ionization MSI (MALDI-MSI) using the instrument's laser beam. Note that the mass reporter in some embodiments can include an attached photocleavable PC-linker moiety. [Figure 2C]Figures 2A-2C. Basic design and use of preferred photocleavable mass tags (PC-MTs) and photocleavable mass-tagged probes (PC-MT-probes). (Figure 2A) Preferred peptide-based PC-MTs. The curved lines represent peptide-based mass units, the ovals represent photocleavable linkers (PC-linkers), and "NHS" represents the probe-reactive moiety, shown in this example as a primary amine-reactive NHS ester leaving group. Note that peptide-based PC-MTs are N-terminally blocked / protected (e.g., acetylated—not shown) to prevent self-reaction / polymerization of the PC-MTs if the probe-reactive moiety is primary amine-reactive (and internal primary amines, such as the s-amine on lysine amino acids, are avoided or protected / blocked). Mass coding of the mass units is achieved using different amino acids (or their isotopes, analogs, derivatives, or modifications, including natural and unnatural amino acids). Note that PC-MTs optionally contain a fluorophore (starburst with an "F"), which can aid in method development by enabling direct comparison of mass spectrometry imaging (MSI) results with conventional fluorescence imaging. (Figure 2B) PC-MTs are conjugated to a probe, such as an antibody or amine-modified nucleic acid, to create a PC-MT-probe. This is typically accomplished in a one-step chemical reaction between the probe-reactive moiety of the PC-MT (e.g., the NHS-ester leaving group, which is lost in this case) and the probe, with or without subsequent purification of the PC-MT-probe. (Figure 2C) The PC-MT probe is then used to "stain" tissue (i.e., the PC-MT probe is bound to the target in the tissue) in a procedure similar to traditional immunohistochemistry (IHC) or in situ hybridization (ISH), followed by MSI. Photocleavage to liberate the mass reporter can be achieved by exogenous UV treatment prior to MSI, or in parallel with matrix-assisted laser desorption / ionization MSI (MALDI-MSI) using the instrument's laser beam. Note that the mass reporter in some embodiments can include an attached photocleavable PC-linker moiety.

[0033] [Figure 3]Detailed chemical structures and uses of preferred photocleavable linkers (PC-linkers), photocleavable mass tags (PC-MTs), and photocleavable mass-tagged probes (PC-MT-probes). The Fmoc-PC-linker is incorporated into a peptide-based PC-MT (Step 1) along with other amino acids (or their isotopes, analogs, derivatives, or modifications, including natural and unnatural amino acids) during conventional Fmoc-based solid-phase peptide synthesis (SPPS). The Fmoc-PC-linker includes an optional linker unit, which can be of various chemical compositions. The exemplary PC-MT shown is oriented with the N-terminus facing left and the C-terminus facing right. "APRLRFYSL" is an example of a mass unit amino acid sequence. The PC-MT includes an optional spacer unit, which is shown as part of the PC-linker + GSGGK amino acid sequence as an example. The PC-MT also includes an optional mass unit linker. The spacer unit and mass unit linker can be of various chemical compositions. The photocleavable core of the PC-linker (PC-core) is a fast and efficient 1-(2-nitrophenyl)ethyl-based moiety. The probe-reactive moiety (e.g., shown as an NHS-ester leaving group) can be generated, for example, on the s-amine of the included lysine amino acid as shown (or, for example, on the C-terminal carboxylic acid group—not shown). "Ac" is the N-terminal acetylation of the a-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 the PC-MT-probe. In the example shown, the NHS-ester leaving group (probe-reactive moiety) of the PC-MT reacts with a primary amine on the antibody (the NHS-ester is lost, forming an amide bond). 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 typically the common structure of all PC-MT-probe species, corresponding to the common structure of all PC-MT species. (Step 3) Finally, the PC-MT-probes are bound to targets in tissues, followed by photocleavage (to release the mass reporter) and mass spectrometry imaging (MSI) (MSI not shown).Note that a small residual portion of the PC-linker (mass unit linker) remains as part of the photocleaved mass reporter, in this example generating a primary amine group, which can assist ionization in positive-mode MSI.

[0034] [Figure 4] Comparison of the structure of the PC-linker used in the example PC-linker of the present invention and the PC-linker used in the study 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-linker is shown as incorporated into the exemplary peptide sequence.

[0035] [Figure 5A] Figures 5A-5B. Exemplary methods for PC-MT signal amplification. (Figure 5A) For example, multiple PC-MTs bearing NHS-ester-based probe-reactive moieties can be conjugated to the surface of amine-terminated gold nanoparticles ("NPs," shown) or dendrimers (not shown). The dendrimer or NP can then be conjugated to a probe ("antibody probe" shown). (Figure 5B) For nucleic acid probes, a primary amine-modified nucleic acid sequence that is not part of the "target binding" sequence (i.e., target hybridization sequence) can preferably be used to facilitate the attachment of many PC-MTs per probe molecule (e.g., again, if the probe-reactive moiety is primary amine reactive). [Figure 5B]Figures 5A-5B. Exemplary methods for PC-MT signal amplification. (Figure 5A) For example, multiple PC-MTs bearing NHS-ester-based probe-reactive moieties can be conjugated to the surface of amine-terminated gold nanoparticles ("NPs," shown) or dendrimers (not shown). The dendrimer or NP can then be conjugated to a probe ("antibody probe" shown). (Figure 5B) For nucleic acid probes, a primary amine-modified nucleic acid sequence that is not part of the "target binding" sequence (i.e., target hybridization sequence) can preferably be used to facilitate the attachment of many PC-MTs per probe molecule (e.g., again, if the probe-reactive moiety is primary amine reactive).

[0036] [Figure 6] 15-plex PC-MT-Ab-based MSI using a bead array as a model system. Fifteen different versions of anti-streptavidin PC-MT-Ab were generated by direct, one-step labeling with 15 novel amine-reactive NHS-activated PC-MT reagents. Each PC-MT-Ab was used separately to probe 20 μm polymeric streptavidin beads. The beads were then pooled and used to form arrays on the footprint of a microscope slide, which were then subjected to MALDI-MSI. The inset shows a color-coded 15-plex MALDI-MS "mass image" of a representative area of ​​the bead array. Color-coded overlaid spectra are from representative single beads within the mass image for each of the 15 different PC-MT-Ab-probed bead versions (black arrows indicate distinct PC-MT mass reporter peaks). Note that the 1 Da separated natural isotopes of the peptide PC-MT are readily resolved by MALDI-MS but are not visible in the provided spectra due to the compact x-axis scaling.

[0037] [Figure 7A]Figures 7A-7E. 5-plex MIHC using PC-MT-Ab on mouse brain FFPE tissue sections. FFPE tissue sections were simultaneously stained with PC-MT-Ab for five different protein targets and then subjected to MALDI-MSI. For MSI, matrix application was by sublimation followed by recrystallization (MSI in positive ion reflectance mode). Standard immunofluorescence staining was also performed on adjacent tissue sections. (Figure 7A) Color-coded 5-plex overlay MALDI-MSI "mass image" for synapsin (blue), MAP-2 (orange), NeuN (green), myelin (red), and Glut-1 (cyan). Different colors correspond to different m / z values ​​of the monoisotopic PC-MT mass reporter mass spectral peaks. Among other structures, the hippocampus (*) and cerebellum (I) are observed. (Figure 7B) Colorized immunofluorescence overlay of the cerebellum for synapsin (blue), NeuN (green), and myelin (red). Immunofluorescence was performed in "singleplex" mode on adjacent tissue sections and overlaid images. (Figure 7C) A standalone MALDI-MS mass image (subregion) of the less prominent Glut-1 biomarker is shown (in this case, magenta), which primarily detects brain capillaries (cross-section). (Figure 7D) The corresponding immunofluorescence of Glut-1 (in this case, green). (Figure 7E) A color-coded overlay MALDI-MS spectrum is shown for selected pixels from the 5-plex mass image (from the pixels indicated by the color-coded arrows in panel a). The black arrow in the spectrum indicates the m / z peak of the PC-MT mass reporter. Note that the 1 Da separated natural isotope of the peptide PC-MT is easily resolved by MALDI-MS but is not visible in the provided spectrum due to the compact x-axis scaling. [Figure 7B]Figures 7A-7E. 5-plex MIHC using PC-MT-Ab on mouse brain FFPE tissue sections. FFPE tissue sections were simultaneously stained with PC-MT-Ab for five different protein targets and then subjected to MALDI-MSI. For MSI, matrix application was by sublimation followed by recrystallization (MSI in positive ion reflectance mode). Standard immunofluorescence staining was also performed on adjacent tissue sections. (Figure 7A) Color-coded 5-plex overlay MALDI-MSI "mass image" for synapsin (blue), MAP-2 (orange), NeuN (green), myelin (red), and Glut-1 (cyan). Different colors correspond to different m / z values ​​of the monoisotopic PC-MT mass reporter mass spectral peaks. Among other structures, the hippocampus (*) and cerebellum (I) are observed. (Figure 7B) Colorized immunofluorescence overlay of the cerebellum for synapsin (blue), NeuN (green), and myelin (red). Immunofluorescence was performed in "singleplex" mode on adjacent tissue sections and overlaid images. (Figure 7C) A standalone MALDI-MS mass image (subregion) of the less prominent Glut-1 biomarker is shown (in this case, magenta), which primarily detects brain capillaries (cross-section). (Figure 7D) The corresponding immunofluorescence of Glut-1 (in this case, green). (Figure 7E) A color-coded overlay MALDI-MS spectrum is shown for selected pixels from the 5-plex mass image (from the pixels indicated by the color-coded arrows in panel a). The black arrow in the spectrum indicates the m / z peak of the PC-MT mass reporter. Note that the 1 Da separated natural isotope of the peptide PC-MT is easily resolved by MALDI-MS but is not visible in the provided spectrum due to the compact x-axis scaling. [Figure 7C]Figures 7A-7E. 5-plex MIHC using PC-MT-Ab on mouse brain FFPE tissue sections. FFPE tissue sections were simultaneously stained with PC-MT-Ab for five different protein targets and then subjected to MALDI-MSI. For MSI, matrix application was by sublimation followed by recrystallization (MSI in positive ion reflectance mode). Standard immunofluorescence staining was also performed on adjacent tissue sections. (Figure 7A) Color-coded 5-plex overlay MALDI-MSI "mass image" for synapsin (blue), MAP-2 (orange), NeuN (green), myelin (red), and Glut-1 (cyan). Different colors correspond to different m / z values ​​of the monoisotopic PC-MT mass reporter mass spectral peaks. Among other structures, the hippocampus (*) and cerebellum (I) are observed. (Figure 7B) Colorized immunofluorescence overlay of the cerebellum for synapsin (blue), NeuN (green), and myelin (red). Immunofluorescence was performed in "singleplex" mode on adjacent tissue sections and overlaid images. (Figure 7C) A standalone MALDI-MS mass image (subregion) of the less prominent Glut-1 biomarker is shown (in this case, magenta), which primarily detects brain capillaries (cross-section). (Figure 7D) The corresponding immunofluorescence of Glut-1 (in this case, green). (Figure 7E) A color-coded overlay MALDI-MS spectrum is shown for selected pixels from the 5-plex mass image (from the pixels indicated by the color-coded arrows in panel a). The black arrow in the spectrum indicates the m / z peak of the PC-MT mass reporter. Note that the 1 Da separated natural isotope of the peptide PC-MT is easily resolved by MALDI-MS but is not visible in the provided spectrum due to the compact x-axis scaling. [Figure 7D]Figures 7A-7E. 5-plex MIHC using PC-MT-Ab on mouse brain FFPE tissue sections. FFPE tissue sections were simultaneously stained with PC-MT-Ab for five different protein targets and then subjected to MALDI-MSI. For MSI, matrix application was by sublimation followed by recrystallization (MSI in positive ion reflectance mode). Standard immunofluorescence staining was also performed on adjacent tissue sections. (Figure 7A) Color-coded 5-plex overlay MALDI-MSI "mass image" for synapsin (blue), MAP-2 (orange), NeuN (green), myelin (red), and Glut-1 (cyan). Different colors correspond to different m / z values ​​of the monoisotopic PC-MT mass reporter mass spectral peaks. Among other structures, the hippocampus (*) and cerebellum (I) are observed. (Figure 7B) Colorized immunofluorescence overlay of the cerebellum for synapsin (blue), NeuN (green), and myelin (red). Immunofluorescence was performed in "singleplex" mode on adjacent tissue sections and overlaid images. (Figure 7C) A standalone MALDI-MS mass image (subregion) of the less prominent Glut-1 biomarker is shown (in this case, magenta), which primarily detects brain capillaries (cross-section). (Figure 7D) The corresponding immunofluorescence of Glut-1 (in this case, green). (Figure 7E) A color-coded overlay MALDI-MS spectrum is shown for selected pixels from the 5-plex mass image (from the pixels indicated by the color-coded arrows in panel a). The black arrow in the spectrum indicates the m / z peak of the PC-MT mass reporter. Note that the 1 Da separated natural isotope of the peptide PC-MT is easily resolved by MALDI-MS but is not visible in the provided spectrum due to the compact x-axis scaling. [Figure 7E]Figures 7A-7E. 5-plex MIHC using PC-MT-Ab on mouse brain FFPE tissue sections. FFPE tissue sections were simultaneously stained with PC-MT-Ab for five different protein targets and then subjected to MALDI-MSI. For MSI, matrix application was by sublimation followed by recrystallization (MSI in positive ion reflectance mode). Standard immunofluorescence staining was also performed on adjacent tissue sections. (Figure 7A) Color-coded 5-plex overlay MALDI-MSI "mass image" for synapsin (blue), MAP-2 (orange), NeuN (green), myelin (red), and Glut-1 (cyan). Different colors correspond to different m / z values ​​of the monoisotopic PC-MT mass reporter mass spectral peaks. Among other structures, the hippocampus (*) and cerebellum (I) are observed. (Figure 7B) Colorized immunofluorescence overlay of the cerebellum for synapsin (blue), NeuN (green), and myelin (red). Immunofluorescence was performed in "singleplex" mode on adjacent tissue sections and overlaid images. (Figure 7C) A standalone MALDI-MS mass image (subregion) of the less prominent Glut-1 biomarker is shown (in this case, magenta), which primarily detects brain capillaries (cross-section). (Figure 7D) The corresponding immunofluorescence of Glut-1 (in this case, green). (Figure 7E) A color-coded overlay MALDI-MS spectrum is shown for selected pixels from the 5-plex mass image (from the pixels indicated by the color-coded arrows in panel a). The black arrow in the spectrum indicates the m / z peak of the PC-MT mass reporter. Note that the 1 Da separated natural isotope of the peptide PC-MT is easily resolved by MALDI-MS but is not visible in the provided spectrum due to the compact x-axis scaling.

[0038] [Figure 8]MISH using PC-MT directly conjugated to an oligonucleotide probe. In situ hybridization was performed on sagittal tissue sections of mouse brain 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 corresponding to the m / z values ​​of the monoisotopic PC-MT mass reporter mass spectral peaks (green) are shown. Yellow arrows highlight cerebellar and hippocampal features detected with U6.

[0039] [Figure 9A]Figures 9A-9D. Nontargeted small molecule MALDI-MSI and targeted PC-MT-Ab-based macromolecular MALDI-MSI on the same tissue section. (Figure 9A) Direct nontargeted MALDI-MSI was performed on a sagittal tissue section of unfixed, fresh-frozen mouse brain. Three well-known lipid species (sulfatide, m / z 888.7; phosphatidylinositol, m / z 885.4; and phosphatidylethanolamine, m / z 790.5) are shown in the colorized MALDI-MS image (red, green, and blue, respectively). (Figures 9B-9C) The same tissue section was then processed for a second round of MALDI-MSI. To do so, the matrix was washed away, the tissue was fixed, and targeted multiplex MALDI-MSI of macromolecular antigens using PC-MT-Ab was performed. For demonstration purposes, images of selected biomolecules from the first and second rounds of MALDI-MSI were overlaid. (Figure 9B) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of NeuN (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9C) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of myelin basic protein (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9D) An exemplary overlay spectrum from the first MALDI-MSI (direct small molecule detection) is shown, color-coded to match the image in Figure 9A (three lipid masses are shown). The color-coded arrows in Figure 9A indicate the region from which the MALDI-MS spectrum was derived. [Figure 9B]Figures 9A-9D. Nontargeted small molecule MALDI-MSI and targeted PC-MT-Ab-based macromolecular MALDI-MSI on the same tissue section. (Figure 9A) Direct nontargeted MALDI-MSI was performed on a sagittal tissue section of unfixed, fresh-frozen mouse brain. Three well-known lipid species (sulfatide, m / z 888.7; phosphatidylinositol, m / z 885.4; and phosphatidylethanolamine, m / z 790.5) are shown in the colorized MALDI-MS image (red, green, and blue, respectively). (Figures 9B-9C) The same tissue section was then processed for a second round of MALDI-MSI. To do so, the matrix was washed away, the tissue was fixed, and targeted multiplex MALDI-MSI of macromolecular antigens using PC-MT-Ab was performed. For demonstration purposes, images of selected biomolecules from the first and second rounds of MALDI-MSI were overlaid. (Figure 9B) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of NeuN (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9C) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of myelin basic protein (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9D) An exemplary overlay spectrum from the first MALDI-MSI (direct small molecule detection) is shown, color-coded to match the image in Figure 9A (three lipid masses are shown). The color-coded arrows in Figure 9A indicate the region from which the MALDI-MS spectrum was derived. [Figure 9C]Figures 9A-9D. Nontargeted small molecule MALDI-MSI and targeted PC-MT-Ab-based macromolecular MALDI-MSI on the same tissue section. (Figure 9A) Direct nontargeted MALDI-MSI was performed on a sagittal tissue section of unfixed, fresh-frozen mouse brain. Three well-known lipid species (sulfatide, m / z 888.7; phosphatidylinositol, m / z 885.4; and phosphatidylethanolamine, m / z 790.5) are shown in the colorized MALDI-MS image (red, green, and blue, respectively). (Figures 9B-9C) The same tissue section was then processed for a second round of MALDI-MSI. To do so, the matrix was washed away, the tissue was fixed, and targeted multiplex MALDI-MSI of macromolecular antigens using PC-MT-Ab was performed. For demonstration purposes, images of selected biomolecules from the first and second rounds of MALDI-MSI were overlaid. (Figure 9B) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of NeuN (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9C) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of myelin basic protein (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9D) An exemplary overlay spectrum from the first MALDI-MSI (direct small molecule detection) is shown, color-coded to match the image in Figure 9A (three lipid masses are shown). The color-coded arrows in Figure 9A indicate the region from which the MALDI-MS spectrum was derived. [Figure 9D]Figures 9A-9D. Nontargeted small molecule MALDI-MSI and targeted PC-MT-Ab-based macromolecular MALDI-MSI on the same tissue section. (Figure 9A) Direct nontargeted MALDI-MSI was performed on a sagittal tissue section of unfixed, fresh-frozen mouse brain. Three well-known lipid species (sulfatide, m / z 888.7; phosphatidylinositol, m / z 885.4; and phosphatidylethanolamine, m / z 790.5) are shown in the colorized MALDI-MS image (red, green, and blue, respectively). (Figures 9B-9C) The same tissue section was then processed for a second round of MALDI-MSI. To do so, the matrix was washed away, the tissue was fixed, and targeted multiplex MALDI-MSI of macromolecular antigens using PC-MT-Ab was performed. For demonstration purposes, images of selected biomolecules from the first and second rounds of MALDI-MSI were overlaid. (Figure 9B) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of NeuN (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9C) Sulfatides (red) from the first MALDI-MSI (direct small molecule detection) are overlaid with an image of myelin basic protein (green) from the second MALDI-MSI (multiplex MIHC). (Figure 9D) An exemplary overlay spectrum from the first MALDI-MSI (direct small molecule detection) is shown, color-coded to match the image in Figure 9A (three lipid masses are shown). The color-coded arrows in Figure 9A indicate the region from which the MALDI-MS spectrum was derived.

[0040] [Figure 10A]Figures 10A-10B. Preferred attachment sites of mass units to the PC-core. (Figure 10A) Two configurations for final attachment of mass units to the phenyl ring of the PC-core are shown (in this case shown after photocleavage—see Figure 3, step 2, for an example of configuration 1 before photocleavage). In this example, a photocleavable peptide was used, which was ultimately attached to the surface as shown (similar to the PC-MT probe ultimately attached to the tissue surface). In preferred configuration 1, the photocleaved phenyl ring of the PC-core (indicated with an *) does not remain attached to the photocleaved mass reporter as measured by mass spectrometry (MS), whereas in configuration 2 it does. (Figure 10B) Obtained overlaid mass spectra of the mass reporter after photocleavage from the surface for the two configurations. The expected monoisotopic peak of the mass reporter is labeled with its respective m / z value for both configurations. [Figure 10B] Figures 10A-10B. Preferred attachment sites of mass units to the PC-core. (Figure 10A) Two configurations for final attachment of mass units to the phenyl ring of the PC-core are shown (in this case shown after photocleavage—see Figure 3, step 2, for an example of configuration 1 before photocleavage). In this example, a photocleavable peptide was used, which was ultimately attached to the surface as shown (similar to the PC-MT probe ultimately attached to the tissue surface). In preferred configuration 1, the photocleaved phenyl ring of the PC-core (indicated with an *) does not remain attached to the photocleaved mass reporter as measured by mass spectrometry (MS), whereas in configuration 2 it does. (Figure 10B) Obtained overlaid mass spectra of the mass reporter after photocleavage from the surface for the two configurations. The expected monoisotopic peak of the mass reporter is labeled with its respective m / z value for both configurations.

[0041] [Figure 11A]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11B]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11C]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11D]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11E]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11F]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11G]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11H]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11I]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11J]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11K]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L. [Figure 11L]Figures 11A-11L. Comparison of different PC linkers and pre-photocleavage versus in-line photocleavage using a MALDI-MS laser beam. (Figure 11A) Exemplary MALDI-MSI mass images of a bead array. The beads were dual-labeled with PC-MT (red) and PC-MT-L (green), both consisting of mass unit 1 (however, the mass reporters are distinguishable by a 43 m / z difference, as only PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter). (Figures 11B-11I) Four different bead species were fabricated, each dual-labeled with PC-MT and PC-MT-L, each with the same mass unit (mass units 1-4 in Table 1). Four separate bead arrays were formed from each of these bead types (one bead array per bead type), and each bead array was subjected to 5 minutes of pre-UV irradiation for photocleavage. Another four bead arrays were fabricated and subjected to 25 minutes of pre-UV irradiation. A total of eight separate bead arrays were then subjected to MALDI-MSI. Representative spectra are shown for a single pixel from each bead array (each of the eight spectra contains peaks corresponding to mass reporters from both PC-MT and PC-MT-L, which are composed of the same mass unit). (Figure 11J) Anti-myelin antibody probes were double-labeled with PC-MT and PC-MT-L, both composed of 1 mass unit. Mouse brain tissue sections were immunostained using the probes and subsequently subjected to MALDI-MSI. Color-coded mass images of the tissues are shown. PC-MT is color-coded red, and PC-MT-L is color-coded green. Pre-UV conditions of 0, 5, and 25 minutes were tested, with pre-UV performed immediately before matrix application and before MALDI-MSI analysis. In the case of 0 minutes of pre-UV, photocleavage can only occur during MALDI-MSI analysis due to the instrument's laser beam. Spectra from representative pixels are shown in Figures 11K and 11L.

[0042] [Figure 12]Fluorescent PC-MT for mass spectrometry-based immunohistochemistry. The "Fluorescence" panel shows fluorescence images (yellow) of three sagittal mouse brain tissue sections probed with fluorescent anti-NeuN Fluor-PC-MT1, non-fluorescent anti-NeuN PC-MT7, and negative control fluorescent anti-Cas9 Fluor-PC-MT1. The "MALDI-MSI" panel shows MALDI-MS images of the same probed tissue sections (red for the mass reporter from Fluor-PC-MT1 and green for the mass reporter from PC-MT7). The blue arrow indicates the hippocampus, and the white arrow indicates the cerebellum. The inset spectrum is from a representative single pixel within the hippocampus; the corresponding mass reporter peak, if detected, is indicated by a black arrow.

[0043] [Figure 13A]Figures 13A-13E. Recombinant antibodies versus conventional antibodies. 37 μm streptavidin PMMA beads were directly conjugated to PC-MTs (bead ID tags), then loaded with biotinylated protein G and subsequently bound to specific PC-MT-Abs (each PC-MT-Ab species was loaded separately onto a specific bead species). (Figure 13A) MALDI-MSI mass image of bead pool 1. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-MBP1, anti-MBP1, and recombinant anti-AB2 (note that the numbers indicate the mass units from Table 1 for the specific PC-MTs), respectively. (Figure 13B) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13A). The spectra are color-coded according to the bead colors observed in (Figure 13A). (Figure 13C) MALDI-MSI mass image of bead pool 2. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-NeuN7, anti-NeuN7, and recombinant anti-AB2, respectively (note that the numbers indicate the mass units from Table 1 for the specific PC-MT). (Figure 13D) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13C). The spectra are color-coded according to the bead colors observed in (Figure 13C). (Figure 13E) Separately, a sagittal tissue section of mouse brain was stained with anti-NeuN7 and recombinant anti-NeuN7 and subjected to MALDI-MSI. Antibody-derived PC-MT7 is colored green in the image. The white arrow indicates the cerebellum, the blue arrow indicates the hippocampus, and the yellow arrow indicates a punctate nuclear staining pattern. In the provided spectra, selected from the most intense pixels in the hippocampus of both images, the red trace shows PC-MT7 from the “recombinant” recombinant anti-NeuN7 antibody probe, and the blue trace shows PC-MT7 from the “normal” non-recombinant anti-NeuN7 antibody probe. [Figure 13B]Figures 13A-13E. Recombinant antibodies versus conventional antibodies. 37 μm streptavidin PMMA beads were directly conjugated to PC-MTs (bead ID tags), then loaded with biotinylated protein G and subsequently bound to specific PC-MT-Abs (each PC-MT-Ab species was loaded separately onto a specific bead species). (Figure 13A) MALDI-MSI mass image of bead pool 1. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-MBP1, anti-MBP1, and recombinant anti-AB2 (note that the numbers indicate the mass units from Table 1 for the specific PC-MTs), respectively. (Figure 13B) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13A). The spectra are color-coded according to the bead colors observed in (Figure 13A). (Figure 13C) MALDI-MSI mass image of bead pool 2. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-NeuN7, anti-NeuN7, and recombinant anti-AB2, respectively (note that the numbers indicate the mass units from Table 1 for the specific PC-MT). (Figure 13D) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13C). The spectra are color-coded according to the bead colors observed in (Figure 13C). (Figure 13E) Separately, a sagittal tissue section of mouse brain was stained with anti-NeuN7 and recombinant anti-NeuN7 and subjected to MALDI-MSI. Antibody-derived PC-MT7 is colored green in the image. The white arrow indicates the cerebellum, the blue arrow indicates the hippocampus, and the yellow arrow indicates a punctate nuclear staining pattern. In the provided spectra, selected from the most intense pixels in the hippocampus of both images, the red trace shows PC-MT7 from the “recombinant” recombinant anti-NeuN7 antibody probe, and the blue trace shows PC-MT7 from the “normal” non-recombinant anti-NeuN7 antibody probe. [Figure 13C]Figures 13A-13E. Recombinant antibodies versus conventional antibodies. 37 μm streptavidin PMMA beads were directly conjugated to PC-MTs (bead ID tags), then loaded with biotinylated protein G and subsequently bound to specific PC-MT-Abs (each PC-MT-Ab species was loaded separately onto a specific bead species). (Figure 13A) MALDI-MSI mass image of bead pool 1. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-MBP1, anti-MBP1, and recombinant anti-AB2 (note that the numbers indicate the mass units from Table 1 for the specific PC-MTs), respectively. (Figure 13B) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13A). The spectra are color-coded according to the bead colors observed in (Figure 13A). (Figure 13C) MALDI-MSI mass image of bead pool 2. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-NeuN7, anti-NeuN7, and recombinant anti-AB2, respectively (note that the numbers indicate the mass units from Table 1 for the specific PC-MT). (Figure 13D) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13C). The spectra are color-coded according to the bead colors observed in (Figure 13C). (Figure 13E) Separately, a sagittal tissue section of mouse brain was stained with anti-NeuN7 and recombinant anti-NeuN7 and subjected to MALDI-MSI. Antibody-derived PC-MT7 is colored green in the image. The white arrow indicates the cerebellum, the blue arrow indicates the hippocampus, and the yellow arrow indicates a punctate nuclear staining pattern. In the provided spectra, selected from the most intense pixels in the hippocampus of both images, the red trace shows PC-MT7 from the “recombinant” recombinant anti-NeuN7 antibody probe, and the blue trace shows PC-MT7 from the “normal” non-recombinant anti-NeuN7 antibody probe. [Figure 13D]Figures 13A-13E. Recombinant antibodies versus conventional antibodies. 37 μm streptavidin PMMA beads were directly conjugated to PC-MTs (bead ID tags), then loaded with biotinylated protein G and subsequently bound to specific PC-MT-Abs (each PC-MT-Ab species was loaded separately onto a specific bead species). (Figure 13A) MALDI-MSI mass image of bead pool 1. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-MBP1, anti-MBP1, and recombinant anti-AB2 (note that the numbers indicate the mass units from Table 1 for the specific PC-MTs), respectively. (Figure 13B) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13A). The spectra are color-coded according to the bead colors observed in (Figure 13A). (Figure 13C) MALDI-MSI mass image of bead pool 2. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-NeuN7, anti-NeuN7, and recombinant anti-AB2, respectively (note that the numbers indicate the mass units from Table 1 for the specific PC-MT). (Figure 13D) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13C). The spectra are color-coded according to the bead colors observed in (Figure 13C). (Figure 13E) Separately, a sagittal tissue section of mouse brain was stained with anti-NeuN7 and recombinant anti-NeuN7 and subjected to MALDI-MSI. Antibody-derived PC-MT7 is colored green in the image. The white arrow indicates the cerebellum, the blue arrow indicates the hippocampus, and the yellow arrow indicates a punctate nuclear staining pattern. In the provided spectra, selected from the most intense pixels in the hippocampus of both images, the red trace shows PC-MT7 from the “recombinant” recombinant anti-NeuN7 antibody probe, and the blue trace shows PC-MT7 from the “normal” non-recombinant anti-NeuN7 antibody probe. [Figure 13E]Figures 13A-13E. Recombinant antibodies versus conventional antibodies. 37 μm streptavidin PMMA beads were directly conjugated to PC-MTs (bead ID tags), then loaded with biotinylated protein G and subsequently bound to specific PC-MT-Abs (each PC-MT-Ab species was loaded separately onto a specific bead species). (Figure 13A) MALDI-MSI mass image of bead pool 1. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-MBP1, anti-MBP1, and recombinant anti-AB2 (note that the numbers indicate the mass units from Table 1 for the specific PC-MTs), respectively. (Figure 13B) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13A). The spectra are color-coded according to the bead colors observed in (Figure 13A). (Figure 13C) MALDI-MSI mass image of bead pool 2. Mass spectral peak intensities of various PC-MTs are colored as indicated above the image. Beads with bead ID tags 9, 10, and 15 were loaded with recombinant anti-NeuN7, anti-NeuN7, and recombinant anti-AB2, respectively (note that the numbers indicate the mass units from Table 1 for the specific PC-MT). (Figure 13D) Three overlaid spectra obtained from three single pixels within the three beads circled in (Figure 13C). The spectra are color-coded according to the bead colors observed in (Figure 13C). (Figure 13E) Separately, a sagittal tissue section of mouse brain was stained with anti-NeuN7 and recombinant anti-NeuN7 and subjected to MALDI-MSI. Antibody-derived PC-MT7 is colored green in the image. The white arrow indicates the cerebellum, the blue arrow indicates the hippocampus, and the yellow arrow indicates a punctate nuclear staining pattern. In the provided spectra, selected from the most intense pixels in the hippocampus of both images, the red trace shows PC-MT7 from the “recombinant” recombinant anti-NeuN7 antibody probe, and the blue trace shows PC-MT7 from the “normal” non-recombinant anti-NeuN7 antibody probe.

[0044] [Figure 14A]Figures 14A-14D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human tonsil FFPE tissue sections. MIHC was performed similarly to Figure 7, except that 12 different biomarkers were used (see Table 1 for PC-MT assignments for each antibody). In addition, a pan-cytokeratin antibody (CK) was labeled with both PC-MTs and fluorophores. (Figure 14A) CK immunofluorescence image of a whole tonsil tissue section captured at 5 micron resolution using a GenePix 4200A fluorescent microarray scanner. (Figure 14B) MALDI-MS image of the same tissue section showing an intensity map of monoisotopic m / z values ​​for PC-MTs derived from the CK antibody (all MALDI-MS images are at 10 micron spatial resolution). (Figure 14C) Multicolor MALDI-MS image overlay of selected biomarkers from a whole tissue section, showing differential structural patterns. Color coding is shown in the key below the images. (Figure 14D) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors for a representative small region of a tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3), is also shown. The gradient color scale is shown at the bottom. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 25 and a minimum display intensity of 2.5, except for CK, CD20, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively. [Figure 14B]Figures 14A-14D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human tonsil FFPE tissue sections. MIHC was performed similarly to Figure 7, except that 12 different biomarkers were used (see Table 1 for PC-MT assignments for each antibody). In addition, a pan-cytokeratin antibody (CK) was labeled with both PC-MTs and fluorophores. (Figure 14A) CK immunofluorescence image of a whole tonsil tissue section captured at 5 micron resolution using a GenePix 4200A fluorescent microarray scanner. (Figure 14B) MALDI-MS image of the same tissue section showing an intensity map of monoisotopic m / z values ​​for PC-MTs derived from the CK antibody (all MALDI-MS images are at 10 micron spatial resolution). (Figure 14C) Multicolor MALDI-MS image overlay of selected biomarkers from a whole tissue section, showing differential structural patterns. Color coding is shown in the key below the images. (Figure 14D) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors for a representative small region of a tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3), is also shown. The gradient color scale is shown at the bottom. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 25 and a minimum display intensity of 2.5, except for CK, CD20, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively. [Figure 14C]Figures 14A-14D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human tonsil FFPE tissue sections. MIHC was performed similarly to Figure 7, except that 12 different biomarkers were used (see Table 1 for PC-MT assignments for each antibody). In addition, a pan-cytokeratin antibody (CK) was labeled with both PC-MTs and fluorophores. (Figure 14A) CK immunofluorescence image of a whole tonsil tissue section captured at 5 micron resolution using a GenePix 4200A fluorescent microarray scanner. (Figure 14B) MALDI-MS image of the same tissue section showing an intensity map of monoisotopic m / z values ​​for PC-MTs derived from the CK antibody (all MALDI-MS images are at 10 micron spatial resolution). (Figure 14C) Multicolor MALDI-MS image overlay of selected biomarkers from a whole tissue section, showing differential structural patterns. Color coding is shown in the key below the images. (Figure 14D) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors for a representative small region of a tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3), is also shown. The gradient color scale is shown at the bottom. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 25 and a minimum display intensity of 2.5, except for CK, CD20, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively. [Figure 14D]Figures 14A-14D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human tonsil FFPE tissue sections. MIHC was performed similarly to Figure 7, except that 12 different biomarkers were used (see Table 1 for PC-MT assignments for each antibody). In addition, a pan-cytokeratin antibody (CK) was labeled with both PC-MTs and fluorophores. (Figure 14A) CK immunofluorescence image of a whole tonsil tissue section captured at 5 micron resolution using a GenePix 4200A fluorescent microarray scanner. (Figure 14B) MALDI-MS image of the same tissue section showing an intensity map of monoisotopic m / z values ​​for PC-MTs derived from the CK antibody (all MALDI-MS images are at 10 micron spatial resolution). (Figure 14C) Multicolor MALDI-MS image overlay of selected biomarkers from a whole tissue section, showing differential structural patterns. Color coding is shown in the key below the images. (Figure 14D) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors for a representative small region of a tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3), is also shown. The gradient color scale is shown at the bottom. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 25 and a minimum display intensity of 2.5, except for CK, CD20, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively.

[0045] [Figure 15A]Figures 15A-15D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human breast cancer FFPE tissue sections. MIHC was performed as in Figure 14 (see Table 1 for PC-MT assignments for each antibody). (Figure 15A) Multicolor MALDI-MS image overlay of a breast cancer tissue section showing monoisotopic m / z intensity maps for PC-MTs from selected antibodies exhibiting differential structural patterns (all MALDI-MS images are at 10 micron spatial resolution). A key on the image indicates color coding. Clinical annotation of this biospecimen from the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma (ductal) of the breast, TNM staging of pTlcpN3apMX, minimum staging IIIC, 75% tumor, and PR- / ER- / HER2+ by conventional IHC. (Figure 15B) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors across the entire tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3) is also shown. The gradient color scale is also shown. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, CD3, CD4, CD68, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively. (Figure 15C) Multicolor MALDI-MS image overlay of the same breast cancer tissue section; in this case, for simplicity, only CK, HER2, and ER (color-coded on the image) are shown, allowing visual identification of the relative distribution of these three important biomarkers. (Figure 15D) Multicolor MALDI-MS image overlay (top panel, color coding of bottom image) and individual MALDI-MS images (gradient scale, bottom panel) of selected biomarkers on different breast cancer tissue sections.In this case, the clinical annotation of this biospecimen according to the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma of the breast, ductal, lobular, metastatic, TNM staging of T2N2aMX, minimum staging IIIA, 95% tumor, and PR+ / ER+ / HER2- by conventional IHC (i.e., the PR / ER / HER2 profile is the reciprocal of the previous histology). For comparison, the display scale of the gradient color image is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, which produced an exceptionally strong signal and therefore was set to 100 and 10, respectively. [Figure 15B]Figures 15A-15D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human breast cancer FFPE tissue sections. MIHC was performed as in Figure 14 (see Table 1 for PC-MT assignments for each antibody). (Figure 15A) Multicolor MALDI-MS image overlay of a breast cancer tissue section showing monoisotopic m / z intensity maps for PC-MTs from selected antibodies exhibiting differential structural patterns (all MALDI-MS images are at 10 micron spatial resolution). A key on the image indicates color coding. Clinical annotation of this biospecimen from the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma (ductal) of the breast, TNM staging of pTlcpN3apMX, minimum staging IIIC, 75% tumor, and PR- / ER- / HER2+ by conventional IHC. (Figure 15B) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors across the entire tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3) is also shown. The gradient color scale is also shown. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, CD3, CD4, CD68, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively. (Figure 15C) Multicolor MALDI-MS image overlay of the same breast cancer tissue section; in this case, for simplicity, only CK, HER2, and ER (color-coded on the image) are shown, allowing visual identification of the relative distribution of these three important biomarkers. (Figure 15D) Multicolor MALDI-MS image overlay (top panel, color coding of bottom image) and individual MALDI-MS images (gradient scale, bottom panel) of selected biomarkers on different breast cancer tissue sections.In this case, the clinical annotation of this biospecimen according to the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma of the breast, ductal, lobular, metastatic, TNM staging of T2N2aMX, minimum staging IIIA, 95% tumor, and PR+ / ER+ / HER2- by conventional IHC (i.e., the PR / ER / HER2 profile is the reciprocal of the previous histology). For comparison, the display scale of the gradient color image is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, which produced an exceptionally strong signal and therefore was set to 100 and 10, respectively. [Figure 15C]Figures 15A-15D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human breast cancer FFPE tissue sections. MIHC was performed as in Figure 14 (see Table 1 for PC-MT assignments for each antibody). (Figure 15A) Multicolor MALDI-MS image overlay of a breast cancer tissue section showing monoisotopic m / z intensity maps for PC-MTs from selected antibodies exhibiting differential structural patterns (all MALDI-MS images are at 10 micron spatial resolution). A key on the image indicates color coding. Clinical annotation of this biospecimen from the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma (ductal) of the breast, TNM staging of pTlcpN3apMX, minimum staging IIIC, 75% tumor, and PR- / ER- / HER2+ by conventional IHC. (Figure 15B) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors across the entire tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3) is also shown. The gradient color scale is also shown. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, CD3, CD4, CD68, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively. (Figure 15C) Multicolor MALDI-MS image overlay of the same breast cancer tissue section; in this case, for simplicity, only CK, HER2, and ER (color-coded on the image) are shown, allowing visual identification of the relative distribution of these three important biomarkers. (Figure 15D) Multicolor MALDI-MS image overlay (top panel, color coding of bottom image) and individual MALDI-MS images (gradient scale, bottom panel) of selected biomarkers on different breast cancer tissue sections.In this case, the clinical annotation of this biospecimen according to the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma of the breast, ductal, lobular, metastatic, TNM staging of T2N2aMX, minimum staging IIIA, 95% tumor, and PR+ / ER+ / HER2- by conventional IHC (i.e., the PR / ER / HER2 profile is the reciprocal of the previous histology). For comparison, the display scale of the gradient color image is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, which produced an exceptionally strong signal and therefore was set to 100 and 10, respectively. [Figure 15D]Figures 15A-15D. Multiplexed mass spectrometry-based immunohistochemistry (MIHC) for 12 biomarkers in human breast cancer FFPE tissue sections. MIHC was performed as in Figure 14 (see Table 1 for PC-MT assignments for each antibody). (Figure 15A) Multicolor MALDI-MS image overlay of a breast cancer tissue section showing monoisotopic m / z intensity maps for PC-MTs from selected antibodies exhibiting differential structural patterns (all MALDI-MS images are at 10 micron spatial resolution). A key on the image indicates color coding. Clinical annotation of this biospecimen from the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma (ductal) of the breast, TNM staging of pTlcpN3apMX, minimum staging IIIC, 75% tumor, and PR- / ER- / HER2+ by conventional IHC. (Figure 15B) Individual MALDI-MS images of all 12 biomarkers (biomarker identity indicated by label) shown as gradient colors across the entire tissue section. An adjacent tissue section, a "blank," stained with isotype control IgG with PC-MT (same PC-MT as CD3) is also shown. The gradient color scale is also shown. For comparison, the display scale for all biomarkers is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, CD3, CD4, CD68, and Ki67, which produced exceptionally strong signals and were therefore set to 50 and 5, respectively. (Figure 15C) Multicolor MALDI-MS image overlay of the same breast cancer tissue section; in this case, for simplicity, only CK, HER2, and ER (color-coded on the image) are shown, allowing visual identification of the relative distribution of these three important biomarkers. (Figure 15D) Multicolor MALDI-MS image overlay (top panel, color coding of bottom image) and individual MALDI-MS images (gradient scale, bottom panel) of selected biomarkers on different breast cancer tissue sections.In this case, the clinical annotation of this biospecimen according to the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma of the breast, ductal, lobular, metastatic, TNM staging of T2N2aMX, minimum staging IIIA, 95% tumor, and PR+ / ER+ / HER2- by conventional IHC (i.e., the PR / ER / HER2 profile is the reciprocal of the previous histology). For comparison, the display scale of the gradient color image is set to a full intensity threshold (arbitrary peak intensity units) of 20 and a minimum display intensity of 2, except for CK, which produced an exceptionally strong signal and therefore was set to 100 and 10, respectively.

[0046] [Figure 16] Comparison of common elements of the protocols of the present invention (MIHC and MISH) with 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 conventional direct MSI (e.g., [Caprioli, Farmer et al. (1997) Anal Chem 69:4751-60]) and bead array MSI (e.g., U.S. Patent No. 9,523,680, incorporated herein by reference). Note that many protocol variations are possible, for example, depending on whether FF or FFPE tissue is used, whether it is an IHC / MIHC or ISH / MISH-based protocol, and / or what type of optical detection method (e.g., directly labeled primary antibody or secondary detection method, and colorimetric vs. fluorescent readout) is used for traditional IHC or ISH; therefore, Figure 16 shows only the common essential elements of the protocols.

[0047] [Figure 17]Examples of damage to mouse brain tissue sections on indium tin oxide (ITO)-coated glass slides subjected to the present mass spectrometry-based immunohistochemistry (MIHC) procedure. The top two images are on ITO-coated glass slides, and the bottom image is on a gold-coated glass slide. Loss or damage to the tissue sections can be variable. The main sites of damage to the tissue sections are indicated by black arrows.

[0048] [Figure 18] Comparison of MIHC results for anti-NeuN antibody detection on mouse brain FFPE tissue sections using DHB matrix sublimation with and without matrix recrystallization.

[0049] [Figure 19A] Figures 19A-19C. MALDI-MSI of sagittal mouse brain FFPE tissue sections stained with PC-MT-labeled lectin probes. (Figures 19A and 19B) Colorized mass images of PC-MT corresponding to three lectins, PHA-E4 (red), PNA (green), and WGA (blue). (Figure 19B) Competitive inhibition (blocking) to demonstrate specificity of lectin binding (in this case, of WGA). The mixture of PC-MT lectin probes was preincubated with the soluble sugar N,N',N"-triacetylchitotriose (TCT), which specifically binds to WGA (the soluble sugar remained present during the tissue staining / probing process). As a control, TCT blocking was omitted. (Figure 19C) Grand-averaged spectra were acquired from the entirety of each mass image in panel b. The monoisotopic PC-MT peaks of the three lectins are labeled (WGA, PNA, and PHA-E4). The orange trace is tissue treated with TCT blocking, and the purple trace is tissue treated without TCT blocking. A 70% decrease in the WGA PC-MT peak intensity is observed with TCT blocking, while the other two lectins remain unchanged. [Figure 19B]Figures 19A-19C. MALDI-MSI of sagittal mouse brain FFPE tissue sections stained with PC-MT-labeled lectin probes. (Figures 19A and 19B) Colorized mass images of PC-MT corresponding to three lectins, PHA-E4 (red), PNA (green), and WGA (blue). (Figure 19B) Competitive inhibition (blocking) to demonstrate specificity of lectin binding (in this case, of WGA). The mixture of PC-MT lectin probes was preincubated with the soluble sugar N,N',N"-triacetylchitotriose (TCT), which specifically binds to WGA (the soluble sugar remained present during the tissue staining / probing process). As a control, TCT blocking was omitted. (Figure 19C) Grand-averaged spectra were acquired from the entirety of each mass image in panel b. The monoisotopic PC-MT peaks of the three lectins are labeled (WGA, PNA, and PHA-E4). The orange trace is tissue treated with TCT blocking, and the purple trace is tissue treated without TCT blocking. A 70% decrease in the WGA PC-MT peak intensity is observed with TCT blocking, while the other two lectins remain unchanged. [Figure 19C]Figures 19A-19C. MALDI-MSI of sagittal mouse brain FFPE tissue sections stained with PC-MT-labeled lectin probes. (Figures 19A and 19B) Colorized mass images of PC-MT corresponding to three lectins, PHA-E4 (red), PNA (green), and WGA (blue). (Figure 19B) Competitive inhibition (blocking) to demonstrate specificity of lectin binding (in this case, of WGA). The mixture of PC-MT lectin probes was preincubated with the soluble sugar N,N',N"-triacetylchitotriose (TCT), which specifically binds to WGA (the soluble sugar remained present during the tissue staining / probing process). As a control, TCT blocking was omitted. (Figure 19C) Grand-averaged spectra were acquired from the entirety of each mass image in panel b. The monoisotopic PC-MT peaks of the three lectins are labeled (WGA, PNA, and PHA-E4). The orange trace is tissue treated with TCT blocking, and the purple trace is tissue treated without TCT blocking. A 70% decrease in the WGA PC-MT peak intensity is observed with TCT blocking, while the other two lectins remain unchanged.

[0050] [Figure 20A] Figures 20A-20C. Direct unlabeled MALDI-MSI followed by MIHC on the same tissue section. FF sagittal mouse brain tissue sections were used. Mass images of directly detected lipids and PC-MT derived from various antibodies are shown. (Figure 20A) MSI-1. Initial direct unlabeled MALDI-MSI of endogenous lipids (see image key for exemplary lipids). (Figure 20B) MSI-2. Subsequent MIHC showing selected antibody PC-MT (see image key for antibody PC-MT). (Figure 20C) Composite. Composite of images of selected analytes from MSI-1 and MSI-2 (see image key for analytes). The expected colocalization of lipid sulfatides (ST) with myelin, but not between ST and NeuN, is described in Example 4. [Figure 20B]Figures 20A-20C. Direct unlabeled MALDI-MSI followed by MIHC on the same tissue section. FF sagittal mouse brain tissue sections were used. Mass images of directly detected lipids and PC-MT derived from various antibodies are shown. (Figure 20A) MSI-1. Initial direct unlabeled MALDI-MSI of endogenous lipids (see image key for exemplary lipids). (Figure 20B) MSI-2. Subsequent MIHC showing selected antibody PC-MT (see image key for antibody PC-MT). (Figure 20C) Composite. Composite of images of selected analytes from MSI-1 and MSI-2 (see image key for analytes). The expected colocalization of lipid sulfatides (ST) with myelin, but not between ST and NeuN, is described in Example 4. [Figure 20C] Figures 20A-20C. Direct unlabeled MALDI-MSI followed by MIHC on the same tissue section. FF sagittal mouse brain tissue sections were used. Mass images of directly detected lipids and PC-MT derived from various antibodies are shown. (Figure 20A) MSI-1. Initial direct unlabeled MALDI-MSI of endogenous lipids (see image key for exemplary lipids). (Figure 20B) MSI-2. Subsequent MIHC showing selected antibody PC-MT (see image key for antibody PC-MT). (Figure 20C) Composite. Composite of images of selected analytes from MSI-1 and MSI-2 (see image key for analytes). The expected colocalization of lipid sulfatides (ST) with myelin, but not between ST and NeuN, is described in Example 4.

[0051] [Figure 21A]Figures 21A-21B. PC-MT probes in non-imaging mass spectrometry applications. PC-MT-Ab was bound to protein G agarose beads, the beads were washed, and PC-MT was photoreleased from the bead-bound PC-MT-Ab. The supernatant was 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 range of PC-MT-Ab concentrations added to the beads is indicated by black arrows). A control peptide was included in the samples at a fixed concentration for data normalization purposes (Figure 21B). The ratio of the photoreleased PC-MT to the control peptide monoisotopic peak intensity was taken and plotted as a function of the PC-MT-Ab concentration added to the protein G beads. [Figure 21B] Figures 21A-21B. PC-MT probes in non-imaging mass spectrometry applications. PC-MT-Ab was bound to protein G agarose beads, the beads were washed, and PC-MT was photoreleased from the bead-bound PC-MT-Ab. The supernatant was 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 range of PC-MT-Ab concentrations added to the beads is indicated by black arrows). A control peptide was included in the samples at a fixed concentration for data normalization purposes (Figure 21B). The ratio of the photoreleased PC-MT to the control peptide monoisotopic peak intensity was taken and plotted as a function of the PC-MT-Ab concentration added to the protein G beads.

[0052] [Figure 22] Figure 22 shows an exemplary schematic showing working with undigested and digested tissue in parallel. FFPE = formalin fixed and paraffin embedded (FFPE). DETAILED DESCRIPTION OF THE INVENTION

[0053] Table 1. Mass unit sequences and mass reporter masses of PC-MTs (see Figure 3 for exemplary PC-MT structures).

[0054] Table 1.1. Amino acid isotopes.

[0055] Description of the Invention

[0056] The present invention relates to immunohistochemistry (IHC) and in situ hybridization (ISH) for targeted detection and mapping of biomolecules (e.g., proteins and miRNAs) in tissues or cells, for example, for research applications and clinical applications by pathologists (e.g., biomarker analysis of resected tumors or tumor biopsies). In particular, the use of mass spectrometry imaging (MSI) as a mode for detecting and mapping biomolecules, for example, within tissues or cells. More specifically, the field of the present invention relates to photocleavable mass tag reagents that are conjugated to probes such as antibodies and nucleic acids and used to achieve multiplexed immunohistochemistry and in situ hybridization, using MSI as a detection / readout modality.

[0057] Current tissue imaging methods, such as fluorescence IHC, lack the multiplexing and / or multi-omic levels necessary to elucidate complex biological systems and human diseases. Mass spectrometry imaging (MSI) is generally limited to untargeted 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 that utilizes novel photocleavable peptide mass tags (PC-MTs) for easy labeling of probes, including antibodies, lectins, and nucleic acids, for highly multiplexed MSI of targeted macromolecules in tissues. When combined with untargeted MSI, both highly multiplexed and multi-omic tissue imaging are achieved on a single specimen. Combined multimodal fluorescence and MS IHC imaging can also be achieved on a single tissue specimen by using dual-labeled antibody probes. What is needed are novel photocleavable mass tags (PC-MTs) and MALDI-MSI procedures that overcome these aforementioned limitations.

[0058] Here, we report a novel photocleavable mass tag (PC-MT) and MALDI-MSI procedure that overcomes these aforementioned limitations. PC-MT is a modified polypeptide containing a mass unit, a highly efficient photocleavable linker (PC-linker) incorporated into the peptide by solid-phase synthesis, a spacer, and an NHS-ester probe-reactive moiety near the C-terminus. PC-MT antibody probes are generated in a single-step reaction. The fast and efficient photonucleation technique used with the novel PC-linker [Olejnik, Sonar et al. (1995) Proceedings of the National Academy of Sciences (USA) 92:7590-7594] provides robust sensitivity in practice, enabling high-plex MSI of a wide range of biomarkers in various tissues, including mouse brain (Example 2), human tonsil, and breast cancer (Example 9), as demonstrated here. Furthermore, a novel dual-labeled antibody combining both PC-MT and a fluorophore enabled direct correlation of MSI with conventional immunofluorescence (Example 9). Finally, the versatility of this approach is demonstrated by the ability to perform both label-free, untargeted small molecule MSI (of lipids), which is not possible with standard IHC, and multiplexed PC-MT-based targeted MSI of macromolecular biomarkers on the same tissue section (Example 4).

[0059] The present invention involves compositions and methods of manufacture and use of novel photocleavable linkers (PC-linkers), photocleavable mass tags (PC-MTs) and photocleavable mass-tagged probes (PC-MT-probes) that overcome the aforementioned limitations of previous targeted mass spectrometry imaging (MSI) methods to enable highly multiplexed MSI of targeted biomolecules in biological specimens such as tissues and cells using probes such as antibodies and nucleic acids.

[0060] The basic design of the peptide-based PC-MTs and the resulting PC-MT-probes and their use are shown in Figure 2.

[0061] As shown in Figure 2a, peptide-based PC-MTs contain i) a probe-reactive moiety, for example, an amine-reactive NHS-ester leaving group (N-hydroxysuccinimidyl ester) as shown, ii) an internal PC-linker (oval in Figure 2a) that is introduced into the peptide chain during solid-phase peptide synthesis (SPPS), iii) a selectively detectable mass unit (curve in Figure 2a) that includes an amino acid or isotope or its analog / derivative that can be incorporated using SPPS chemistry, and iv) an optional fluorophore (starburst with "F" in Figure 2a).

[0062] The PC-MT is covalently attached to the probe by chemical reaction between the probe-reactive portion of the PC-MT and the probe to produce the 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 an aptamer.

[0063] Cells / tissues are "stained" with PC-MT probes (i.e., the PC-MT probes are bound to their targets in the cells / tissues) and photocleaved, liberating (photoreleased) the mass reporter region for detection by MSI (Figure 2c; note that the mass reporter comprises the mass unit and, in some embodiments, also comprises a portion of the photocleaved PC-linker). Note that photocleavage can be achieved by a MALDI-MS laser beam and / or any light, e.g., UV or near-UV light with a wavelength in the range of 200 nm to 400 nm.

[0064] Improvements in PC-MT, the resulting PC-MT-probes, and their use are due to the following: i) the rapid and efficient photocleavable nuclei (PC nuclei, see step 1 in Figure 3) that have previously been demonstrated in a variety of other applications when incorporated into other compounds such as photocleavable biotin (PC-biotin) and photocleavable phosphoramidites (PC-phosphoramidites) [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, Krzymancka-Olejnik et al. (1998) Nucleic Acids Res 26:3572-3576; 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 [U.S. Patent Nos. 5,643,722, 5,986,076, 6,218,530, 8,906,700, and 10,060, which are incorporated herein by reference.912], ii) a novel Fmoc-protected photocleavable linker (Fmoc-PC-linker, see Figure 3) containing the PC-core and capable of being incorporated into peptide chains using standard Fmoc-based SPPS chemistry (see Figure 3), iii) facile one-step probe labeling using a probe-reactive moiety on the PC-MT (e.g., an NHS-ester probe-reactive moiety, see steps 1-2 in Figure 3), iv) an optional fluorescent label incorporated on the PC-MT to aid in method development by enabling conventional fluorescent imaging in addition to MSI (see Figure 2a), and v) the ability to perform untargeted, unlabeled small molecule MSI and multiplex PC-MT-based targeted MSI of macromolecules on the same tissue section (see Example 4).

[0065] The present invention is not limited to tissues alone. For example, digested tissues can be used. Furthermore, the compositions and methods described herein can be applied to cells grown or deposited on a surface, or biofilms grown or deposited on a surface. For example, the use of MSI for rapid identification of microorganisms in clinical microbiology is rapidly growing [(2019) Nat Commun 10:4029; Oviano and Bou (2019) Clin Microbiol Rev 32]. Bacterial cells can be grown or deposited on a substrate, followed by MALDI-MSI. In a further example, cells derived from cancer biopsies and deposited on a surface can be analyzed using the compositions and methods described herein. An additional example is the analysis of both single and multiple species of bacteria grown on a surface to form complex, heterogeneous patterns. Another example is MALDI-MSI of biofilms. Recent progress has been made in applying this method to Bacillus subtilis biofilms grown on agar using a sprayer to deposit specific matrix compounds, such as 2,5-dihydroxybenzoic acid solutions compatible with MALDI-MSI [Li, Comi et al. (2016) J Mass Spectrom 51:1030-1035]. This method can also be applied to complex multicellular whole organisms deposited on surfaces. For example, C. elegans is a free-living, transparent nematode worm approximately 1 mm long that inhabits soft soil environments. MSI has previously been applied to C. elegans, demonstrating the feasibility of applying 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 intracellular or molecular assemblies grown or deposited on surfaces, including both organic and inorganic nanostructures. Examples of MSI profiling of single cells and subcellular structures are described in a recent review by Lanni et al. [Lanni, Rubakhin et al. (2012) J Proteomics 75:5036-5051].Recently, subcellular resolution has been achieved using MALDI-MSI by using specialized techniques such as transmission mode geometry [Niehaus, Soltwisch et al. (2019) Nat Methods 16:925-931] that are compatible with the compositions and methods described in this invention.

[0066] In another embodiment, PC-MT and PC-MT probes may be used for coding and / or detection in microarrays and bead arrays (e.g., U.S. Patent Nos. 9,523,680, 9,513,285, and 10,060,912, which are incorporated herein by reference).

[0067] Detailed Description of the Invention

[0068] Photocleavable mass tag (PC-MT)

[0069] The chemical structure of a preferred Fmoc-protected photocleavable linker (Fmoc-PC-linker) is shown in Figure 3 and is a key component in the synthesis of PC-MTs. The compound minimally contains an Fmoc-protected primary amine terminus, a free carboxyl terminus, and a 1-(2-nitrophenyl)ethyl-based photocleavable core (PC-core) in between. Optionally, the Fmoc-PC-linker also contains the linker unit shown in Figure 3, which connects the 1-(2-nitrophenyl)ethyl-based photocleavable core to the Fmoc-protected primary amine and carboxyl moieties. Overall, the minimal configuration of the Fmoc-PC-linker allows for the incorporation of the PC-linker into a peptide to create a photocleavable mass tag (PC-MT) (Step 1 in Figure 3). The PC-linker is incorporated in the same manner as an amino acid using standard Fmoc-based solid-phase peptide synthesis (SPPS), 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, such as the Boc protecting group and related peptide synthesis chemistries, and other peptide synthesis methods are possible [Stawikowski and Fields (2012) Curr Protoc Protein Sci Chapter 18:Unit 18 1].

[0070] As shown in Figure 3, the obtained PC-MT consists of, but is not limited to, the following characteristics:

[0071] i) A 1-(2-nitrophenyl)ethyl-based PC-core is disclosed in step 1 of Figure 3. The photocleavage site is indicated by the black arrow in step 1 of Figure 3 and the photocleavage reaction shown in step 3 of Figure 3. Note that this PC-core used in the PC-linker of the present invention provides fast and efficient photocleavage and is shown herein (Example 6) to provide superior sensitivity in mass spectrometry compared to the PC-linker used in the aforementioned work by Lemaire et al., which contains an additional methoxy moiety on the nitrophenyl ring of the PC-core (see Figure 4 for a comparison of PC-linker structures). For previous work by Lemaire et al., see [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67] and U.S. Patent No. 8,221,972.

[0072] ii) Probe-reactive moieties, such as primary amine-reactive N-hydroxysuccinimidyl ester (NHS-ester) leaving groups. For example, NHS-esters can be generated on the s-amine of the lysine (K) side chain using, for example, N,N'-disuccinimidyl carbonate (DSC) or DSS (disuccinimidyl suberate) for conversion [Morpurgo, Bayer et al. (1999) J. Biochem. Biophys. Methods 38:17-28] (see step 1, NHS-ester, in Figure 3). Probe-reactive moieties can also be generated, for example, by chemical conversion / modification of the carboxylic acid functional group at the C-terminus of peptide-based PC-MTs (not shown in Figure 3). The probe-reactive moiety allows for easy conjugation of PC-MTs to targeting probes, such as antibodies or nucleic acids. It should be understood that a variety of probe reactive moieties can be used, including, but not limited to, amine-reactive N-hydroxysuccinimidyl (NHS) esters, sulfo-N-hydroxysuccinimidyl (sulfo-NHS) esters, succinimidyl esters (SEs), sulfo-succinimidyl esters (SSEs), aldehyde or tetrafluorophenyl (TFP) esters, sulfhydryl-reactive maleimides or iodoacetamides, or poly-reactive epoxy moieties. 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].

[0073] iii) An optional spacer unit linking the PC-nucleic acid to the probe-reactive moiety, for example, as shown as part of the PC-linker+GSGGK amino acid sequence in step 1 of Figure 3. It should be understood that this spacer unit is not necessary, as the probe-reactive moiety can be attached to the 1-(2-nitrophenyl)ethyl-based PC-nucleus at the same position without a spacer unit. It should also be understood that this spacer unit can be a variety of chemical structures to promote hydrophilicity / water solubility, such as a polyethylene glycol (PEG) spacer.

[0074] iv) A selectively detectable mass unit, shown as an example in Figure 3 as the APRLRFYSL amino acid (peptide) sequence. It should be understood that any amino acid used can be, for example, natural and unnatural amino acids, as well as modified amino acids, isotopic amino acids, amino acid analogs / derivatives, and any combination thereof. Peptide-based mass units are a preferred embodiment due to their ease of synthesis, robust performance in mass spectrometry, and the ability to gain additional specificity in mass spectrometric identification using established methods of tandem MS-based fragmentation analysis (e.g., MS / MS). However, the selectively detectable mass unit need not be a peptide and can be, for example, any chemical entity that can be detected by mass spectrometry. Polymeric mass units are preferred due to their general ease of synthesis and the ability to easily adjust the mass simply by changing the number and type of monomer subunits. Although peptides are considered a form of biopolymer (in addition to, for example, nucleic acids), polymers other than peptides may also be used, such as polyethylene glycol, which is easily synthesized and detected by MALDI-MS [Enjalbal, Ribiere et al. (2005) J Am Soc Mass Spectrom 16:670-8].

[0075] v) An optional mass unit linker, shown in step 1 of Figure 3, connects the PC-core to the mass unit. It should be understood that this mass unit linker is not necessary, as the mass unit can be attached to the 1-(2-nitrophenyl)ethyl-based PC-core at the same position without the mass unit linker. It should also be understood that this mass unit linker can be a variety of chemical structures to promote hydrophilicity / water solubility, such as a polyethylene glycol (PEG) linker.

[0076] vi) To prevent self-reaction or polymerization of the PC-MT, if the probe-reactive moiety is primary amine reactive, a blocking group, such as acetylation ("Ac" in Figure 3), may be used on the N-terminal a-amine of the peptide-based PC-MT. Internal amino acids with free primary amines (e.g., lysines with s-amines) may be avoided or blocked / protected in these cases. If the probe-reactive moiety reacts with a different functional group, such as sulfhydryl, any such functional group present on the peptide (e.g., from cysteine) may also be blocked / protected to prevent self-reaction or polymerization of the PC-MT.

[0077] vii) A fluorophore or other detectable label (e.g., a chromophore or affinity ligand such as biotin) is optionally included in the PC-MTs in addition to a selectively detectable mass unit (fluorophore not shown in Figure 3; see Figure 2a for one embodiment). This can aid in method development, for example, by enabling conventional fluorescence imaging in addition to MSI on the same cell / tissue sample. The fluorophore can be attached to the PC-MTs, for example, on the 8-amine of included lysines, using a variety of commercially available amine-reactive dyes (e.g., Cy5-NHS or Sulfo-Cy5-NHS). This attachment can be achieved during or after SPPS. Fluorophore attachment is also possible using other chemistries, such as click chemistry [McKay and Finn (2014) Chem Biol 21:1075-101]. In a preferred embodiment, the fluorophore is attached to a spacer unit. In other embodiments, the fluorophore may be attached to a mass unit or a mass unit linker. However, the fluorophore may be attached to any part of the PC-MT structure, as long as it does not interfere with the reaction between the probe-reactive moiety and the probe, the photocleavage reaction, or MSI detection of the mass unit, and does not impair the solubility of the PC-MT labeling reagent in the solvent environment selected for probe labeling. This approach has the advantage that the mass reporter and fluorophore are part of the same PC-MT labeling reagent, thereby allowing for assessment of successful PC-MT probe labeling by, for example, fluorescence means (in addition to mass spectrometry). Alternatively, a PC-MT lacking a fluorescent label (or other detectable label such as a chromophore or affinity ligand such as biotin) may be attached to the probe, or, further, a fluorescent label (or other detectable label such as a chromophore or affinity ligand such as biotin) may be attached to the same probe at a site different from the PC-MT. This can be easily achieved, for example, by labeling a probe (e.g., an antibody) with both a PC-MT bearing an NHS-ester probe-reactive moiety and the aforementioned NHS-activated fluorophore.Labeling of the probe with PC-MT and fluorophore can be performed simultaneously or sequentially (either PC-MT followed by the fluorophore first, or the fluorophore followed by PC-MT). In this embodiment, which uses a primary amine-reactive NHS group for probe labeling, labeling with both PC-MT and fluorophore is readily possible, for example, due to the presence of numerous primary amines in antibody probes. Overall, this approach may have advantages over fluorescent PC-MT because it avoids the detrimental effects of fluorophores on the PC-MT labeling reaction or PC-MT solubility during probe labeling, which is typically performed under aqueous conditions. It also avoids the detrimental effects of fluorophores on mass spectrometric readout (e.g., potentially low ionization efficiency if the fluorophore is attached to a mass reporter). Furthermore, this alternative approach allows for independent tuning of the fluorescent and PC-MT labeling ratios.

[0078] Finally, it should be understood that the optional chemical linkers shown in Figure 3, i.e., linker units, mass unit linkers, and spacer units, are not intended to limit the scope of the present invention. They serve as bridges between the PC-core and key moieties, such as the probe reactive moiety and the mass unit. These chemical linkers may be of various chemical compositions. For example, the chemical linker may simply be a hydrocarbon chain, or, for example, 2,2'-(ethylenedioxy)-bis-(ethylamine) chemical linker may be used for better solubility in aqueous environments [Pandori, Hobson et al. (2002) Chem Biol 9:567-73]. Polyethylene glycol (PEG) is another example, recognized by those skilled in the art as an excellent chemical linker that is relatively stable, water-soluble, and biocompatible.

[0079] PC-MT-Probe

[0080] As shown in step 2 of Figure 3, PC-MT is conjugated to the probe via the probe-reactive moiety. In the example shown, the NHS-ester leaving group of PC-MT is lost after reaction with a primary amine on the antibody, forming an amide bond between PC-MT and the antibody. While an antibody is shown, the probe can be of any type, 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., camelid 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. The probe can also be a nucleic acid, such as DNA, RNA, or locked nucleic acid (LNA), for example, in oligonucleotide hybridization probes or DNA / RNA aptamers [Nielsen, Singh et al. (1999) J Biomol Struct Dyn 17:175-91]. Probes may also be other organic or biomolecules, such as lipids, carbohydrates, steroids, or drugs. Labeling of probes with PC-MTs can be random (e.g., nonspecifically at any primary amine site on a protein, as can occur with PC-MTs containing NHS-esters as the probe-reactive moiety), or site-specific (e.g., carbohydrate regions present at specific sites on the heavy chains of some antibodies). In some cases, it may be necessary to use modified probes, such as primary amine-modified nucleic acid probes (e.g., when the probe-reactive moiety is an NHS-ester), to facilitate PC-MT labeling.

[0081] Probes can have a variety of targets, i.e., the moiety (molecular structure) to which the probe binds. The following examples are not intended to limit the types of probe targets; different probes can target different biomolecules or biomarkers (e.g., different proteins), or 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 portions thereof, including proteins, post-translational modifications of proteins, glycoproteins, nucleic acids, lipids and their derivatives, drugs, metabolites, carbohydrates, glycans, proteoglycans, gangliosides, and organic compounds.

[0082] In PC-MT probes, the core structure is defined as the structure that connects the mass unit to the probe (see, for example, "Core Structure" in Step 2 of Figure 3). The core structure is composed of, but is not limited to, a PC core, optional spacer units, and optional mass unit linkers, and is typically a common structure among all PC-MT probe species. In preferred embodiments, the core structure is non-neutral (e.g., containing ionizable groups such as sulfonates, phosphates, amines, or carboxylic acids). This improves the water solubility of the PC-MT labeling reagent, thereby enhancing the probe labeling reaction, which is typically performed in an aqueous environment. This can also improve the water solubility of the PC-MT probe. In one embodiment, a sulfonated fluorophore is included on the core structure (e.g., Sulfo-Cy5, as used in Example 7). Because fluorophores are typically polycyclic compounds that are not water soluble without such a sulfonate moiety, a sulfonated version of the fluorophore is used. In another embodiment, the amino acid moiety of the spacer unit of the PC-MT contains aspartic acid and / or glutamic acid to improve the water solubility of the PC-MT labeling reagent and / or PC-MT-probe.

[0083] In a preferred embodiment, multiple PC-MT molecules are attached to each probe molecule to facilitate improved sensitivity in the mass spectrometry process. Carriers can be added to the probe to further increase the number of PC-MT labels, thereby facilitating further signal amplification. In one embodiment, polyamine-modified dendrimers or nanoparticles (NPs) are labeled with PC-MT and then attached 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 in electron microscopy, for example, for immunolabeling of target biomolecules [Ackerson, Powell et al. (2010) Methods Enzymol 481:195-230]. In one example, polyamine-terminated gold NPs or branched dendrimers are commercially available (e.g., from Dendritech and Nanovex Biotechnologies) and can be conjugated to PC-MT (via an amine-reactive NHS-ester probe-reactive moiety). Alkyne groups can also be introduced into polyamine dendrimers or NPs using available NHS-activating reagents (e.g., NHS-DIBO-alkyne, Thermo Scientific, Waltham, MA). Similarly, antibodies or amine-modified nucleic acid probes can be modified with azide groups using commercially available NHS-activating reagents (e.g., NHS-PEG4-azide, Thermo Scientific, Waltham, MA). Finally, PC-MT / alkyne-modified dendrimers or NPs can be coupled with azide-modified probes using well-established, highly selective, mild, and bioorthogonal click chemistry (Kolb, Finn et al. (2001) Angew Chem Int Ed Engl 40:2004-2021). (Azides and alkynes spontaneously and selectively form covalent bonds under physiological aqueous conditions, even in complex mixtures; in some cases, copper catalysis is required, depending on the type of alkyne used.)

[0084] In another embodiment, 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 nucleic acid probe's target binding (hybridization) sequence; these "tails" have multiple amine modifications to provide multiple PC-MT labeling sites (Figure 5b). This configuration reduces potential interference from PC-MT with the target binding region of the nucleic acid probe. Amine modifications can be introduced into the nucleic acid probe, for example, using modified nucleotides such as 5'-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-uridine, an amino modifier commonly known as C6-U phosphoramidite, and 2'-O-triisopropylsilyloxymethyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, which is introduced during phosphoramidite-based chemical DNA synthesis. Modified nucleotides can also be enzymatically (e.g., by DNA polymerase) introduced into nucleic acid probes using, for example, 5-[3-aminoallyl]-2'-deoxyuridine-5'-triphosphate, commonly referred to as aminoallyl-dUTP. Modifications other than amines for use as sites for PC-MT labeling can also be introduced into nucleic acid probes.

[0085] In a preferred embodiment, the PC-MT is attached to the probe in a one-step chemical reaction between the PC-MT and the probe (e.g., by mixing the PC-MT containing an NHS-ester as the probe-reactive moiety with the probe, such as an antibody or an amine-modified nucleic acid). The labeling of probes with PC-MT in the present invention differs significantly from the complex multistep process in Lemaire et al.'s previous work [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67] [see also U.S. Pat. No. 8,221,972], in which the probe (e.g., antibody) must first be conjugated to the non-photocleavable heterobifunctional crosslinker MBS (3-maleimidobenzoic 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 linker); the sulfhydryl-reactive maleimide moiety generated on the antibody by the attached MBS linker is then reacted with a peptide containing an internal photocleavable site and a cysteine ​​amino acid (the cysteine ​​on the photocleavable peptide thereby provides a free sulfhydryl moiety for reaction with the maleimide on the antibody-bound MBS linker).Similarly, the previously mentioned imaging mass cytometry approach [Giesen, Wang et al. (2014) Nat Methods 11:417-22] also uses a highly complex, multi-step probe (i.e., antibody) labeling procedure, which involves preloading the polymer with metal ions, partially reducing the antibody, coupling 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-fl dm-00015-rev01 / fluidigm%3Afile].

[0086] In one embodiment of the present invention, further purification of the probe after the PC-MT labeling reaction may be avoided for simplicity (so as not to remove unreacted PC-MT), while in other embodiments, removal of unreacted PC-MT by, for example, 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) may be desirable to avoid background in downstream biomarker detection.

[0087] Processing tissues with PC-MT probes and mass spectrometry imaging (MSI)

[0088] The final step in the process is the use of PC-MT probes to process ("stain") cells or tissues (i.e., the PC-MT probes are bound to targets in the cells / tissues), followed by MSI to image the photocleaved mass reporters (see step 3 in Figure 2c and Figure 3). Two exemplary embodiments, referred to herein as mass spectrometry-based immunohistochemistry (MIHC) and mass spectrometry-based in situ hybridization (MISH), are similar to traditional immunohistochemistry (IHC) and in situ hybridization (ISH), respectively, when antibodies and nucleic acid probes are used. Essentially, MIHC and MISH differ in the use of PC-MT-labeled probes instead of fluorophore- or chromogen-labeled probes and in the use of MSI instead of optical imaging (e.g., microscopy). The MIHC and MISH processes, illustrated in detail later in the experimental examples, typically include the basic steps described in the following paragraphs (although as with standard IHC and ISH, many protocol variations are possible, as will be recognized by those skilled in the art). See also Figure 16 for a comparison of the essential common elements of the protocols of the present invention (MIHC and MISH) with 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 conventional direct MSI (e.g., [Caprioli, Farmer et al. (1997) Anal Chem 69:4751-60]) and bead array MSI (e.g., U.S. Patent No. 9,523,680, incorporated herein by reference).Note that many protocol variations are possible, for example, depending on whether FF or FFPE tissue is used, whether it is an IHC / MIHC or ISH / MISH-based protocol, and / or what type of optical detection method (e.g., directly labeled primary antibody or secondary detection method, and colorimetric vs. fluorescent readout) is used for traditional IHC or ISH; therefore, Figure 16 shows only the common essential elements of the protocols. The basic steps of MIHC are: i) mounting thin FFPE or fresh-frozen tissue sections (e.g., 5-10 μm thick, cut from FFPE or fresh-frozen tissue blocks by a microtome or cryostat) onto conductive slides (e.g., metal-coated glass slides). Note that glass slides coated with indium tin oxide (ITO) as a conductive surface are almost universally used in MSI (see, for example, [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]). In the present invention, considering the extensive processing steps of MIHC and MISH described below, gold-coated glass slides are not suitable for the conductive surface required for MSI (e.g., gold-coated glass slides with a 10 nm gold layer and a 2 nm titanium adhesive underlayer, manufactured by Platypus Technologies LLC, Madison, Wisconsin, or Substrata Thin Film Transducers, Ontario, Canada). It has been found to be beneficial to avoid tissue lifting and / or tissue damage during processing while still providing a suitable substrate (glass slides with a 50 nm gold layer and a 5 nm chromium adhesive underlayer manufactured by Solutions / Angstrom Engineering Inc.), and after mounting the tissue, ii) deparaffinization (e.g., with xylene) in the case of FFPE, iii) rehydration (if deparaffinization was performed) typically using a series of ethanol / water mixtures and aqueous saline buffers, iv) fixation in formalin or paraformaldehyde in the case of fresh-frozen tissue, v) antigen retrieval and reversal of some of the adverse effects of formalin / paraformaldehyde fixation (e.g., heating in citrate buffer at pH 6 or use of formic acid), vi) treatment with a blocking buffer (typically a saline buffer containing a non-ionic detergent such as Tween®-20 and a protein blocker such as bovine serum albumin [BSA] and animal serum) to reduce background.vii) simultaneous staining with a mixture of different PC-MT antibodies (PC-MT-Abs) (typically diluted in blocking buffer) for multiplexing; viiii) washing in a saline buffer containing a non-ionic surfactant such as Tween®-20 to remove any unbound PC-MT-Abs, followed by washing in a volatile aqueous buffer such as ammonium bicarbonate to remove non-volatile salts that may interfere with some forms of mass spectrometry; and ix) drying the tissue slides prior to MSI. Using an immunohistochemistry-style mass spectrometry imaging (MSI) protocol (flowchart in Figure 16), we discovered that tissues mounted on gold slides (e.g., slides with a gold layer) had better results. More specifically, mounting on gold slides helped avoid tissue damage or loss during the required slide processing steps (conductive slides for MSI). Previous use of gold slides in tissue MSI was for direct MSI, which lacked all of the processing steps of the present invention (see flowchart in Figure 16), and therefore did not pose a problem with tissue loss / damage. The present invention uses gold slides to improve tissue adhesion (more commonly, ITO conductive slides are used for direct MSI, but have shown poor results in our protocol; see Example 10). Tissue damage and loss can occur during any of the liquid-phase processing steps of the slides.

[0089] The steps of MISH include i) mounting the tissue, ii) deparaffinization, iii) rehydration, and iv) formalin / paraformaldehyde fixation as described for MIHC, which is typically followed by v) partial protein digestion with proteinase K, vi) fixation of nucleic acids with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), vii) acetylation of the tissue to cap free amines and reduce background (caused by nonspecific binding of the probe), viii) removal of at least unrelated nucleic acids (e.g., yeast) from the tissue. ix) simultaneous staining (hybridization) with a mixture of different PC-MT nucleic acids (PC-MT-NAs) (typically diluted in blocking buffer or similar) for multiplexing; x) washing in saline buffer to remove any unbound PC-MT-NAs, followed by washing in a volatile aqueous buffer such as ammonium bicarbonate to remove non-volatile salts that may interfere with some forms of mass spectrometry; and xi) drying the tissue slides prior to MSI.

[0090] However, as discussed above, the probes of the present invention need not be limited to antibodies (used in MIHC) and nucleic acids (used in MISH), but may be, for example, lectins, receptors, or ligands. Thus, the present invention is not limited to MIHC and MISH methodologies, but also includes other embodiments in which tissue is treated ("stained") with, for example, PC-MT-conjugated lectins, receptors, or ligands, or any probe type or combination thereof.

[0091] Mass Spectrometry Imaging (MSI) (and embodiments including MIHC and MISH). MSI is performed according to the procedures outlined above, and in one embodiment uses MALDI-MSI.

[0092] For MALDI-MSI, matrix compounds are typically applied to tissue in a thin, uniform layer. Examples of matrix compounds include alpha-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 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] is used to achieve both excellent spatial resolution (i.e., provided by sublimation that limits analyte delocalization during matrix application) and high sensitivity (i.e., provided by recrystallization that allows the mass reporter to sufficiently cocrystallize with the matrix but without sufficient analyte delocalization). Other methods of matrix application may also be used, such as using commercially available sprayers (eg, HTX™ sprayer, HTX Technologies, LLC, Chapel Hill, NC).

[0093] Various mass spectrometry techniques and instruments can be used for the MSI process, including but not limited to MALDI-MSI. Other MSI methods include desorption electrospray ionization mass spectrometry imaging (DESI-MSI) [Takats, Wiseman et al. (2004) Science 306:471-3], laser ablation electrospray 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 was originally best suited for small molecules such as metabolites and lipids, but has since been adapted for protein / peptide detection [Takats, Wiseman et al. (2004) Science 306:471-3; Takats, Wiseman et al. (2008) CSH Protoc 2008:pdb prot4994; 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 directing electrosprayed, charged solvent droplets toward a surface (e.g., tissue) to extract (desorb) and ionize analytes from the surface, which are then introduced into the mass spectrometer inlet (e.g., transfer capillary) of a mass spectrometer [Takats, Wiseman, et al. (2004) Science 306:471-3]. However, variations of DESI-MSI, such as nanoDESI-MSI, exist that allow for separation of desorption and ionization events [Roach, Laskin, et al. (2010) Analyst 135:2233-6]. DESI-MSI can be easily performed under ambient conditions rather than under vacuum, and therefore requires minimal sample preparation and is amenable to automation.Furthermore, as an ESI (electrospray ionization)-based method, DESI-MSI does not require matrix application as in MALDI-MSI, which is an additional difficult and tedious step that can lead to reproducibility issues as well as the risk of analyte delocalization.

[0094] Regardless of the type of MSI technique and instrument, the PC-MT is photocleaved to liberate the mass reporter from the PC-MT-probe for MSI analysis (see Figure 2c and step 3 in Figure 3). Note that, as exemplified in step 3 in Figure 3, the mass reporter consists of a mass unit and, in some cases, a portion of the photocleaved PC-linker. (In other cases not shown in Figure 3, the mass reporter may not contain the photocleaved portion of the PC-linker, and thus the mass reporter and mass unit are equivalent.) To avoid matrix absorption of the incident UV, the PC-MT can be pre-photocleaved by UV treatment before MSI, preferably also before matrix application in the case of MALDI-MSI. Photocleavage is also preferably performed on a dry slide to prevent delocalization of the photocleaved mass reporter due to diffusion. Pre-photocleavage can be achieved, for example, by using a Model XX-15 self-filtering 365 nm peak lamp (UVP / Analytik Jena US LLC, Upland, CA), although many other light sources are possible. In a preferred embodiment, the preliminary photocleavage is performed using relatively low intensity UV light (e.g., 3-10 mW / cm 2This can be achieved in a short time (e.g., 5 minutes) using a laser beam (e.g., 500 kJ / s). Alternatively, PC-MTs can be photocleaved inline with MALDI-MSI analysis by the instrument's laser beam (or by the laser beam of any other laser-based mass spectrometer, such as LAESI-MSI). This alternative approach can be expected to improve the spatial resolution of MSI, since in the case of MALDI-MSI, the mass reporter is not photocleaved prior to matrix application (which could otherwise cause diffusion of the pre-photocleaved mass reporter). However, results herein demonstrate that this approach of inline photocleavage provides insufficient sensitivity for MALDI-MSI (Example 6). The advantages of pre-photocleavage before matrix application and MALDI-MSI can be explained by the following: i) pre-photocleavage avoids the light-shielding effect of the matrix compound (indeed, it is the function of the matrix to absorb the impinging UV laser light during MALDI-MSI and convert it to heat so that the mass reporter is vaporized and ionized for analysis); ii) having the mass reporter pre-photocleaved allows the liberated 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-MS); conversely, photocleavage in-line with MALDI-MSI analysis by the instrument's laser beam necessarily occurs after matrix application / crystallization and likely does not allow efficient co-crystallization of the matrix and mass reporter (because at the time of matrix application / crystallization, the mass reporter is still attached to the probe, and the probe 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, can be expected to provide efficient in-line photocleavage and sufficient sensitivity, assuming the laser wavelength is well matched to the photocleavage wavelength.

[0095] Note that in the preferred PC-linker (Figure 3), photocleavage leaves a small residual moiety of the PC-linker attached to the mass reporter, thereby generating a free primary amine on the mass reporter upon photocleavage (see step 3 in Figure 3), which may aid in mass reporter ionization in positive-mode MALDI-MSI, and thus this design may increase sensitivity.

[0096] Also note that Figure 3 shows the mass reporter of the PC-MT linked (prior to photocleavage) to the phenyl ring of the PC-core via a photocleavage site (see step 3 in Figure 3 for the photocleavage site indicated by a lightning bolt), and the probe ultimately linked to the phenyl ring of the PC-core at a position distinct from the photocleavage site. Importantly, the photocleaved 1-(2-nitrophenyl)-ethyl moiety of the PC-linker does not remain attached to the mass reporter (instead, it remains attached to the probe) upon photocleavage, as shown in step 3 in Figure 3, and this is a preferred embodiment of the present invention. Conversely, it is possible to completely reverse this orientation, as taught by Olejnik et al. [Olejnik, Ludemann et al. (1999) Nucleic Acids Res 27:4626-31] and Levy and Caprioli (U.S. Patent No. 7,569,392). However, in this configuration, the photocleaved 1-(2-nitrophenyl)-ethyl moiety of the PC-linker remains attached to the mass reporter (not shown in Figure 3), resulting in the observation of a highly complex mass spectrum containing multiple peaks associated with mass reporters attached to various side reaction by-products of the photocleaved 1-(2-nitrophenyl)-ethyl moiety (see Example 5). This reduces the sensitivity of MSI (by splitting the signal for a single mass reporter among the multiple mass spectral peaks, i.e., the by-products) and confounds the identification of different mass reporters (e.g., by peak overlap).

[0097] If a fluorophore is optionally used on the PC-MT, it is preferable that it not be located on the mass reporter region. This avoids potential interference from the fluorophore with mass spectrometry detection of the mass reporter (e.g., the fluorophore may be structurally altered by the photocleavage light, thereby confounding the analysis). However, in some cases, it may be useful to locate the fluorophore on the mass reporter, such as for quantifying the photocleavage efficiency of the PC-MT by fluorescence.

[0098] Finally, as discussed in the preceding paragraph, the present invention is not limited to the type of probe and the type of mass spectrometry used for MSI. Thus, more generally, the PC-MT-probe-based MSI techniques of the present invention are referred to as PC-MT-MSI (of which the aforementioned mass spectrometry-based immunohistochemistry [MIHC] and mass spectrometry-based in situ hybridization [MISH] methods are some of the many possible methodological subtypes).

[0099] Multi-omic tissue imaging using PC-MT-probes

[0100] A key advantage of the present invention is the ability to perform multi-omic imaging of tissue specimens. "Omics" refers to the measurement of several features of large families 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, the measured "features" may be, for example, spatial mapping, morphological analysis, and colocalization 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 combines the measurement and analysis of different omic groups (e.g., combining proteomics and glycomics). In the context of tissue imaging, multi-omics can be achieved, for example, by using a) different PC-MT probe types or classes (e.g., antibodies and lectins) and / or b) different MSI "modes" (e.g., untargeted direct MSI of endogenous tissue biomolecules and MSI of biomolecules targeted by specific PC-MT probes). In a preferred embodiment, different omic measurements are performed on the same tissue section. However, these measurements can also be performed on separate, preferably consecutive / adjacent, tissue sections sliced ​​from the same tissue specimen / block. Furthermore, different omic measurements can be performed on tissue sections / specimens simultaneously (e.g., by treating the tissue with a mixture of different PC-MT probe classes, such as antibodies and lectins) or sequentially (one omic measurement followed by another). For example, in fluorescence imaging, this has been achieved through various methodological permutations using lectins and antibodies [Zupancic, Kreft et al. (2020) Eur J Histochem 64].When different omic measurements are performed sequentially on a tissue sample, 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 become apparent in the following paragraphs.

[0101] Finally, when combining carbohydrate-binding probes, such as lectin probes, with antibody probes for simultaneous processing of tissues, it may be important to use antibody probes that lack glycosylation to avoid cross-reactivity between the lectin and antibody probes, which may confound tissue imaging results (e.g., generating artifacts that do not represent the tissue's true intrinsic biomarker pattern). This can be done, for example, using antibodies that lack a glycosylated Fc domain, such as, but not limited to, Fab fragment antibodies, F(ab')2 fragment antibodies, nanobodies, singlekine fragment variable (scFv) antibodies, and VHH single-domain antibodies (e.g., camelid single-domain VHH antibodies). As long as antibody glycosylation is absent, whole antibody probes containing an Fc domain can still be used simultaneously with carbohydrate-binding probes, such as lectins, including, but not limited to, the use of recombinant antibodies (e.g., when produced in prokaryotic expression systems) or chemically or enzymatically deglycosylated antibodies (e.g., using the enzyme PNGase F). More broadly, when different probe classes are combined for simultaneous tissue processing, it is important to take steps to avoid probe cross-reactions in order to avoid artifacts in tissue imaging.

[0102] The multi-omics approach of the present invention is facilitated by the use of MSI methods that employ "soft" ionization for molecular analysis, because these methods generally do not or only cause limited molecular fragmentation and, more specifically, do not atomize molecules for detection (e.g., allowing for the detection of intact peptide or polymer-based mass tags, as well as intact endogenous biomolecules in tissues, 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), silicon desorption / ionization (DIOS), fast atom / ion bombardment (FAB), and electrospray ionization (ESI), including derivatives of the aforementioned ESI such as DESI and LAESI [Siuzdak (2004) JALA 9:50-63]. The foregoing list is not intended to limit the present invention to any particular type of "soft" ionization mass spectrometry. For purposes of this invention, methods based on "soft" ionization are defined as methods that ionize and detect molecular ions, i.e., charged molecules consisting of two or more atoms held together by chemical bonds.

[0103] Dual sequential MSI: In a simple multi-omic embodiment, it is useful to first perform label-free direct MSI of endogenous small biomolecules (e.g., metabolomics), followed by MSI of targeted biomolecules (e.g., protein targets, such as proteomics) using PC-MT-MSI, preferably in the same tissue section. Targeted PC-MT-MSI is necessary to detect biomolecules that are generally inaccessible to direct MSI, such as macromolecular targets that themselves are poorly ionized, poorly detected, may fragment in undesirable ways even with soft ionization, and / or are not sufficiently resolved by the mass spectrometer. It is also important to include PC-MT-MSI in a multi-omic workflow if targeting at least some known biomarkers is desirable or necessary. Overall, this embodiment of multi-omic tissue imaging can be important for colocalizing, for example, drug compounds (small molecule detection by direct label-free MSI) and drug targets (polymer detection by PC-MT-MSI, since drug targets are typically proteins). In a preferred embodiment, direct label-free MALDI-MSI is performed first, using fresh-frozen (FF) tissue sections, whereby the tissue has not yet undergone tissue fixation (formalin-fixed, paraffin-embedded [FFPE] tissue is possible but more ideal for direct label-free MSI of endogenous molecules [Wisztorski, Franck et al. (2010) Methods Mol Biol 656:303-22]). In a preferred embodiment, the tissue section is then washed and fixed if not previously done, and a PC-MT-MSI method, such as MIHC and / or MISH, is then used on the same tissue section, including another cycle of MSI.

[0104] In some embodiments, it is useful to first perform unlabeled direct MSI on tissue sections to directly detect, for example, small biomolecules, followed by targeted MIHC and / or MISH on the same tissue sections to detect macromolecular and / or nucleic acid targets. This can be important, for example, for colocalizing drug compounds (small molecule detection by direct unlabeled MSI) and drug targets (macromolecular detection by targeted PCMT-probe-based MSI). In a preferred embodiment, unlabeled direct MALDI-MSI is performed first using fresh-frozen tissue sections, whereby the tissue has not yet undergone tissue fixation (FFPE is possible, but is more ideal for direct unlabeled MSI of endogenous molecules [Wisztorski, Franck et al. (2010) Methods Mol Biol 656:303-22]). In a preferred embodiment, the tissue sections are then washed (e.g., in organic solvent) to remove residual matrix compounds, and then another cycle of MIHC and / or MISH followed by MALDI-MSI is performed on the same tissue sections as described above. See Example 4 for more details.

[0105] It should be noted that the present invention is facilitated by the use of mass spectrometry methods that employ "soft" ionization for molecular analysis, since these methods generally do not or only cause limited molecular fragmentation and, more specifically, do not atomize molecules for detection (e.g., allowing for the detection of peptide- or polymer-based mass tags and endogenous biomolecules in tissues). Such methods include, but are not limited to, laser desorption / ionization (LDI), matrix-assisted laser desorption / ionization (MALDI), silicon desorption / ionization (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 the present invention to a particular type of "soft" ionization mass spectrometry. For the purposes of the present invention, "soft" ionization-based methods are defined as methods that can detect molecular ions, i.e., charged molecules consisting of two or more atoms held together by chemical bonds.

[0106] In another preferred multi-omic embodiment, targeted PC-MT-MSI methods are combined with non-targeted "bottom-up" omics methods.

[0107] Bottom-up omics refers to the fact that biomolecules are first broken down (digested) into smaller fragments that are more accessible to mass spectrometry. The identity and structure of the entire biomolecule can then be inferred from mass spectrometry. In other bottom-up scenarios, the identity of the biomolecule does not need to be known, as only the fingerprints of various mass species (measured with high precision) can be correlated with, for example, disease state or stage. In the context of bottom-up tissue MSI, tissue is treated with a digestion agent to liberate biomolecular fragments that are more accessible to mass spectrometry. See, for example, Figure 22. Typically, the digestion agent is sprayed onto the tissue in a thin film to promote digestion without delocalization of the analytes, known as in situ digestion. However, achieving a balance between sufficient digestion and minimal delocalization is difficult, and a perfect balance likely does not exist. These digestion agents 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., peptide N-glycosidase F [PNGase F]), or chemical agents such as cyanogen bromide (CNBr) or hydroxylamine [Gundry, White et al. (2009) Curr Protoc Mol Biol Chapter 10:Unit 10 25; Barrett, Wither et al. (2017) J Proteome Res 16:4177-4184]. In situ tissue digestion followed by MSI has 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 enzymatically cleave N-linked glycans from proteins in the tissues, followed by MALDI-MSI of the released glycans.Other examples of using proteases as digestion agents in this case include the use of collagenase to digest specific protein types or trypsin to achieve general protein digestion [Angel, Schwamborn et al. (2019) Proteomics Clin Appl 13:e 1700152, Lazova, Smoot et al. (2020) J Cutan Pathol 47:226-240].

[0108] When bottom-up MSI is combined with PC-MT-MSI for a multi-omic tissue imaging approach, the present invention is not intended to be limited to a specific order of operations. For example, PC-MT-MSI may be performed before or after bottom-up MSI (both options require two rounds of MSI). Conversely, tissues can be stained with PC-MT-probes, then in situ digestion can be performed, followed by a single round of MSI. If PC-MT-MSI is performed first, it may be desirable to remove the PC-MT-probes later if they may interfere with subsequent bottom-up MSI. To do so, the probes can be first separated from the tissue by a denaturing treatment, and then washed away. Such denaturing treatments may include, but are not limited to, chaotropic agents, solutions with a pH of 5 or less, solutions with a pH of 10 or more, 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.

[0109] Note that while the above encompasses a wide range of multi-omic methodological permutations, two specific examples using in situ protease or glycosidase digestion are provided in Example 15.

[0110] [Table 1-1] [Table 1-2] [Table 1-3]

[0111] Shaded (bold) rows are mass units 1 or variations of mass units 1 used to minimize variable MALDI-MS ionization efficiency. Letters in brackets represent stable isotope amino acids as shown in Table 1.1.

[0112] * The mass unit is N-terminally acetylated on the a-amine, and the mass reporter mass comprises this acetylation plus the mass unit and a small portion of the photocleaved PC-linker (see step 3 in Figure 3).

[0113] [Table 1.1]

[0114] experiment

[0115] Materials for example experiments.

[0116] Water (LCMS grade) and xylene (semiconductor grade) were obtained from Acros Organics (Pittsburgh, PA). Methanol (LCMS grade) was obtained from J.T. Baker (Avantor, Radnor, PA). Ethanol (Molecular Biology Bioreagents), acetone (HPLC grade), N,N-dimethylformamide (anhydrous, ≥99.8%), 1,5-diaminonaphthalene (DAN, 97%), 2,5-dihydroxybenzoic acid (DHB, 98.0%), isopropyl alcohol (Molecular Biology Bioreagents), sodium chloride (BioXtra, ≥99.5%), sodium bicarbonate (99.7%–100.3%, tested molecular biology), ammonium bicarbonate (BioUltra, ≥99.5%), bovine serum albumin (heat shock fraction, protease-free, fatty acid-free, essentially globulin-free, pH 7, ≥98%), phosphate-buffered saline (PBS) (BioPerformance Certified, pH 7.4, P5368), glycine (Molecular Biology Ultra, ≥99%, Fluka Biochemika), paraformaldehyde (powder, 95%), citrate buffer (pH 6.0, 10×, Antigen Retriever), 5-(ethylthio)-1H-tetrazole (5-ETT, 95%), 1-methylimidazole (ReagentPlus, 99%), formamide (BioReagents for Molecular Biology, ≥99.5%), RNA from baker's yeast, tRNA, Denhardt's solution (50 × 5 mL), Atto-647N-NHS ester and octyl BD-glucopyranoside (OBG) (50% [w / v] stock solution) were obtained from Sigma Aldrich (St. Louis, MO), and 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), DyLight 650 NHS ester, anti-streptavidin antibody clone S3E11, hydroxylamine, Slide-A-Lyzer™ 0.1 mL MINI Dialysis Devices with 20 kDa membranes, and biotinylated protein G were obtained 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 × 53 mm × 0.6 mm deep) were obtained from Fisher Scientific (Hampton, NH). Tris-HCl and Tris base (molecular biology grade), 5 N 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 obtained from Promega (Madison, WI). Antigen Retrieval Reagent-Basic (CTS013) was obtained from R&D Systems / BioTechne (Minneapolis, MN). Gold-coated microscope slides (Au-500A) were obtained from Angstrom Engineering Inc. (Kitchener, Ontario, Canada). Sagittal FFPE sections of mouse C57 brain (5 μm thick) and sagittal fresh-frozen (FF) sections of mouse C57 brain (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 thin sectioning (5 μm thick) and mounting on slides.Antibodies for multiplex imaging of mouse brain sections were obtained from various suppliers as follows: anti-myelin basic protein antibody (MAB42282) from R&D Systems / BioTechne (Minneapolis, MN); anti-NeuN antibody (MAB377) and anti-synapsin 2 antibody (MABN1573) from Millipore Sigma (Burlington, MA); anti-GLUT1 antibody (PA146152) from Fisher Scientific. The antibodies were obtained from Biosciences Scientific (Hampton, NH) and included anti-MAP2 antibody (ab11268), recombinant anti-NeuN antibody (ab209898) and recombinant anti-Cas9 (ab218654, BSA and azide free), recombinant anti-pan-cytokeratin antibody [C-11]-BSA and azide free (ab264485), recombinant anti-CD3 repsilon antibody [CAL57]-BSA and azide free (ab251607), recombinant anti-CD4 antibody [EPR6855]-BSA and azide free (ab181724), recombinant anti-CD8 alpha antibody [CAL66]-BSA and azide free (ab251596), recombinant anti-CD20 antibody [EP459Y]-BSA and azide free (ab214282), and anti-CD45RO antibody [ UCH-L1 (ab23), recombinant anti-estrogen receptor alpha antibody [SP1] -BSA and azide-free (ab187260), recombinant anti-progesterone receptor antibody [YR85] -BSA and azide-free (ab206926), recombinant anti-ErbB2 antibody [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 obtained from Abeam (Cambridge, MA).Mouse IgG whole molecule (015-000-003), normal mouse serum (015-222-001), rabbit IgG whole molecule (011-000-003), and normal rabbit serum (011-000-001) were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). For specific bead array experiments, recombinant anti-β-amyloid 1-42 antibody (ab224275) and recombinant anti-myelin basic protein antibody (ab230378) were BSA- and azide-free and obtained from Abeam (Cambridge, MA). U6-amine custom LNA oligonucleotide (339406 YC00191704), sense miR-159-amine custom LNA oligonucleotide (339406 YC00191705), and miRCURY LNA miRNA ISH buffer set for FFPE were obtained from Qiagen (Germantown, MD). Streptavidin-coated 20 μm and 37 μm PMMA beads (microspheres) were obtained from PolyAn GmbH (Berlin, Germany). 0.5 mL Ultrafree-MC Centrifugal 0.45 μm filter devices were obtained from Millipore Sigma (Burlington, MA). NAP-5 Sephadex G-25 and PD SpinTrap G-25 columns were obtained from GE Healthcare Life Sciences (Pittsburgh, PA), and 4-[4-[1-(9-fluorenylmethyloxycarbonylamino)ethyl]-2-methoxy-5-nitrophenoxy]butanoic acid, hereafter referred to as 4-[4-[1-(9-Fmoc-amino)ethyl]-2-methoxy-5-nitrophenoxy]butanoic acid, was obtained from Santa Cruz Biotechnology (Dallas, TX). FlexWell™ 16-chamber self-adhesive gaskets (204916) were obtained from Grace Bio-Labs (Bend, OR).

[0117] Example 1. 15-plex PC-MT-Ab-based MSI using bead arrays as a model system.

[0118] PC-MT.

[0119] Peptide-based PC-MTs were generated using standard Fmoc amino acid solid-phase peptide synthesis (SPPS) [Behrendt, White et al. (2016) J Pept Sci 22:4-27]. An Fmoc-protected photocleavable amino acid linker (see Fmoc-PC-linker in Figure 3) was introduced into the peptide chain in the same manner as other amino acids, and the N-terminal α-amine of the peptide was acetylated with acetic anhydride using standard procedures. An NHS-ester probe-reactive moiety was created on the s-amine of the lysine (K) moiety contained in the spacer unit (see Step 1, NHS-ester in Figure 3). Conversion of the s-amine of the lysine (K) moiety to the NHS-ester was achieved using disuccinimidyl suberate (DSS). The use of bifunctional succinimidyl esters, such as DSS or DSC (disuccinimidyl carbonate), has been previously reported for the conversion of primary amines to NHS esters [Morpurgo, Bayer et al. (1999) J Biochem Biophys Methods 38:17-28]. After synthesis, PC-MTs were purified by HPLC using an Onyx Monolithic C18 column and an acetonitrile solvent system: 0.05% TFA in HO / 0.05% TFA in acetonitrile. The chemical structure of an exemplary PC-MT is shown in Figure 3. Fifteen different PC-MTs were made, differing only in the amino acid sequence of the mass unit shown in Figure 3. The 15 different mass unit sequences are listed in Table 1 (IDs 1, 2-3, and 5-16) as well as the monoisotopic masses of the mass reporters, which contain N-terminal acetylation, a peptide mass unit, and a small portion of the photocleaved PC-linker, as shown in Figure 3 (step 3).

[0120] In some cases, an additional lysine was included in the spacer unit of the PC-MT and modified with a fluorophore using a commercially available amine-reactive reagent (e.g., Sulfo-Cy5-NHS, Lumiprobe, Hunt Valley, MD). In this case, the amino acid sequence of the spacer unit was GSGG[K-NHS] for the non-fluorescent PC-MT. (SEQ ID NO: 31) Instead of GS[K-Sulfo-Cy5]GG[K-NHS] (SEQ ID NO: 30) It was.

[0121] Preparation of PC-MT antibody (PC-MT-Ab)

[0122] A 100 pL antibody solution (1 μg / μL in PBS) was supplemented with 1 / 9 volume of 1 mM sodium bicarbonate, followed by the addition of sufficient PC-MT from a 1 mM stock in anhydrous DMF to achieve a 10-fold molar excess over the antibody. The reaction was allowed to proceed for 1 hour with gentle mixing and protection from light. The reaction was then quenched with 1 / 9 volume of 1 mM glycine, followed by 15 minutes of mixing and protection from light. Finally, 1 / 199 volume of 10% (w / v) aqueous BSA carrier stock solution was added, resulting in a final concentration of 0.05% BSA (w / v). To remove unreacted PC-MT, the resulting PC-MT antibody (PC-MT-Ab) was loaded onto a PD SpinTrap G-25 column according to the manufacturer's instructions using TBS (50 mM Tris, pH 7.5, 200 mM NaCl) as the pre-equilibration buffer. An additional 1 / 9 volume of 10x TBS was added to the resulting PC-MT-Ab. In some cases, PC-MT-Ab was purified by extensive dialysis against TBS using Slide-A-Lyzer™ 0.1 mL MINI Dialysis Devices with 20 kDa membranes.

[0123] Bead array

[0124] Streptavidin-coated 20-micron PMMA beads were processed in 0.5 mL Ultrafree-MC Centrifugal 0.45 μm filter devices unless otherwise noted. (Washing was performed by vortexing the bead suspension in the filter device for 3 seconds, followed by filtration to separate the beads from the solution at 15,000 rpm for 5 seconds in a standard microcentrifuge.) For each PC-MT-Ab version, 10,000 beads were used and processed separately unless otherwise noted. Beads were first washed four times with 400 μL of bead-blocking buffer (1% BSA [w / v] in TBS-T; note that TBS-T is TBS supplemented with 0.05% [v / v] Tween®-20). The beads were then separately probed with 15 different versions of anti-streptavidin PC-MT-Ab, each carrying a different PC-MT species (i.e., with different mass units—IDs 1, 2–3, and 5–16—see Table 1 for mass units). PC-MT-Ab was diluted to 1 μg / mL in bead blocking buffer, and 100 μL was used for probing for 1 hour with gentle mixing. The beads were then washed four times with 400 μL of TBS-T, after which all 15 different bead versions were pooled. The pooled beads were then washed four more times with 400 μL of mass spectrometry-grade water (MS-Water). Bead array formation on indium tin oxide (ITO)-coated microwell substrates with the footprint of a standard microscope slide was performed 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].

[0125] light cutting

[0126] The substrates were finally dried in a vacuum drying chamber for 45 min and then irradiated with 365 nm light at approximately 3 mW / cm at a distance of 5 cm using a Model XX-15 lamp (UVP / Analytik Jena US LLC, Upland, CA). 2 Alternatively, the substrates were illuminated with 365 nm light at approximately 30 mW / cm using an LED Cube 100 IC (Honle UV Technology, Marlborough, MA). 2 A 5 minute light treatment was used unless otherwise stated.

[0127] Matrix Application

[0128] The DHB or DAN matrix was then applied to the dried substrate by sublimation, followed by recrystallization according to published 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].

[0129] MALDI-MS Imaging (MALDI-MSI)

[0130] MALDI-MS imaging (MALDI-MSI) was achieved on a rapifleX MALDLTOF-MS instrument (Bruker Daltonics, Billerica, MA) using the following parameters: reflector mode, laser spot size of 10 μm or 20 μm with continuous raster scanning of 10 μm or 20 μm, respectively, 300–500 laser shots / pixel, and, in some cases, normalization to total ion counts (TIC). Image and spectral analysis was performed using fleximaging and flexAnalysis software (Bruker Daltonics, Billerica, MA).

[0131] result

[0132] The results in Figure 6 (inset image) show a MALDI-MS "mass image" of the bead array. Different colors in the inset image correspond to different m / z values ​​of the monoisotopic mass spectral peak for a specific PC-MT mass reporter (see Table 1 for the expected masses of mass reporters with IDs 1, 2-3, and 5-16). Fifteen different mass-resolution mass reporter species are observed, derived from PC-MT-Ab probes bound to separate 20 µm beads in the array. Color-coded overlay MALDI-MSI spectra from a representative single bead in the array (Figure 6, black arrow) show no "crosstalk" between beads.

[0133] Example 2. 5-plex MIHC using PC-MT-Ab on sagittal FFPE tissue sections of mouse brain.

[0134] Immunostaining with PC-MT-Ab

[0135] PC-MT and PC-MT-Ab were prepared as in Example 1 and used in MIHC as follows: FFPE tissue sections were deparaffinized and hydrated as follows (each step in a separate staining jar): 3 times with xylene for 5 minutes, 1 time with xylene:ethanol (1:1) for 3 minutes, 2 times with 100% ethanol for 2 minutes, 2 times with 95% ethanol for 3 minutes, 1 time with 70% ethanol for 3 minutes, 1 time with 50% ethanol for 3 minutes, and 1 time with TBS for 10 minutes.

[0136] Antigen retrieval was achieved by preheating 200 mL of 1x citrate buffer (pH 6.0, see Materials) in a beaker in a 95°C water bath for 1 h and then cooling in the same beaker at room temperature for 30 min. Slides were then blocked 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 TBS supplemented with 0.05% [v / v] Tween®-20) for 1 h. For PC-MT-Ab staining, slides were treated overnight at 4°C with 200 pL / section of a solution containing 2.5 μg / mL of each antibody diluted in tissue blocking buffer (incubation was performed protected from light, in a humidified chamber to avoid evaporation, and with each tissue section surrounded by a hydrophobic barrier pen to maintain fluidity).

[0137] The slides were then washed three times for 5 minutes each in TBS, followed by three times for 2 minutes each in 50 mM ammonium bicarbonate as follows (note that all solutions were in LCMS-grade water, all washes were performed using excess solution, and the slides were placed horizontally in a Petri dish and gently shaken).

[0138] Finally, photocleavage, matrix application and MALDI-MSI were carried out as in Example 1.

[0139] Note that in some cases, immunofluorescence was performed instead of staining with PC-MT-Ab. In these cases, antibody labeled with a 15-fold molar excess of DyLight 650 NHS ester reagent (added from a 5 mM stock in DMF) was used instead of PC-MT-labeled antibody (otherwise, the same labeling procedure described previously for PC-MT-Ab in Example 1 was used). Furthermore, photocleavage, matrix application, and MALDI-MSI were not performed; instead, imaging of dried slides was performed at 5 μm resolution on a GenePix 4200A fluorescence scanner (Molecular Devices, San Jose, CA). All other procedures were the same as those described for MIHC in this example.

[0140] result

[0141] Five-plex MIHC was performed on FFPE sagittal sections of mouse brain. For this purpose, different PC-MTs were directly conjugated to antibodies in one step. The five antibodies targeted myelin basic protein (a well-known axon sheath marker, e.g., [van Tilborg, van Kammen et al. (2017) Sci Rep 7:16492]), NeuN (a neuronal nucleus marker, e.g., [Gusel'nikova and Korzhevskiy (2015) Acta Naturae 7:42-7]), synapsin (a synaptic protein, e.g., [Mason (1986) Neuroscience 19:1319-33]), Glut-1 (enriched in capillaries in 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 achieved on an in-house Bruker rapifleX MSI instrument with a spatial resolution of 10 μm. Figure 7a shows a five-color MALDI-MS "mass image" of a whole-brain section. Different colors correspond to different m / z values ​​of the monoisotopic mass spectral peak of a particular PC-MT. Myelin (red), NeuN (green), and synapsin (blue) are the most dominant and produce the most distinct structural patterns. For example, NeuN produces a distinct "swirl" pattern ( *Furthermore, myelin, NeuN, and synapsin highlight three distinct layers of the cerebellum (denoted by I). Less obvious biomarkers hidden in the composite color image are best seen as monochromatic images. For example, Figure 7c shows a monochromatic standalone MALDI-MS image of the Glut-1 biomarker (showing primarily a cross-section of brain capillaries). MAP-2 results in fairly uniform staining of the brain section (not shown as a standalone image). Furthermore, a species-matched non-immune isotype control immunoglobulin with the same PC-MT shows no MALDI-MS signal (not shown). Color-coded overlay mass spectra are shown for selected pixels of the MALDI-MS image (these pixels are indicated by arrows in Figure 7a) (Figure 7e). Finally, MIHC results clearly agree with conventional immunofluorescence. For example, using myelin, NeuN, and synapsin antibodies directly labeled with fluorophores alone produces the same pattern as MIHC in the cerebellum (Figure 7b). The MALDI-MS pattern of Glut-1 is also consistent with that by immunofluorescence (Fig. 7d).

[0142] Example 3. MISH using PC-MT-NA miRNA hybridization probes on FFPE tissue sections

[0143] Preparation of PC-MT nucleic acids (PC-MT-NA)

[0144] Amine-modified locked nucleic acid (LNA) probes (see Materials) were labeled with PC-MT as follows: To 100 μL of LNA probe solution (10 μM in 200 mM sodium chloride and 200 mM sodium bicarbonate), sufficient PC-MT was added from a 10 mM stock in anhydrous DMF to achieve a 200-fold molar excess over the LNA probe (2 μL of the 10 mM stock was added every 30 min for a total of five additions). The reaction was allowed to proceed for a total of 2.5 h, protected from light and with gentle mixing. To remove unreacted PC-MT, the resulting PC-MT-NA was loaded onto a NAP-5 Sephadex G-25 column according to the manufacturer's instructions, using TE-150 mM NaCl (10 mM Tris, pH 8.0, 1 mM EDTA, and 150 mM NaCl) as the pre-equilibration buffer.

[0145] In situ hybridization with PC-MT-NA

[0146] For deparaffinization and hydration, FFPE tissue sections were treated as described in Example 2. The following procedure was 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 proteinase K digestion, EDC fixation, and acetylation. For proteinase K digestion, 1.5 μL of proteinase K stock solution from the miRCURY LNA miRNA ISH Buffer Set for FFPE (see Materials) was diluted to 2.0 mL with proteinase K buffer (5 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM NaCl), and 300 μL was incubated with each tissue section for 10 minutes at 37°C. After proteinase K digestion, tissue sections were washed twice with 0.2% (w / v) glycine in PBS for 10 minutes each. For EDC fixation, tissue sections were pretreated twice in 0.1 M 1-methylimidazole solution for 3 min each, then treated for 1 h with 200 μL of EDC fixation solution (freshly prepared by adding 1.0 mL of 0.1 M 5-ETT and 0.1 M 1-methylimidazole to a 10 mg EDC HCl vial, adjusting the pH to 8.0 with 10 M NaOH). After EDC fixation, tissue sections were washed with 0.2% (w / v) glycine in PBS for 10 min. For acetylation, 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 min. After acetylation, tissue sections were washed in excess PBS for 3 min. Sections were then prehybridized in 150 μL of hybridization buffer consisting of 50% formamide, 1.0 M NaCl, 75 mM Tris-HCl (pH 8.5), 1× Denhardt's solution, 250 μg / mL baker's yeast tRNA, 500 μg / mL salmon sperm DNA, 5 mM CHAPS, and 0.1% Tween®-20 in a humidified chamber for 1 hour at 25° C. After prehybridization, self-adhesive hybridization chambers were placed on the slides (22 mm × 53 mm × 0.6 mm per chamber).For hybridization, the PC-MT U6 and 159 LNA probes were diluted to 200 nM in hybridization buffer, and the tissue sections were incubated overnight at 55°C in a humidified chamber (protected from light). The tissue sections were then washed once for 5 min in 5x SSC buffer, twice for 5 min in 1x SSC buffer, and once for 5 min in 0.2x SSC buffer at 55°C, and once for 5 min in 0.2x SSC buffer at room temperature.

[0147] The slides were then washed three times for 5 minutes each in TBS, followed by three times for 2 minutes each in 50 mM ammonium bicarbonate as follows (note that all solutions were in LCMS-grade water, all washes were performed using excess solution, and the slides were placed horizontally in a Petri dish and gently shaken).

[0148] Finally, photocleavage, matrix application and MALDI-MSI were carried out as in Example 1.

[0149] Note that in some cases, FISH was performed instead of staining with PC-MT-NA. In these cases, nucleic acids labeled with the Atto-647N-NHS ester reagent were used instead of PC-MT-labeled nucleic acids (otherwise, the same labeling procedure as described above in this example for PC-MT-NA was used). Furthermore, photocleavage, matrix application, and MALDI-MSI were not performed; instead, imaging of dried slides was performed with a GenePix 4200A fluorescence scanner (Molecular Devices, San Jose, CA) at 5 μm resolution. All other procedures were the same as those described for MISH in this example.

[0150] result

[0151] To demonstrate MISH, an amine-reactive NHS-activated PC-MT labeling reagent was directly conjugated to a 5' amine-terminated LNA hybridization probe (LNA = locked nucleic acid for 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]. Sagittal tissue sections of mouse brain were then stained with the probe 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]. The results in Figure 8 show that MISH using the U6 probe produced a tissue staining pattern similar to that of conventional FISH, while the negative control miR-159 probe did not produce a noticeable staining pattern.

[0152] Example 4. Multi-omic MSI: Untargeted, unlabeled MSI and targeted MIHC of lipids on the same FF tissue section

[0153] Non-targeted, unlabeled direct MSI on FF tissue sections

[0154] Unprocessed fresh-frozen (FF) tissue sections were first analyzed directly by MALDI-MSI in a non-targeted manner (after thawing the tissue sections and only matrix application as in Example 2 [in this case DAN matrix]).

[0155] MIHC on the same FF tissue section

[0156] Next, while deparaffinization was not required for FF tissue sections, paraformaldehyde (PFA) fixation was performed (each step in a separate staining jar) as follows: two 3-minute prewashes in cold acetone (note this helps remove residual matrix compounds from previous MALDI-MSI while providing solvent fixation), followed by 10-minute air-drying, followed by 10-minute fixation using 1% PFA in PBS, pH 7.4 (note this solution was freshly prepared by dissolving 1.0 g of PFA in 60 mL of PBS containing 1.0 mL of 1 M NaOH on a heating block at approximately 60 °C with constant stirring, followed by adjusting the pH to 7.4 with approximately 1 mL of 1 M HCl, and adjusting the final volume to 100 mL with PBS), followed by one 10-minute wash with PBS. The remaining steps of the PC-MT-Ab-based MIHC, from the antigen retrieval step to the end, were performed as in Example 2.

[0157] result

[0158] It would be highly advantageous to be able to detect both untargeted, unlabeled small molecules and PC-MT-Ab-targeted macromolecules on the same tissue section. For example, this would enable the colocalization of drug targets, such as small molecule drugs and their receptors, as well as related biomolecules involved in the cellular response to the drug. To demonstrate the basic feasibility of this capability, we first performed direct MALDI-MSI analysis on sagittal tissue sections of fresh-frozen mouse brain (fresh-frozen, instead of FFPE, is optimal to facilitate small molecule detection without tissue fixation or preclearance). We used negative-ion mode MALDI-MSI with a DNA matrix. Next, the tissue was washed / fixed with cold acetone to remove the MALDI-MS matrix compound, and then further fixed with paraformaldehyde. Then, we performed MIHC, which constitutes two rounds of MALDI-MSI (so-called double MSI). The results of the first direct MALDI-MSI run are shown in Figure 9a. The image is color-coded for the m / z values ​​of three well-known lipids identified from the METLIN database at the Scripps Center for Metabolomics [Smith, OMaille et al. (2005) Ther Drug Monit 27:747-51], consistent with a previous MALDI-MSI analysis of lipids from mouse brain tissue sections [Wang, Wang et al. (2018) Anal Chim Acta 1000:155-162] (sulfatide [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 clearly enriched in different brain structures, and sulfatide (red) in particular shows a distinct pattern from the other two lipids. However, as expected for two of the major structural lipids in eukaryotic cell membranes, there is also significant colocalization with phosphatidylethanolamine (blue) and phosphatidylinositol (green) [van Meer, Voelker et al. (2008) Nat Rev Mol Cell Biol 9:112-24] (the colocalized blue and green appear as cyan in Figure 9a).For demonstration purposes, Figures 9b and 9c show two-color overlays of sulfatide lipids detected by direct MALDI-MSI and selected macromolecular biomarkers detected by MIHC in a second MALDI-MSI run. Figure 9b shows sulfatide (red) overlaid with the neuronal nuclear biomarker NeuN (green), detected using PC-MT-Ab as previously demonstrated in Example 2. These two biomolecules generally do not colocalize. Conversely, Figure 9c shows the same lipid sulfatide (red) overlaid with myelin basic protein (green), again detected using PC-MT-Ab in a multiplex MIHC run. In this case, there is strong colocalization of sulfatide with myelin (evidenced by the yellow color resulting from the colocalization of green and red). The colocalization of sulfatide and myelin is consistent with previous literature showing that sulfatide is found primarily in the myelin sheath (Schwann cells / oligodendrocytes) of neuronal axons [Eckhardt (2008) Mol Neurobiol 37:93-103; Hirahara, Wakabayashi et al. (2017) J Neurochem 140:435-450]. Conversely, sulfatide would not be expected to colocalize with the neuronal nuclear biomarker NeuN, as observed here (note that myelin and NeuN also generally do not colocalize, as previously shown in Example 2). Finally, example spectra from the first direct MALDI-MSI run are shown in Figure 9d. These spectra are from selected image pixels chosen from three distinct colored "layers" observed in the medulla / neuronal layer of the mouse brain (see the colored arrows in Figure 9a for these layers). In the future, the use of MS / MS or higher resolution FTICR mass spectrometers will provide more accurate small molecule identification.

[0159] Example 5. Mass unit preferred binding site to PC-nuclei.

[0160] In the present invention, the preferred mode of attachment of the mass unit to the phenyl ring of the PC-core (see Figure 3 for the PC-core) is ultimately via a photocleavage site (see Configuration 1 in Figure 10a, also see Figure 3). In this configuration, the photocleaved phenyl ring of the PC-core does not remain attached to the photocleaved mass reporter detected by mass spectrometry (MS). Alternatively, as taught by Olejnik et al. [Olejnik, Ludemann et al. (1999) Nucleic Acids Res 27:4626-31] and Levy and Caprioli (U.S. Patent No. 7,569,392), it is possible to ultimately attach the mass unit to the phenyl ring of the PC-core via a site other than the photocleavage site, but this is not preferred because the photocleaved phenyl ring of the PC-core remains attached to the photocleaved mass reporter detected by MS (see Configuration 2 in Figure 10a).

[0161] To demonstrate the advantages of preferred configuration 1, photocleavable peptides with the same peptide sequence and mass unit were attached to a surface according to the two configurations shown in Figure 10a. (See also Figure 10a for the structure and sequence of the photocleavable peptide.) The mass reporter was then photocleaved from the surface and measured by mass spectrometry. Preferred configuration 1 provides a clean monoisotopic peak at the expected mass of the mass reporter (light gray trace in Figure 10b, 1,194.7 m / z, configuration 1) and only an accompanying peak cluster corresponding to the natural isotope of the mass reporter, separated by 1 m / z (present but not discernible in Figure 10b due to the x-axis scaling). In configuration 2, the photocleaved mass reporter provides 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 likely due to side-reaction byproducts of the photocleaved phenyl ring of the PC-nucleus that remains attached to the mass reporter (see Figure 10b, "Byproducts of the PC-nucleus in configuration 2"). These by-products may include, in part, oxygen adducts and losses due to 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 (due to peak overlap) in multiplexed analyses.

[0162] Example 6. Comparison of different PC linkers and preparative photocleavage versus in-line photocleavage using the MALDI-MS laser beam.

[0163] PC-MT

[0164] In the same manner as in Example 1, PC-MTs having the configuration shown in FIG. 3 were prepared (four PC-MTs in this example consisting of mass units 1 and 2 to 4 listed in Table 1).

[0165] Furthermore, so-called PC-MTs-L were prepared in the same manner, differing only by using the PC-linker of Lemaire et al. [Lemaire, Stauber et al. (2007) J Proteome Res 6:2057-67] [U.S. Patent No. 8,221,972] instead of the PC-linker shown in Figure 3. To achieve this, the PC-linker was introduced into PC-MTs-L during peptide synthesis using 4-[4-[1-(9-Fmoc-amino)ethyl]-2-methoxy-5-nitrophenoxy]butanoic acid (see Materials). (See Figure 4, "Lemaire's PC-linker," for this PC-linker incorporated into a plain peptide—note that in Figure 4, the plain peptide is shown to show the PC-linker itself, but the plain peptide lacks other features of PC-MTs and PC-MTs-L.) Importantly, although the PC-linker of PC-MTs-L was the one used by Lemaire et al., other features of PC-MTs-L that are identical to the PC-MT features shown in Figure 3 were not taught by Lemaire et al. These features (Figure 3) include, but are not limited to, the probe-reactive moiety (in this case, an NHS-ester), the N-terminal blocking of peptide-based PC-MTs (in this case, N-terminal acetylation), the peptide sequence of the mass unit (Table 1) and spacer unit, and the specific attachment of the mass unit to the phenyl ring of the PC-core via a photocleavage site.

[0166] Bead array

[0167] PC-MT-L was used in equimolar concentrations with PC-MT to simultaneously double-label streptavidin-coated 37-micron PMMA beads. To accomplish this, four bead species were created by simultaneously double-labeling each bead with the same mass units of PC-MT-L and PC-MT in four separate reactions. This is possible because PC-MT leaves a small residual portion of the PC-linker attached to the photocleaved mass reporter (see step 3 in Figure 3), whereas PC-MT-L does not (thus, even though the mass units of each bead species are the same, the masses of the photocleaved mass reporters are distinguishable in the mass spectrometer).

[0168] Mass tagging of beads was performed as follows: Streptavidin-coated 37-micron PMMA beads were processed in 0.5 mL Ultrafree-MC Centrifugal 0.45 μm filter devices unless otherwise noted. (Washing was performed by vortexing the bead suspension in the filter device for 3 seconds, followed by filtration to separate the beads from the solution at 15,000 rpm for 5 seconds in a standard microcentrifuge.) 100,000 beads were used for each of the four bead types (processed separately unless otherwise noted). The beads were washed four times with 400 μL of conjugation buffer (200 mM sodium bicarbonate containing 200 mM NaCl). Each bead pellet was then resuspended in 100 μL of conjugation buffer, followed by the addition of 2 μL of 500 μM each of PC-MT-L and PC-MT in DMF. The reaction was mixed for 30 min, then the beads were washed three times for 15 min each with 400 μL of glycine-hydroxylamine quench (1 M glycine and 100 mM hydroxylamine in 10x concentrated TBS, freshly prepared). The beads were then washed four times for 15 min each with 400 μL of OBG saline (25 mM ammonium bicarbonate, 0.05% (w / v) octyl-BD-glucopyranoside, and 50 mM NaCl) and resuspended at 250 beads / μL in the same buffer.

[0169] Finally, bead arrays were formed as follows: the beads were washed four times with 400 pL of mass spectrometry-grade purified water and resuspended in mass spectrometry-grade purified water (20,000 beads / 100 pL). The beads were then arrayed at 400 beads / mm using a FlexWell™ 16-chamber self-adhesive gasket (see Materials) attached to a gold-coated microscope slide (see Materials) in a chemical fume hood. 2 After drying, the slides were gently washed in a tray containing excess mass spectrometry-grade purified water (the beads remained attached to the slides), and then the slides were dried again in a vacuum drying chamber for 45 minutes.

[0170] Photocleavage of bead arrays, matrix application and MALDI-MSI

[0171] Performed as in Example 1 (note that the beads directly mass tagged as above were not probed with PC-MT-Ab as was done in Example 1). In some cases, UV irradiation was increased to 25 min.

[0172] C-MT-Ab

[0173] The preparation was similar to that in Example 1, except that the anti-myelin antibody was simultaneously double-labeled with equimolar concentrations of PC-MT-L and PC-MT, consisting of 1 mass unit as listed in Table 1 (i.e., both 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 for labeling]).

[0174] Tissue immunostaining with PC-MT-Ab, photocleavage, matrix application and MALDI-MSI

[0175] The procedure was carried out as in Example 2. In some cases, UV irradiation was increased to 25 min. In other cases, UV irradiation was not performed, and only in-line photocleavage was allowed during MALDI-MSI using the instrument's laser beam.

[0176] result

[0177] We first compared PC-MT with PC-MT-L for mass units 1 and 2-4 listed in Table 1 using a bead array. Figure 11a shows an example mass image of the bead array for mass unit 1, representing a pixel map of the mass spectral peak intensities of the mass reporters 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). In the image, PC-MT is color-coded red, and PC-MT-L is color-coded green. If the peaks were of similar intensity, a two-color overlay would be expected to produce a yellow color; however, because PC-MT produces a much stronger signal, the beads appear reddish-orange. Representative mass spectra from a single pixel from a bead in the array are shown in Figures 11b-i for all four mass units at 5 and 25 minutes of pre-UV irradiation (i.e., UV photocleavage before matrix application and MALDI-MSI). Note that because each of the four bead species is dual-labeled, each spectrum contains both PC-MT and PC-MT-L peaks for a given mass unit. (PC-MT yields an m / z of +43 compared to the equivalent mass unit from PC-MT-L, because PC-MT leaves a small residual portion of the photocleaved PC-linker attached to the mass reporter, whereas PC-MT-L does not.) The PC-MT to PC-MT-L ratio of mass spectral peak intensities was then calculated for each mass unit and each pre-UV time point, averaged from five pixels (five spectra) for each sort. These ratios were 5:1, 8:1, 10:1, and 6:1 at 5 min of pre-UV for mass units 1 and 2-4, respectively. Thus, PC-MT exhibited a 5- to 10-fold higher signal compared to PC-MT-L. The relative PC-MT-L signal improved modestly over 25 min of UV, resulting in ratios of 2:1, 3:1, 7:1, and 5:1, but the improvement was never proportional to increasing pre-UV time (a 5-fold increase in pre-UV time) and there were diminishing returns, as PC-MT always remained superior to PC-MT-L.

[0178] Second, we used MIHC (mass spectrometry-based immunohistochemistry) to compare PC-MT and PC-MT-L at 1 mass unit to stain mouse brain tissue sections using a dual-labeled antibody probe for myelin. In this case, we tested 5 and 25 minutes of pre-UV treatment of the tissue (immediately before matrix application and before MALDI-MSI), as well as 0 minutes of pre-UV. In this case, photocleavage was only consistent with MALDI-MSI analysis using the instrument's laser beam. Similar to the bead array, a two-color mass image for tissue myelin imaging is shown in Figure 11j. Again, PC-MT (red) provides a much higher signal than PC-MT-L (green), thereby dominating the image. Spectra from representative pixels are shown in Figures 11k and 11l, confirming this result. In this case, the PC-MT to PC-MT-L ratio of peak intensity was 6:1 for both 5 and 25 minutes of pre-UV treatment. Finally, 0 min of pre-UV treatment yielded virtually 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 regions of interest (see dotted outlines in Figure 11j) for each tissue section. From these average spectra, the monoisotopic peak intensities of PC-MT were 2.5, 15.8, and 23.3 for 0, 5, and 25 min of pre-UV treatment, respectively. Importantly, this demonstrates that photocleavage (0 min of pre-UV treatment), consistent with MALDI-MSI analysis using the instrument's laser beam, is much less sensitive (approximately 6-9 times less sensitive in this example) than pre-UV treatment for photocleavage.

[0179] Example 7. Fluorescent PC-MTs for mass spectrometry-based immunohistochemistry.

[0180] Mass spectrometry-based immunohistochemistry (MIHC)

[0181] MIHC on sagittal tissue sections of mouse brain was performed as in Example 2, except that the following recombinant anti-NeuN antibody (see Materials) was conjugated to a PC-MT configured as in Figure 3 (with mass unit 1 in Table 1), but further containing a Sulfo-Cy5 fluorescent label attached to the s-amine of the lysine amino acid appended to the spacer unit. Thus, the complete sequence of this fluorescent PC-MT with mass unit 1, designated Fluor-PC-MT1, was (N- to C-terminally): Acetyl-APRLRFYSL-[PC-Linker]-GS[K-Sulfo-Cy5]GG-[K-NHS]-COOH (SEQ ID NO: 32) The resulting antibody probe, called anti-NeuN Fluor-PC-MT1, was used for MIHC.

[0182] As a negative control, a recombinant anti-Cas9 antibody (see Materials) was also conjugated to Fluor-PC-MT1 (note that Cas9 is a bacterial-specific protein that is not present in mammals). The resulting antibody probe, designated anti-Cas9 Fluor-PC-MT1, was used for MIHC.

[0183] Finally, as a positive control, recombinant anti-NeuN antibodies were conjugated only to non-fluorescent PC-MTs constructed as in Figure 3, except for mass unit 7 in Table 1, termed PC-MT7. The resulting antibody probe, termed anti-NeuN PC-MT7, was used for MIHC.

[0184] Because Fluor-PC-MT1 contains a fluorophore, in addition to MALDI-MSI, fluorescence imaging of the same tissue (on a gold-coated slide) was performed (immediately before UV pretreatment for photocleavage, before matrix application, and before MALDI-MSI analysis). Fluorescence imaging was performed as in Example 2.

[0185] result

[0186] Fluorescence images of mouse brain tissue are shown in yellow in Figure 12 (see "Fluorescence"). When tissue is probed with anti-NeuN Fluor-PC-MT1, a strong, specific fluorescent signal is observed. In comparison, tissue probed with the negative control anti-Cas9 Fluor-PC-MT1 shows only weak background fluorescence (Cas9 is a bacterial protein not present in mammals). Tissue probed with non-fluorescent anti-NeuN PC-MT7 shows only very weak background fluorescence, corresponding to tissue autofluorescence.

[0187] MALDI-MS images of mouse brain tissue are displayed in red (mass reporter from Fluor-PC-MT1) and green (mass reporter from PC-MT7) in Figure 12 (see "MALDI-MSI"). Tissue probed with anti-NeuN Fluor-PC-MT1 shows a strong and specific MALDI-MSI signal compared to tissue probed with the negative control anti-Cas9 Fluor-PC-MT1, which provides only weak background (see red in the "MALDI-MSI" panel of Figure 12).

[0188] The "staining" patterns of anti-NeuN Fluor-PC-MT1 are similar in the fluorescence and MALDI-MS images in Figure 12. Specifically, as in Example 2, anti-NeuN detects distinct features, such as the hippocampus (blue arrow in Figure 12) and cerebellum (white arrow in Figure 12). However, in MALDI-MSI, the nonfluorescent anti-NeuN PC-MT7 probe produces a clearer pattern (green in the "MALDI-MSI" panel in Figure 12) than the anti-NeuN Fluor-PC-MT1 probe. Based on the spectra in the inset of Figure 12, which show the Fluor-PC-MT1 and PC-MT7 mass reporter peaks (black arrows in the spectrum) from a representative single pixel within the hippocampus (blue arrow in Figure 12), the superiority of the anti-NeuN PC-MT7 probe does not appear to be due to higher peak intensity (same y-axis scaling of the spectra). The superior results may be explained by the lower nonspecific binding (and therefore less diffuse background) of the anti-NeuN PC-MT7 probe compared to the anti-NeuN Fluor-PC-MT1 probe. This can be resolved by using a different fluorophore to reduce nonspecific binding. Furthermore, increasing the length of the spacer unit to further separate the fluorophore from the probe reactive moiety (the NHS-ester on PC-MT in this example) can improve probe labeling efficiency.

[0189] Finally, note that the spectrum shown for the negative control anti-Cas9 Fluor-PC-MT1 probe shows no discernible mass reporter peaks.

[0190] Example 8. Recombinant antibodies versus conventional antibodies.

[0191] PC-MT-Ab

[0192] The following PC-MT-Abs were prepared as in Example 1 (antibodies are non-recombinant unless otherwise specified - see Materials for antibodies) (note that the numbers below indicate the mass units from Table 1 used in the particular PC-MT): recombinant anti-amyloid beta (referred to as recombinant anti-AB2), recombinant anti-myelin basic protein (referred to as recombinant anti-MBP1), anti-myelin basic protein (referred to as anti-MBP1), recombinant anti-NeuN (referred to as recombinant anti-NeuN7), and anti-NeuN (referred to as anti-NeuN7).

[0193] Bead array

[0194] The procedure was carried out as in Example 6 with the following exceptions: Streptavidin-coated 37 μm PMMA beads were used for conjugation to PC-MTs (these PC-MTs are referred to as bead ID-tags). The beads were then used to capture the PC-MT-Ab described above as follows: The beads were processed in a filter device as in Example 6. Unless otherwise noted, each PC-MT bead type was processed separately. The beads were washed four times with 400 μL of OBG saline (see Example 6 for formulation). 200 μL of 100 μg / mL biotinylated protein G in OBG saline was added to each bead pellet and mixed for 30 minutes. The beads were washed eight times with 400 μL of OBG saline. 200 μL of a 1 μg / mL PC-MT-Ab solution in OBG saline was added to the bead pellet and mixed for 30 minutes. The beads were washed twice with 400 μL of OBG saline and twice with 400 μL of OBG buffer (same as OBG saline but without NaCl). At this stage, the different bead species were pooled. Next, bead array formation, photocleavage, matrix application, and MALDI-MSI were performed as in Example 6.

[0195] As a result of the above procedure, two bead pools were created and analyzed separately on a bead array by MALDI-MSI. Each bead species in the pool had a unique directly attached bead ID tag and a bound PC-MT-Ab. All numbers below refer to the mass units listed in Table 1 used for the specific PC-MT.

[0196] Bead Pool 1:

[0197] Bead ID-tag 9 / recombinant anti-MBP1

[0198] Bead ID-tag 10 / anti-MBP1

[0199] Bead ID-tag 15 / recombinant anti-AB2

[0200] Bead Pool 2:

[0201] Bead ID-tag 9 / recombinant anti-NeuN7

[0202] Bead ID-tag 10 / anti-NeuN7

[0203] Bead ID-tag 15 / recombinant anti-AB2

[0204] Mass spectrometry-based immunohistochemistry (MIHC)

[0205] MIHC on sagittal tissue sections of mouse brain was performed as in Example 2 using the following previously described PC-MT-Abs: recombinant anti-NeuN7 and anti-NeuN7.

[0206] result

[0207] Figure 13a shows a MALDI-MS mass image of the bead array for bead pool 1. The beads are color-coded in the image as blue, green, and yellow, based on the mass spectral peak intensities of bead ID tags 9, 10, and 15, which correspond to beads loaded with recombinant anti-MBP1, anti-MBP1, and recombinant anti-AB2, respectively. All PC-MTs derived from PC-MT-Ab are color-coded red. Therefore, the colocalization (overlay) of the blue bead ID tag 9 with the conjugated red recombinant anti-MBP1 appears as pink in the mass image. Conversely, while the green bead ID tag 10 is detected, little or no signal is observed for the corresponding red anti-MBP1, and therefore these beads appear primarily green. Finally, the colocalization (overlay) of the yellow bead ID tag 15 with the conjugated red recombinant anti-AB2 appears as orange in the mass image. In conclusion, both recombinant antibodies show strong PC-MT-Ab signals (observed as pink and orange beads) on their respective beads, whereas non-recombinant anti-myelin basic protein PC-MT-Ab (anti-MBP1) is barely detectable. This is confirmed by the overlay spectra shown in Figure 13b. The three spectra correspond to three single pixels selected from the centers of the three beads circled in Figure 13a and are color-coded according to the bead colors observed in Figure 13a. The pink trace in Figure 13b shows a strong peak for the corresponding antibody PC-MT1 for bead ID-tag 9 and the bound recombinant anti-MBP1. Conversely, the green trace in Figure 13b shows a strong peak for bead ID-tag 10 but only a weak peak (black arrow) for the corresponding antibody PC-MT1 for the bound anti-MBP1. Note that the antibody PC-MT1 peak for the recombinant anti-MBP1 is 10 times stronger than that for the (non-recombinant) anti-MBP1. Finally, the orange trace in Figure 13b shows a strong peak for the bead ID-tag 15 and the corresponding antibody PC-MT2 for the bound recombinant anti-AB2.

[0208] Bead pool 2 shows similar results. Figure 13c shows a MALDI-MS mass image of the bead array from bead pool 2. The beads are color-coded in the image as blue, green, and yellow, based on the mass spectral peak intensities of bead ID tags 9, 10, and 15, which correspond to beads loaded with recombinant anti-NeuN7, anti-NeuN7, and recombinant anti-AB2, respectively. All PC-MTs derived from PC-MT-Ab are color-coded red. Therefore, the colocalization (overlay) of the blue bead ID tag 9 with the conjugated red recombinant anti-NeuN7 appears as pink in the mass image. Conversely, while the green bead ID tag 10 is detected, little or no signal is observed for the corresponding red anti-NeuN7, and therefore these beads appear primarily green. Finally, the colocalization (overlay) of the yellow bead ID tag 15 with the conjugated red recombinant anti-AB2 appears as orange in the mass image. In conclusion, both recombinant antibodies show strong PC-MT-Ab signals (observed as pink and orange beads) on their respective beads, whereas the non-recombinant anti-NeuN7 PC-MT-Ab (anti-NeuN7) is barely detectable. This is confirmed by the overlay spectra shown in Figure 13d. The three spectra correspond to three single pixels selected from the centers of the three beads circled in Figure 13c and are color-coded according to the bead colors observed in Figure 13c. The pink trace in Figure 13d shows a strong peak for the corresponding antibody PC-MT7 for bead ID-tag 9 and the bound recombinant anti-NeuN7. Conversely, the green trace in Figure 13d shows a strong peak for bead ID-tag 10 but only a very weak peak (black arrow) for the corresponding antibody PC-MT7 for the bound anti-NeuN7. Note that the antibody PC-MT7 peak for the recombinant anti-NeuN7 is 60-fold stronger than that for the (non-recombinant) anti-NeuN7. Finally, the orange trace in Figure 13d shows a strong peak for the bead ID-tag 15 and the corresponding antibody PC-MT2 for the bound recombinant anti-AB2.

[0209] The superior performance of the recombinant PC-MT-Ab in the bead array was reproduced in mass spectrometry-based immunohistochemistry (MIHC) on sagittal tissue sections of mouse brain. Figure 13e shows MALDI-MSI mass images of tissue stained with anti-NeuN7 and recombinant anti-NeuN7. NeuN immunostaining exhibits the typical pattern of this biomarker, similar to that observed in Example 2. Notably, the hippocampus (blue arrow) and cerebellum (white arrow) are strongly stained, with a speckled pattern characteristic of nuclear staining (NeuN is a neuronal nuclear biomarker) (yellow arrow). However, the recombinant anti-NeuN7 exhibits substantially higher sensitivity than the non-recombinant anti-NeuN7, as evident from the mass image in Figure 13e. This is confirmed by the spectra shown in Figure 13e. When spectra were taken from the most intense pixel within the hippocampus in each image, the peak from the "recombinant" antibody (red trace) was approximately 7-fold more intense than the peak from the "regular" non-recombinant antibody (blue trace).

[0210] Overall, the superior performance of recombinant antibodies is likely derived from the generally higher purity of these antibodies, free from contaminating proteins, azide- and amine-containing buffers, which enhances PC-MT labeling efficiency.

[0211] Example 9. Demonstration of 12-plex MIHC on FFPE tonsil and breast cancer tissues

[0212] method

[0213] The same procedure was followed as in Example 2, with the following exceptions: FFPE human tonsil and breast cancer tissues were used. The antigen retrieval step was performed as follows: Basic antigen retrieval was performed in 60 mL of 1x Antigen Retrieval Reagent-Basic (see Materials) at 95°C for 30 minutes, followed by cooling at room temperature for 30 minutes in the same Coplin staining jar. Furthermore, in this case, 12 different PC-MT antibodies were used (see Table 1 for PC-MT antibody assignments), with 0.5 μg / mL of antibody used for the tissue probe. Finally, for double labeling of pan-cytokeratin antibody (CK) with both PC-MT and fluorescence, PC-MT labeling reagent was added and allowed to react for 1 hour, followed by the addition of DyLight 650 NHS ester reagent (a 15-fold molar excess from a 5 mM stock in DMF) and an additional 1 hour of reaction, followed by the remaining antibody labeling procedure detailed in Example 1.

[0214] result

[0215] The inventors developed a 12-plex biomarker panel suitable for assessing the breast cancer tumor microenvironment, specifically, breast cancer-associated 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); T cell subset biomarkers CD3 (T cells), CD4 (T helper), CD8 (cytotoxic T cells), and CD45RO (memory T cells); B cell biomarkers CD20 and CD68; and biomarkers for tumor-infiltrating lymphocytes (TILs) and other immune-related cells, including biomarkers for macrophages and other mononuclear phagocytes [Blom, Paavolainen et al. (2017) Sci Rep 7:15580; Chistiakov, Killingsworth et al. (2017) Lab Invest 97:4-13, Haise, Colebatch et al. (2018) Sci Rep 8:11158, Poh and Ernst (2018) Front Oncol 8:49; and finally, a pan-cytokeratin (CK) antibody as a general epithelial cell biomarker [Karantza (2011) Oncogene 30:127-38] and a histone H2A.X antibody as a nuclear biomarker [Rogakou, Pilch et al. (1998) J Biol Chem 273:5858-68]. Importantly, it should be clarified that the purpose of these studies was not to validate a given biomarker for the detection of specific cell types or cancer types, nor to determine the effectiveness of the biomarker in identifying specific disease states. The purpose was to demonstrate the functionality and utility of the novel MIHC method presented herein.

[0216] Each antibody was directly labeled with a unique PC-MT (see Table 1 for PC-MT assignments for each antibody and mass reporter mass). To eliminate bias from variable MALDI-MS ionization efficiencies, which would otherwise arise with different PC-MT amino acid sequences, eight PC-MTs were constructed either from the same sequence consisting of mass unit 1 (see Table 1 for all mass units) or various stable isotopes (mass units Iso-1.1 to Iso-1.5, Iso-1.7, and Iso-1.8). The remaining four PC-MTs were also constructed from a core sequence of mass unit 1, but with terminal extensions of one to three glycine and / or serine amino acids (mass units 1.2 to 1.5), which are not expected to significantly alter MALDI-MS ionization efficiencies.

[0217] Furthermore, to enhance the MIHC procedure, we dual-labeled the CK antibody with PC-MT and a fluorophore. The ability to combine traditional 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-plex. First, such a combination would aid in method development and antibody probe validation. Furthermore, because the spatial resolution of MALDI-MSI has yet to match that of optical imaging, even high-resolution non-multiplexed, registered fluorescence images can be used to aid in the deconvolution of observed structures within highly multiplexed MALDI-MS images. This can be achieved by staining adjacent tissues (one for fluorescence and one for MALDI-MSI) or by sequential procedures on the same tissue section; however, both methods are tedious because they introduce many additional steps and variables that can lead to inaccurate registration of MALDI-MS and fluorescence images. To overcome this limitation, we sequentially labeled the CK antibody with two different amine-reactive NHS-ester-activating reagents: first, PC-MT, and then a fluorescent reagent. The PC-MT labeling reagent was used in a 10-fold molar excess over the antibody, and the fluorescent reagent was used in a 15-fold excess. (Note that antibodies contain up to 80 available lysine amino acids for labeling with NHS-ester-activating reagents [Mueller, Wrasidlo, et al. (1988) Hybridoma 7:453-6].) Using this dual-labeled antibody as part of a panel, tissue sections were stained once with the multiplex antibody mixture, followed by fluorescent imaging, PC-MT photocleavage, MALDI-MS matrix application, and MALDI-MSI on the same tissue sections.

[0218] First, we validated most of the antibodies in the 12-plex panel using human tonsillar tissue. Tonsillar tissue is frequently used as a positive control for immune cell CD markers, including B cells (Kalina, Fiser et al. (2019) Front Immunol 10:2434) and T cells (Sada-Ovalle, Talayero et al. (2012) Clin Exp Immunol 168:200-6; Geissler, Markwart et al. (2017) PLoS One 12:e0183214) (Kap, van Meurs et al. (2009) J Histochem Cytochem 57:1159-67; Kalina, Fiser et al. (2019) Front Immunol 10:2434), and is known to be strongly positive for Ki67 (Hsu, Yang et al. (2013) Histopathology 63:810-6). Figure 14a shows a CK immunofluorescence image (5 μm image resolution using a GenePix 4200A microarray scanner) of a whole tissue section. As expected, the CK antibody selectively stains the squamous epithelial layer that covers the tonsil and lines its many invaginations and crypts. Figure 14b shows the corresponding MALDI-MS image of CK PC-MTs on the same tissue section, producing an identical pattern. (The Caprioli and Spengler groups have developed techniques that can achieve resolutions of approximately 1-2 μm, but a resolution of 10 μm is used here, which is the limit of the Bruker rapifleX instrument used in this study [Zavalin, Todd et al. (2012) J Mass Spectrom 47:i; Kompauer, Heiles et al. (2017) Nat Methods 14:90-96].)

[0219] Figure 14c shows a multicolor MALDI-MS image corresponding to PC-MTs from selected biomarkers (because it is impractical for the human eye to distinguish between multiple overlaid colors, we show selected biomarkers that generate distinctive patterns here as an example; instead, see Figure 14d separately 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 visualization of this biomarker in germinal centers). This is expected, as germinal centers are known to contain proliferating B cells [MacLennan (1994) Annu Rev Immunol 12:117-39]. The strong Ki67 staining in germinal centers is also consistent with previously reported results using standard H4C staining [Hsu, Yang et al. (2013) Histopathology 63:810-6]. In contrast, T cells (e.g., CD3 and CD45RO, red and orange in Figure 14c, respectively) predominate in the extrafollicular region, again 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 distinct regions within the tonsillar crypts, in the periepithelial and intraepithelial regions (green in Figure 14c). This is understandable, as tonsillar crypts are known to harbor or capture microorganisms and pathogens [Jensen, Fago-Olsen et al. (2013) PLoS One 8:e56418; Rieth, Gill et al. (2018) JAMA Otolaryngol Head Neck Surg 144:231-237]. Figure 14d shows all 12 antibodies separately on a representative small area of ​​a tissue section using a gradient color scale.The "blank" corresponds to a PC-MT-labeled isotype control IgG (the same PC-MT as the CD3 antibody) used to stain a separate but adjacent tissue section, providing no detectable signal. Conversely, all CD antibodies were positive to varying degrees, as were CK, Ki67, and histone antibodies, as expected, many of which showed distinct patterns. HER2 and PR were negative. ER was weakly positive, which was not unexpected in a previous report using standard IHC [Shirasaki, Watanabe et al. (2003) International Congress Series 1257:115-118]. ER was detected in all four tonsillar tissues evaluated, whereas PR was not. (It is also worth noting that the tonsils shown in Figure 14 were of female origin.)

[0220] Next, we applied a 12-plex antibody panel to breast cancer FFPE tissue sections to demonstrate the utility and further validate the PR, ER, and HER2 antibodies, which were generally negative in tonsillar tissue (except for the bright ER staining discussed above). For the tissue shown in Figures 15a-c, the clinical annotation from the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma (ductal) of the breast, TNM staging of pTlcpN3apMX, minimum staging IIIC, 75% tumor, and PR- / ZER- / HER2+ by conventional IHC. Figure 15a again shows a multicolor MALDI-MS image overlay of selected biomarkers displaying differential patterns. The tumor is identified by areas staining positive for both CK (red, an epithelial marker) and HER2 (light green), with colocalization of the two colors often appearing yellow-orange. Distinct patches of CD20B cells (dark green) are observed in the extratumoral and peritumoral regions. As in the tonsils, CD8+ cytotoxic T cells (cyan) are not widely distributed throughout the tissue, but bright staining is observed in distinct areas, including those infiltrating within 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 some forms of breast cancer [Vihervuori, Autere 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]. There is also abundant CD68 staining (purple) in the extratumoral and peritumoral regions, 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 often comprise up to 50% of the tumor mass [Poh and Ernst (2018) Front Oncol 8:49].The presence of tumor-associated macrophages (TAMs) can indicate a positive or negative prognosis for various solid tumors, but is usually negative due to their tumor-promoting activities, such as immunosuppression and promotion of angiogenesis and inflammation (reviewed in 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]).

[0221] Figure 15b again shows MALDI-MS images of all 12 antibodies separately, as well as a "blank" performed as previously described (in this case, on an adjacent breast cancer tissue section). HER2 is positive as previously noted, but PR is negative, both of which are consistent with the pathology report of the biospecimen. However, ER shows positive staining, which is in the extratumoral region, but the tumor itself is not ER+, again consistent with the pathology report. To illustrate this, for simplicity, Figure 15c shows a multicolor overlay of only three biomarkers (CK, HER2, and ER) shown in primary colors. The tumor is indicated by colocalized CK (colored blue in this case) and HER2 (green) staining, while ER staining (red) is almost exclusively restricted to the extratumoral region of the tissue section.

[0222] Finally, to further validate the PR, ER, and HER2 antibodies, a second breast cancer tissue specimen was analyzed. In this case, the clinical annotation from the pathology report provided by the biospecimen vendor (OriGene) was as follows: adenocarcinoma of the breast, ductal, lobular, metastatic, TNM staging of T2N2aMX, minimum staging IIIA, 95% tumor, and PR+ / ER+ / HER2- by conventional IHC (i.e., the PR / ER / HER2 profile is the reciprocal of the previous tissue). The MALDI-MS image in the top panel of Figure 15d again shows a three-color image overlay using primary colors for simple visualization of PR, ER, and CK in this case. Both PR (green) and ER (red) are strongly positive, and colocalization with the CK epithelial biomarker (blue) produces a white color in many areas (occurring when all three colors are of similar intensity). Figure 15d (bottom panel) also shows CK, PR, ER and HER2 separately, again each with a gradient color, showing PR+ / ER+ / HER2- in perfect agreement with the pathology report.

[0223] Example 10. Gold-coated microscope slides for improved tissue adhesion in mass spectrometry imaging

[0224] background

[0225] Conductive slides are required for most forms of MSI, including most forms of MALDI-MSI. Indium tin oxide (ITO)-coated glass slides are commonly used, and gold-coated slides are also used for their conductive properties; in some cases, gold is suitable for chemical modification (Chaurand, Cornett, et al. (2011) Mol Cell Proteomics 10:O110 004259; Yang and Caprioli (2014) J Mass Spectrom 49:417-22). However, these are used for direct MSI applications, whereby tissue is mounted, a matrix compound is optionally applied (for MALDI-MS), and MSI is performed. Therefore, there is little or no liquid-phase processing of the slides. In contrast, our methods (mass spectrometry-guided immunohistochemistry and mass spectrometry-guided in situ hybridization, MIHC and MISH) are similar to traditional IHC or ISH, but use PC-MT probes instead of fluorogenic or chromogenic probes, and MSI (and related procedures) instead of light microscopy (see Figure 16 for a comparison of IHC / ISH, direct MSI, and MIHC / MISH protocols). Therefore, MIHC and MISH require the slide to both be conductive (unlike IHC and ISH) and provide stronger tissue adhesion than is required for direct MSI. We found that this is best achieved with a gold surface, which, to our knowledge, has not previously been reported for use in IHC-style and ISH-style procedures using PC-MT probes and MSI.

[0226] method

[0227] The procedure was carried out as in Example 2, except that the tissues were mounted on glass slides (standard microscope slide dimensions) with different conductive coatings. These were the following indium tin oxide (ITO) slides (Bruker Daltonics, Billerica, MA), ITO slides silanized using (3-aminopropyl)triethoxysilane as the silanizing reagent according to published protocols [Qin, Hou et al. (2007) Colloids Surf B Biointerfaces 60:243-9], ITO slides coated with poly-L-lysine solution according to the manufacturer's instructions (0.01%, sterile filtered, bioreagent for molecular biology, suitable for cell culture, Millipore-Sigma, St. Louis, MO), ITO slides coated with chrome aluminum 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), commercially available silver-coated slides (Platypus Technologies LLC, Madison, WI), and two types of gold-coated slides (Platypus Technologies, Madison, WI). The samples were prepared using glass slides with a 10 nm gold layer and a 2 nm titanium adhesive sublayer from Angstrom Engineering Inc., LLC, or a 50 nm gold layer and a 5 nm chromium adhesive sublayer from Substrata Thin Film Solutions / Angstrom Engineering Inc., Ontario, Canada. Tissue damage or loss was assessed visually throughout the antigen retrieval process.

[0228] result

[0229] Severe tissue damage or tissue loss was observed to varying degrees in all cases, except for both types of gold-coated slides, where little or no tissue damage was observed. While damage could occur during any of the solution-phase processing steps, it was more likely to occur or initiate during the heat-mediated antigen retrieval step. Figure 17 shows visible light images of damaged mouse brain tissue sections on ITO slides after processing, as well as an example of an intact tissue section on a gold slide.

[0230] Example 11. Requirement of matrix sublimation and subsequent matrix recrystallization in mass spectrometry-based immunohistochemistry (MIHC)

[0231] background

[0232] Those skilled in the art will recognize that co-crystallization of the analyte with an excess of an exogenously added matrix compound to facilitate absorption and transfer of the mass spectrometer's laser energy to the analyte is generally required for efficient analyte vaporization / ionization and detection in most forms of laser-based mass spectrometry (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]. This is referred to as matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). Although matrix-free LDI methods 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], their use is generally limited to specific applications (e.g., drug detection using DIOS, for which non-laser-based methods are generally preferred). Furthermore, these matrix-free methods lack sensitivity. Many methods of matrix application in MALDI mass spectrometry imaging (MALDI-MSI) exist, some examples being airbrushing, automated commercial sprayers, and sublimation [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 one method of matrix application, or even the need for a matrix at all, a preferred embodiment uses 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 affords both excellent spatial resolution (i.e., provided by sublimation, which limits analyte delocalization during matrix application due to the lack of a liquid phase) and high sensitivity (i.e., provided by vapor-based recrystallization, which allows the analyte [mass reporter of PC-MT] to sufficiently cocrystallize with the matrix but without significant analyte delocalization).

[0233] method

[0234] The same procedures as in Example 2 were carried out except that only the PC-MT anti-NeuN antibody (probing concentration: 1 μg / mL) was used, and the presence or absence of recrystallization was compared.

[0235] result

[0236] MIHC with PC-MT-labeled anti-NeuN antibodies using MALDI-MSI on mouse brain tissue sections, using matrix (DHB) sublimation with and without recrystallization, is shown in Figure 18 (showing colorized MALDI-MS images) (see Examples 1-2 for methods). Figure 18 shows that a strong signal and a typical anti-NeuN pattern are observed in mouse brain tissue sections (see Example 2) when recrystallization is applied. Without recrystallization, no signal is visually detected in the image. To quantify the effect of matrix recrystallization after sublimation, average spectra were generated from each of the entire regions of interest (see dotted outlines in Figure 18) for each tissue section. From these average spectra, the monoisotopic peak intensities of PC-MT were 55.4 and 2.5 with and without recrystallization, respectively. Importantly, this demonstrates that for the MALDI-MSI step of MIHC, matrix recrystallization after sublimation is necessary for robust PC-MT signals.

[0237] Example 12. Mass spectrometry-based immunohistochemistry (MIHC) for multiplexed imaging of biomarkers associated with anti-cancer immune checkpoint inhibitor drugs

[0238] background

[0239] The normal biological function of immune checkpoint pathways is, for example, the maintenance of peripheral immune tolerance by suppressing T cell responses. Evidence of this is that mice lacking key proteins in the immune checkpoint pathway (CTLA-4 or PD-1) develop 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 suppress T cell signaling and activation as a form of negative feedback [Sharma and Alison (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 immune suppression can also block beneficial antitumor immune responses, and in some cases, tumors themselves can hijack immune checkpoints to their advantage. For example, tumor cells can express the PD-L1 ligand, which binds to PD-1 on activated T cells and suppresses antitumor immune responses [Sharma and Alison (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]. Currently, there are several antibody therapeutics, such as pembrolizumab (sold as Keytruda® by Merck, Kenilworth, New Jersey), which bind to PD-1 and prevent its interaction with PD-L1 / 2, thereby preventing immunosuppression (similarly, therapeutic antibodies against PD-L1, for example, 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].Current FDA-approved immune checkpoint inhibitor drugs include ipilimumab, which is specific for CTLA-4; nivolumab, pembrolizumab, and cemiplimab, which are specific for PD-1; and atezolizumab, avelumab, and durvalumab, which are specific for PD-L1 [Vaddepally, Kharel et al. (2020) Cancers (Basel) 12]. Although these drugs have achieved groundbreaking status [Darvin, Toor et al. (2018) Exp Mol Med 50:1-11] and are effective against a variety of cancers [Sharma and Alison (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, understanding and predicting which patients will respond and to which of the available treatments remains problematic. Therefore, it is important to understand the expression of various known immune checkpoints and related molecules both in the tumor itself and in infiltrating immune cells within 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, methods for highly multiplexed IHC, such as the MIHC technology of the present invention, will be important clinical tools.

[0240] method

[0241] MIHC is performed similarly to Examples 2 and 9, except that the 12-plex antibody panel detailed in Example 9 for breast cancer biomarkers and biomarkers of infiltrating immune cells is further expanded to include PC-MT antibodies against the following biomarkers related to 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 the panel, these novel antibodies are labeled with PC-MT (see Table 1) containing 7 to 14 mass units, for a total 20-plex antibody panel, using the methods from Examples 1, 2, and 9.

[0242] Antibodies for PC-MT labeling were commercially available from ABCAM (Cambridge, MA) and included the following: recombinant anti-PDl 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 (ab215255). ab251599), human OX40 antibody pair - BSA and azide-free (TNFRSF4) (ab244076) (test both antibodies and select the best), recombinant anti-CD27 antibody [EPR8569] - BSA and azide-free (ab256583), recombinant anti-CD28 antibody [EPR22076] - BSA and azide-free (ab243557), recombinant anti-TIM3 antibody [EPR22241] - BSA and azide-free (ab242080).

[0243] As an example, MIHC is performed on FFPE breast cancer tissue as described in Example 9, except using the entire 20-plex antibody panel described above. However, the present invention is applicable to any tumor type, including but not limited to tissue prepared as FFPE (formalin-fixed paraffin-embedded) or FF (fresh frozen).

[0244] result

[0245] This approach is expected to enable simultaneous multiplexed imaging of cancer biomarkers, biomarkers for infiltrating immune cells, and biomarkers for immune checkpoints (similar to Example 9, but with the additional biomarkers mentioned above), providing pathologists with important information for cancer diagnosis, prognosis, and guidance of therapy, such as immune checkpoint inhibitors.

[0246] Example 13. PC-MT lectin for targeted multiplexed mass spectrometry imaging of glycans.

[0247] background

[0248] This example describes the use of PC-MT-labeled lectin probes for tissue MSI. Lectins are a type of carbohydrate-binding protein typically derived from plants (see above). PC-MT-labeled lectin probes can be used for MSI of N- and O-glycans in tissues, but O-glycans are particularly important because, unlike N-glycans, no glycosidases are suitable for in situ tissue digestion followed by direct, label-free MSI. Rather, O-glycans must be successively chemically degraded until only the core Gal-β(1→3)-GalNAc carbohydrate remains, at which point O-glycosidases can be used to remove the core; however, these procedures are not suitable for in situ digestion and direct, label-free MSI of tissues [Poiroux, Barre et al. (2017) Int J Mol Sci 18; Wilkinson and Saidova (2020) J Proteome Res 19:3890-3905]. PC-MT-labeled lectin probes circumvent this problem, thus facilitating highly multiplexed MSI targeting both N- and O-glycans in tissues. Importantly, lectins exist not only to bind to N- and O-glycans [Tsaneva and Van Damme (2020) Glycoconj J 37:533-551], but also to differentiate O-glycans from N-glycans. For example, peanut (Arachis hypogaea) agglutinin 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]. Jacalin lectin from Artocarpus integrifola and Agaricus bisporus lectin are also specific for the O-glycan T / Tn antigen [Poiroux, Barre et al. (2017) Int J Mol Sci 18].O-glycans are particularly important because alterations in 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, may be combined, and all types of PC-MT probes may be combined with other "omic" MSI approaches (e.g., direct label-free metabolomics MSI).

[0249] method

[0250] The lectins were labeled with PC-MT in the same manner as the antibodies in Example 1, with the following modifications: A 10-fold molar ratio of PC-MT labeling reagent to probe was maintained; therefore, the amount of PC-MT labeling reagent was adjusted accordingly, since the lectin has a different molecular weight than the antibody. Instead of using a PD SpinTrap G-25 column, removal of unreacted PC-MT labeling reagent in this case was achieved by washing six cycles with 450 μL of TBS using an Amicon Ultra-0.5 centrifugal filter unit (Millipore-Sigma) consisting of an Ultracel regenerated cellulose membrane according to the manufacturer's instructions. The nominal molecular weight limit (NMWL) of the filter unit was 10 kDa for wheat germ agglutinin (WGA, Millipore-Sigma) and Phaseolus vulgaris agglutinin (PHA-E4, amsbio) lectins, and 50 kDa for peanut agglutinin (PNA, Millipore-Sigma) and Dolichos biflorus agglutinin (DBA, Millipore-Sigma) lectins. Each lectin was labeled with a unique PC-MT as follows: PC-MT mass units ID1, Iso-1.2, Iso-1.3, and 1.2 for WGA, PNA, PHA-E4, and DBA lectins, respectively (see Table 1 for details).

[0251] Subsequent tissue processing of sagittal FFPE sections of mouse brain was performed similarly to Example 2, except that the antigen retrieval step was omitted because it was not necessary in this case. Therefore, the processing steps performed were deparaffinization, hydration, blocking, probing / staining (in this case, with PC-MT lectin), washing, drying, photocleavage, DHB matrix sublimation / recrystallization, and MALDI-MSI (immunofluorescence was not performed). Note that antigen retrieval can still be used, for example, when combining a PC-MT lectin probe with a PC-MT antibody probe. For multiplex probing / staining of tissue sections, all four aforementioned PC-MT-labeled lectins were mixed together, with concentrations ranging from 1 to 20 μg / mL. In some cases, the PC-MT lectin probe / staining mixture was supplemented with the soluble sugar N,N',N"-triacetylchitotriose (TCT, which binds to WGA lectin) at a concentration of 1 mM and preincubated for 30 min before tissue probing / staining (the soluble sugar remained present during tissue probing / staining). This was to confirm the specificity of PC-MT lectin (WGA) staining by competitive inhibition.

[0252] result

[0253] Figure 19a shows colorized mass images of the monoisotopic mass spectral peaks of the PC-MT reporter corresponding to three lectins: PHA-E4, PNA, and WGA. The results are consistent with a previous literature report 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 predominant in the choroid plexus, along with some WGA staining, as observed here using PC-MT lectin and MSI (the choroid plexus is shown in Figure 19a; see also Figure 19b). On the other hand, in Kitada et al., PNA lectin preferentially stained the white matter of the brain, i.e., myelinated axons. Thus, the PC-MT PNA lectin staining here (green in Figure 19a) is consistent with the previously observed PC-MT myelin antibody staining, as expected (see Example 2, Figure 7a, red for previous PC-MT myelin antibody staining).

[0254] Finally, to demonstrate the specificity of PC-MT lectin binding to tissue, the PC-MT lectin probe / stain mixture was preincubated with 1 mM of the soluble sugar N,N',N"-triacetylchitotriose (TCT), which remained present during the tissue probe / stain step for competitive inhibition (blocking) of WGA binding (because TCT specifically binds WGA [Damm, Mikkat et al. (2004) Pancreas 28:31-7]). As observed in the PC-MT mass image in Figure 19b, when TCT blocking was not used, predominant WGA staining was observed (blue in Figure 19b). When TCT blocking was used, WGA staining was selectively inhibited, but staining with the other lectins, PHA-E4 (red) and PNA (green), was still observed. To quantify this result, ensemble average spectra were obtained from the entire tissue section, with and without TCT blocking (Figure 19c). With TCT blocking (orange trace), the WGA PC-MT signal intensity was reduced by 70% compared to without TCT blocking (purple trace), whereas the peak intensities of the other two lectins, PNA and PHA-E4, were virtually identical with and without TCT blocking, thereby demonstrating specificity.

[0255] Example 14. Improved multi-omic MSI: Untargeted unlabeled MSI and targeted MIHC of lipids on the same FF tissue section

[0256] method

[0257] This example was performed identically to Example 4, using sagittal FF tissue sections of mouse brain, with some enhancements for better tissue fixation and better removal of unfixed small endogenous organic compounds after the initial direct unlabeled MALDI-MSI (in this case of endogenous lipids) before the subsequent MIHC step with a PC-MT labeled antibody (PC-MT-Ab). Thus, after the initial direct unlabeled MALDI-MSI of endogenous lipids, the tissue sections were subjected to the following (each processing step in a separate staining jar): Prewashing was performed twice with -80 °C acetone for 3 min each (note that this helps remove residual matrix compounds from previous MALDI-MSI while providing solvent fixation), followed by vacuum application for 10 min, followed by fixation for 30 min using 1% PFA in PBS, pH 7.4 (note that this solution was freshly prepared by dissolving 1.0 g of PFA 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 [approximately 1 mL] and adjusting the final volume to 100 mL with PBS), followed by one wash for 10 min with PBS, two washes for 3 min each with room temperature acetone, and one wash for 3 min with Carnoy's solution (6:3:1 EtOH / chloroform / acetic acid) for further fixation to further remove unfixed endogenous organic compounds. The remaining steps of the PC-MT-Ab-based MIHC were performed as in Example 2, from the antigen retrieval step to the end.

[0258] result

[0259] Mass images are shown in Figure 20. Figure 20a is the initial direct unlabeled MALDI-MSI of endogenous lipids (see Figure 20a where exemplary lipids are displayed), Figure 20b is the subsequent MIHC showing selected antibody PC-MT (see Figure 20b where antibody PC-MT is displayed), and Figure 20c is an image composite between selected analytes from the initial direct unlabeled MALDI-MSI and subsequent MIHC (see Figure 20c). The expected colocalization of the lipid sulfatide (ST) with myelin, but not between ST and NeuN, is described in Example 4.

[0260] Example 15. Multi-omic tissue imaging: MIHC combined with bottom-up proteomic MSI.

[0261] The detailed description of the invention under the section headed "Multi-omic Tissue Imaging Using PC-MT-Probes" describes a wide range of permutations of multi-omic imaging approaches incorporating PC-MT probe technology. The following examples illustrate some permutations of such approaches, but are not intended to limit the scope of the invention.

[0262] method

[0263] Breast cancer FFPE tissue sections are used as in Example 9.

[0264] In one embodiment, in situ tissue glycan digestion using glycosidase PNGase F followed by direct, unlabeled MALDI-MSI of the released glycans is performed according to Drake et al. [Drake, Powers et al. (2018) Curr Protoc Protein Sci 94:e68]. After MALDI-MSI, the MALDI-MS matrix is ​​then removed by washing twice with room temperature acetone (in a staining jar) for 3 minutes each, and the tissue is then completely dried under vacuum for 10 minutes. Next, 12-plex MIHC is performed to detect macromolecular protein biomarkers of breast cancer and tumor-infiltrating lymphocytes / immune cells, as in Example 9 (except that the deparaffinization step in Example 9 is omitted, as it has already been performed according to the procedure in Drake et al.).

[0265] In another embodiment, (targeted) MIHC is performed first, followed by in situ protease digestion of the tissue sections and direct untargeted, unlabeled MALDI-MSI of the released proteolytic fragments (derived from endogenous tissue proteins). See, for example, Figure 22. In this case, MIHC is performed again as in Example 9, with MALDI-MSI as the final step. The MALDI-MS matrix is ​​then removed by washing twice with room temperature acetone (in a staining jar) for 3 minutes each, and the tissue is then completely dried under vacuum for 10 minutes. Subsequent in situ protease (trypsin) digestion and direct unlabeled MALDI-MSI of the released proteolytic fragments are performed according to Lazova et al. [Lazova, Smoot et al. (2020) J Cutan Pathol 47:226-240], except that the deparaffinization and antigen retrieval steps are omitted because they are already performed in each MIHC process. Finally, in another embodiment, it may be desirable to remove the antibody (and proteinaceous blocking agent) from the initial MIHC process prior to subsequent in situ proteolytic digestion for direct, unlabeled MALDI-MSI (so that only proteolytic fragments derived from endogenous tissue proteins are detected). In this case, the MALDI-MS matrix from the final step of the MIHC process is again removed by washing twice with room temperature acetone for 3 minutes each (as in all steps in staining jars). However, the tissue is then subjected to a 5-minute denaturation treatment at 65°C in a solution of 1% (w / v) SDS detergent in 50 mM Tris, pH 7.4, containing 1 mM dithiothreitol (DTT). This denaturation treatment helps to separate the antibody probe (and proteinaceous blocking agent) from the tissue. This is followed by two 3-minute washes in plain 50 mM Tris, pH 7.4, followed by two 3-minute washes in room temperature acetone (which further aids in complete removal of the SDS detergent from the tissue). The tissue is then dried completely under vacuum for 10 minutes.Subsequent in situ protease (trypsin) digestion and direct unlabeled MALDI-MSI of the proteolytic fragments is again performed according to Lazova et al. [Lazova, Smoot et al. (2020) J Cutan Pathol 47:226-240] (but again, the deparaffinization and antigen retrieval steps of Lazova et al. are omitted as they are already performed per the MIHC process).

[0266] result

[0267] Combining MIHC results with bottom-up MSI approaches yields much greater spatial information content from tissues compared to either approach alone. This is expected to yield better biomarker "signatures," as determined by, for example, Lazova et al.'s machine learning statistical approach, that can be used, for example, in cancer diagnosis, staging, prognosis, subtyping, and predicting optimal treatment pathways for improved patient outcomes in tumor tissue.

[0268] Example 16. PC-MT probes in non-imaging mass spectrometry applications.

[0269] background

[0270] PC-MT probes are not limited to mass spectrometry imaging applications and can also be used in conjunction with non-imaging mass spectrometry (MS) applications. For example, a group of PC-MT probes can be applied to heterogeneous biological samples, including, but not limited to, tissues or excised tissue sections, tumors, cells derived from tumors (or from any tissue), liquid biopsies, cell cultures, blood and other bodily fluids, bacterial cells, and cultured infectious pathogens, to identify the presence of components in the heterogeneous sample. For example, pathogens present in a biological sample can be identified using one or more PC-MT antibody (PC-MT-Ab) probes targeting antigens characteristic of the pathogen or pathogens. Readout is by detection of photocleaved (light-released) PC-MTs 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 (e.g., LC-MS). It will be recognized by those skilled in the art that further purification (e.g., desalting) of the analyte may be required prior to mass spectrometry analysis. In one embodiment, antigens / epitopes specific to specific variants of the SARS-CoV-2 virus responsible for causing the COVID-19 pandemic can be identified using PC-MT-Ab probes targeting these antigens / epitopes, such as spike protein antigens and / or epitopes thereof. In a second embodiment, specific antigens / epitopes present on tumor-infiltrating lymphocytes present 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 within the tumor, which can provide, for example, prognostic and therapeutically useful information. While spatial information is lost due to this non-imaging approach, those skilled in the art of immunodiagnostics, including ELISA and flow cytometry, are well aware that 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 targeted approach to 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 a biological mixture). The following experimental example demonstrates the binding of a PC-MT probe (PC-MT-Ab) to a surface, followed by photorelease of the PC-MT and non-imaging mass spectrometry of the photoreleased PC-MT. In this case, the surface to which the PC-MT-Ab was bound was a bead, but ultimately, any type of PC-MT probe can be bound to any sample, such as those exemplified above.

[0271] method

[0272] PC-MT (mass unit Iso-1.1 in Table 1) and PC-MT-Ab were prepared in the same manner as in Example 1. In this case, purified rabbit IgG was used as a model PC-MT-Ab (see the "Materials for Experimental Examples" section).

[0273] The resulting 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 then the PC-MTs were photoreleased from the bead-bound PC-MT-Ab. Non-imaging MALDI-MS analysis was performed on the photoreleased PC-MTs present in the supernatant. The complete procedure was as follows: Unless otherwise noted, protein G agarose beads were processed in a 0.5 mL Ultrafree-MC Centrifugal 0.45 μm filter device (see the "Materials" section of the Example Experiment). (Note that washing was performed by briefly vortexing the beads in the wash solution, followed by a short spin at 15,000 rpm in a standard microcentrifuge, and then discarding the filtrate in the bottom chamber of the filter device while the washed beads were retained in the upper chamber of the filter device.) A bead pellet volume of 1 μL was used for each sample (each sample was processed in parallel in a separate filter device). The beads for each sample were first pre-washed four times briefly with 400 μL of OBG-saline (see Example 6 for formulation). Each washed bead pellet was then resuspended in 100 μL of PC-MT-Ab solution (prepared in OBG-saline at PC-MT-Ab concentrations of 0.625, 1.25, 2.5, 5, 7.5, and 10 μg / mL). The beads were gently mixed (protected from light) for 30 minutes to allow binding of the PC-MT-Ab to the protein G on the beads. The beads were then briefly washed four times with 400 μL of OBG-saline, followed by four times with 400 μL of mass spectrometry-grade water (MS-water) to remove unbound PC-MT-Ab.

[0274] The beads were then resuspended and transferred to a clear, thin-walled polypropylene PCR-style microcentrifuge tube in 100 μL of MS-water. The beads were then briefly spun down in a standard microcentrifuge at 15,000 rpm, after which approximately 80 μL of the supernatant was discarded, leaving approximately 20 μL of MS-water in the tube and the bead pellet. The beads were resuspended in the tube by brief mixing and spun at 25% power (30 mW / cm at 360 nm). 2 The beads were exposed to UV light for 5 minutes through the sidewall of the tube (the tube was placed on its side) using a Honle LED Cube 100IC (Honle UV Technology, Marlborough, MA). While the beads remained in the tube, 20 μL of the following solution was then added to each sample: 10 mg / mL alpha-cyano-4-hydroxycinnamic acid (CHCA, Sigma-Aldrich, St. Louis, MO), 80% acetonitrile, 0.2% (v / v) trifluoroacetic acid (TFA), and 25 femtomoles / μL of the sequence APRLRFYSL. (SEQ ID NO: 33) An unmodified control peptide with the formula (custom synthesized by GenScript, Piscataway, NJ) was then mixed for 15 minutes to allow for complete extraction of the photoreleased PC-MTs. The beads were then briefly spun down at 15,000 rpm in a standard microcentrifuge, and 2 μL of each sample supernatant (without beads) was then 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).

[0275] result

[0276] Figure 21a shows the spectra from non-imaging MALDI-MS analysis of photoreleased PC-MT from six samples corresponding to six different concentrations of PC-MT-Ab added to Protein G beads. The photoreleased PC-MT (reporter) and control peptide monoisotopic peaks are observed at m / z 1,210.6 and 1,122.6, respectively, as expected. To quantify the results, the ratio of the monoisotopic peak intensities of the photoreleased PC-MT relative to the control peptide (which was at a fixed concentration) was calculated and graphed in Figure 21b. A linear response as a function of PC-MT-Ab concentration is observed (R 2 =0.9779).

[0277] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention 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 connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art and related fields are intended to be within the scope of the following claims. In certain embodiments, for example, the following are provided: (Item 1) 1. A multiplex method for the simultaneous detection of five or more different types of biomarkers in a tissue sample on a single slide, comprising: a) providing a tissue sample on a single slide; b) contacting the tissue sample with five or more different antibodies to effect binding of the antibodies to the tissue sample, each of the antibodies reactive with a different biomarker, and each of the antibodies conjugated to a unique mass tag; c) detecting said mass tag or a fragment thereof as a molecular ion using mass spectrometry imaging. (Item 2) 2. The method of claim 1, further comprising performing direct mass spectromet...

Claims

1. 1. A multiplex method for the simultaneous detection of multiple different types of biomarkers in a tissue sample on a single slide, 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 reactive with a different biomarker, each of the antibodies conjugated to a unique photocleavable mass reporter comprising a mass tag, the mass reporter linked to a phenyl ring of the photocleavable core via a photocleavage moiety, such that the mass reporter does not remain bound to the photocleavable core upon photocleavage; c) irradiating the photocleavable mass tag with light prior to step d) to photocleave at least a portion of the mass tag; d) detecting said mass tag or a fragment thereof as a molecular ion using mass spectrometry imaging.

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

3. 10. The method of claim 1, wherein the plurality of different antibodies is 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 multiple amino acids.

6. The antibody conjugated to a photocleavable mass tag has the following general structure: 【Chemistry 11】 2. The method of claim 1, comprising:

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

8. 8. The method of claim 7, wherein the matrix compound is selected from the group consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), and 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid).

9. 8. The method of claim 7, wherein the tissue sample is subjected to a treatment after contacting the sample with a matrix compound, the treatment comprising matrix recrystallization.

10. The photocleavable mass tag conjugated to the antibody comprises a core structure conjugated to a mass unit, and the antibody has the following general structure: Mass Unit - Core Structure - Antibody 2. The method of claim 1, comprising:

11. The core structure has the structure: 【Chemistry 12】 11. The method of claim 10, comprising:

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

13. 1. A multiplex method for the simultaneous detection of multiple different types of biomarkers in a tissue sample, comprising: a) providing a tissue sample; 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 reactive with a different biomarker, and each of the antibodies conjugated to a unique photocleavable mass tag; c) irradiating the photocleavable mass tag with light prior to step d) to photocleave at least a portion of the mass tag; d) detecting said mass tag or a fragment thereof as a molecular ion using mass spectrometry imaging; wherein the antibody conjugated to a mass tag has the following general structure: 【Chemistry 13】 and wherein X and Y are optional chemical linkers and Z is a probe-reactive moiety after conjugation with said antibody, and wherein said mass tag does not remain attached to said photocleavable core upon photocleavage.

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