Mass tag labeling of cell secretome

Abstract

The present disclosure provides methods for mass tag labeling of cell secretomes and their soluble components, as well as mass-tagged soluble components of cell secretomes. In certain embodiments, the present disclosure provides methods for mass tagging of extracellular vesicles (EVs) and mass-tagged EVs. Methods of using mass-tagged soluble components of cell secretomes, such as mass-tagged EVs, mass-tagged viruses, or mass-tagged soluble proteins and peptides, are also provided. These can be combined with other labeling strategies, such as cell barcoding, that facilitate multiplex analysis and / or multidimensional analysis of the distribution, uptake, and effects of secretome components (such as EVs).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Patent Application No. 63 / 339,897, filed May 9, 2022, and U.S. Patent Application No. 63 / 345,781, filed May 25, 2022, which are hereby incorporated by reference in their entirety for all purposes.

[0002] The present disclosure generally relates to the field of mass - tag - labeled soluble cell - derived components having elemental isotope compositions. In particular, the present disclosure relates to the labeling of components of the cell secretome, such as extracellular vesicles (EVs), and related compositions and methods.

Background Art

[0003] The cell secretome encompasses a group of soluble proteins, extracellular vesicles, and other biomolecules secreted by cells into the extracellular environment. These molecules play important roles in cell - cell communication, tissue development, and disease progression. In recent years, there has been an increasing interest in the study of the cell secretome to better understand cell - to - cell communication and extracellular functions and to identify potential therapeutic targets.

[0004] Extracellular vesicles (EVs) are membrane - enclosed biological nanoparticles secreted by substantially all known cell types (1). As important actors in cell - to - cell communication and essential components of the cell secretome, EVs are increasingly being studied as therapeutic agents in several diseases [2, 3]. Furthermore, EVs can carry several layers of information such as RNA species, extracellular and intracellular proteins, and lipids (4). However, their exact biological functions, targets or recipient cells, and in - vivo distribution after in - vivo application remain mostly unknown (5). Due to the physical (small size, low refractive index) and biochemical (weak protein and RNA expression) properties of EVs (6), the investigation of the aforementioned unknowns mainly relies on EV labeling strategies (7).

[0005] Mass cytometry and imaging mass cytometry have emerged as powerful methods for analyzing cell phenotypes within heterogeneous cell populations. Approaches include antibody-based and mass tag-dependent specificities, as well as quantification of protein targets in complex biological samples using mass spectrometry of heavy metal isotopes. Single-cell technologies based on mass spectrometry, such as cytometry by time of flight (CyTOF) or imaging mass cytometry, are widely used for cell and tissue analysis but have not yet been used for receptor cell analysis of various components of the cell secretome.

[0006] (Imaging) mass cytometry analysis, several mass tagging techniques have been developed to label whole cells or to measure their diverse intracellular functions. Mass tag labeling is mainly used to examine the phenotype or cell function of target cells.

Prior Art Documents

Patent Documents

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Non-Patent Documents

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[0009] To study the interaction and effect of cell secretome components such as EVs on the phenotype and function of recipient cells, a mass - tagging approach requires uniform and normalized labeling of various secretome components such as extracellular vesicles (EVs), soluble proteins (e.g., antibodies, hormones, cytokines, and enzymes), and viruses. A prerequisite for one approach to this uniform labeling is metabolic labeling of secretome - producing cells.

[0010] For EVs, in most cases, lipophilic fluorescent dyes, protein-labeling probes, or gene-labeling approaches are used to analyze the effects of EV uptake on cell function and signaling in several in vivo and in vitro models (7). None of the tagging strategies are suitable for high-dimensional single-cell analysis of recipient cells in vitro and in vivo, and exhibit even broader limitations that complicate the study of the functional effects of EVs on recipient cells (8): Lipophilic dyes and fluorescent probes often self-aggregate, causing the artificial formation of EV-like structures taken up by cells and tissues and resulting in false-positive signals (9). Gene-labeling strategies are generally limited to a single protein that is not present in all secreted EV subpopulations (e.g., CD9, CD63, or CD81) and cannot be used in most primary cell types (7). Furthermore, fluorescent gene tags are large and can spatially interfere with protein-protein interactions and thus interfere with EV uptake and function. Other labeling strategies, particularly those for use in high-dimensional single-cell analysis, are desired.

[0011] Cell components can be labeled by mass tagging. However, it has never been demonstrated that mass-tagged components of the cell secretome are actively secreted after being mass-tagged intracellularly and can then be detected in the extracellular space and recipient cells. The secretion process may be disrupted by the incorporation of mass tags, which can cause protein binding and phenotypic changes. Furthermore, for the same reason, it remains unclear whether mass-tagged components of the cell secretome, an additional requirement for performing cell secretome recipient cell analysis by mass cytometry and respective imaging techniques, can be internalized by recipient cells.

[0012] In the case of EVs, we aimed to overcome most of the aforementioned limitations by developing a mass tag labeling approach for the cellular secretome, such as EVs, that can be tracked by high-dimensional single-cell mass cytometry and mass cytometry imaging according to the MISEV2018 EV standard (10). We demonstrated that EVs can be mass-tagged without substantially altering the MISEV2018 characteristics. We also found that our mass tag labeling is suitable for labeling all parts of the cellular secretome, including EVs, secreted soluble proteins (e.g., antibodies, cytokines, hormones, etc.), viruses, and intracellular pathogens.

[0013] Direct labeling of extracellular vesicle subpopulations has been carried out based on nucleic acid intercalation (e.g., intercalator-Ir / Rh, IdU) or alkylation (e.g., DDP, cisplatin), but the reported labeling approaches have significant drawbacks that are avoided by proteomic and metabolic mass tag labeling approaches for the cellular secretome, including the following:

[0014] Existing labeling approaches for extracellular vesicles (EVs) are not suitable for the simultaneous labeling of various components of the cellular secretome and label only a small part of a single component of the cellular secretome - DNA+EV-. In contrast, our mass tag labeling approach can uniformly co-label various components of the cellular secretome, such as EVs and soluble proteins, and label all EV subpopulations, in contrast to DNA-based labeling methods.

[0015] Previously reported direct labeling methods require EVs that are permeabilized by electroporation, are time-consuming, expensive, impractical, affect morphology, and cause EV aggregation. Our mass tag labeling approach for the cellular secretome does not require electroporation or any other EV modification prior to metabolic labeling in the producer cells.

[0016] Analysis of a single component of the secretome is often biased by the presence of contaminants from other components of the cellular secretome. (For example, the) biological effects of antibodies or biologically active peptides cannot be compared among several components of the cellular secretome when only a single component of the cellular secretome is labeled. By using our mass tag labeling approach for the cellular secretome, proteins within the secretome are uniformly labeled and normalized based on the Gaussian distribution of mass tag incorporation within the producer cells.

[0017] The novel mass tag labeling of the cellular secretome described herein addresses the limitations of existing methods, as outlined above. This new approach offers the following advantages:

[0018] Improved labeling efficiency: Our method ensures more complete labeling of secreted peptides and proteins, resulting in more accurate quantification and comparison.

[0019] Enhanced multiplexing ability: Our approach enables simultaneous analysis of multiple samples, increasing throughput.

[0020] Simplification of operations and cost reduction: The proposed method streamlines the labeling process and reduces associated costs.

[0021] Compatibility with mass spectrometry, imaging mass spectrometry, electron microscopy-based techniques, and other mass spectrometry-based single-cell and imaging technologies: Our method is specially designed to work smoothly with mass spectrometry-based single-cell and imaging technologies as well as electron microscopy, providing solutions tailored to the recipient cells and mass tag-labeled components of the cellular secretome.

[0022] Adaptability: The novel mass tag labeling approach can be easily adapted to various experimental settings and sample types, providing an easy-to-apply option for researchers as opposed to electroporation-based techniques for labeling EVs.

[0023] Enhanced sensitivity and specificity: Our method provides increased sensitivity and specificity, enabling the simultaneous detection of low-protein-containing components such as EVs and secreted soluble proteins.

[0024] The various embodiments contemplated herein include, but are not limited to, one or more of the following:

[0025] Embodiment 1: Extracellular vesicles (EVs) in which the components of the EVs are labeled with at least one mass tag.

[0026] Embodiment 2: A plurality of EVs according to Embodiment 1.

[0027] Embodiment 3: The plurality of EVs of Embodiment 2, including EVs from two or more samples.

[0028] Embodiment 4: The plurality of EVs of Embodiment 3, wherein the EVs from each different sample are distinguished by different detectable labels or combinations of detectable labels.

[0029] Embodiment 5: The plurality of EVs of Embodiment 4, wherein the different labels or combinations of labels include different mass tags or combinations of mass tags.

[0030] Embodiment 6: A method for producing a mass-tagged soluble component from a production cell, the method comprising exposing at least one production cell to a mass-tagged component that can be taken up by the production cell, and purifying the mass-tagged soluble component produced by the production cell.

[0031] Embodiment 7: The method of Embodiment 6, wherein the mass-tagged soluble component is selected from extracellular vesicles (EVs), virus particles, cell secretomes, EV proteomes or secretomes, or components of any of the foregoing.

[0032] Embodiment 8: The method of Embodiment 7, wherein the mass-tagged component is a mass-tagged EV.

[0033] Embodiment 9: A method according to any one of Embodiments 6-8, wherein the production cells are exposed to mass-tagged components under serum-free conditions.

[0034] Embodiment 10: A method according to any one of Embodiments 6-9, wherein the production cells are derived from a cell line optionally selected from HEK293T, HeLa, OSU-CLL, and PANC-1.

[0035] Embodiment 11: A method according to Embodiment 7 or Embodiment 9, wherein the production cells are primary cells, optionally derived from chronic lymphocytic leukemia cells.

[0036] Embodiment 12: A method according to any one of Embodiments 7-11, wherein the EVs are purified by a method comprising filtration, ultrafiltration, and size exclusion chromatography.

[0037] Embodiment 13: The method of Embodiment 12, wherein the filtration comprises 0.2 μM filtration, the ultrafiltration comprises 10 kDa ultrafiltration, and the size exclusion chromatography comprises qEV / 35nm chromatography.

[0038] Embodiment 14: A method for producing mass-tagged EVs, the method comprising contacting the EVs with a mass tag functionalized to bind to the EVs and components thereof under conditions appropriate for binding to occur.

[0039] Embodiment 15: The method of Embodiment 14, further comprising purifying the EVs from a body fluid or tissue prior to contacting the EVs with the functionalized mass tag.

[0040] Embodiment 16: EVs produced by a method according to any one of Embodiments 7-14.

[0041] Embodiment 17: The extracellular vesicle proteome or secretome of the EVs of Embodiment 16, wherein the proteome or secretome comprises mass-tagged components.

[0042] Embodiment 18: A method of using the EV of Embodiment 1, comprising contacting an EV with a recipient cell, whereby the recipient cell takes up the EV.

[0043] Embodiment 19: The method of Embodiment 18, which is an in vitro method and the EV is used for diagnosis or treatment.

[0044] Embodiment 20: The method of Embodiment 18, wherein the EV is used in non-diagnostic and non-treatment methods.

[0045] Embodiment 21: The method of Embodiment 18, which is an in vitro method.

[0046] Embodiment 22: The method of Embodiment 18, including in vivo distribution studies.

[0047] Embodiment 23: The method of Embodiment 19, including analyzing a single recipient cell.

[0048] Embodiment 24: The method of Embodiment 19, including analyzing a plurality of recipient cells.

[0049] Embodiment 25: The method of Embodiment 24, wherein the plurality of recipient cells includes cells of different cell types.

[0050] Embodiment 26: The method of Embodiment 18, further including measuring changes in cell function after EV uptake compared to before EV uptake, and the changes in cell function are optionally selected from apoptosis, DNA damage response, migration, proliferation, and tyrosine kinase signaling.

[0051] Embodiment 27: The method according to any one of Embodiments 18 to 26, wherein the recipient cell is labeled with at least one detectable label.

[0052] Embodiment 28: The method of Embodiment 27, wherein the detectable label indicates the characteristics of the recipient cell.

[0053] Embodiment 29: The method of Embodiment 28, wherein the characteristics of the recipient cells, alone or in combination with other characteristics, distinguish the recipient cell type from at least one other cell type.

[0054] Embodiment 30: The method of Embodiment 29, wherein the characteristics of the recipient cells, alone or in combination with other characteristics, identify the recipient cell type.

[0055] Embodiment 31: The method of any one of Embodiments 27 to 30, wherein the detectable label comprises a mass tag.

[0056] Embodiment 32: The method of any one of Embodiments 27 to 30, wherein live cell barcoding based on CD45 or fixed cell barcoding based on palladium is performed on the recipient cells.

[0057] Embodiment 33: The method of Embodiment 32, wherein the barcoding identifies cells of different samples and / or cells of different cell types.

[0058] Embodiment 34: For characterizing EV uptake and / or EV-mediated effects, identifying recipient cells, and / or in multiplex analysis, using detectably labeled recipient cells and / or one or more detectable label reagents, optionally wherein one or more detectable label reagents are one or more antibodies, the method of any one of Embodiments 18 to 33.

[0059] Embodiment 35: The method of Embodiment 34, wherein the detectably labeled recipient cells are labeled using a metal-labeled antibody panel and / or one or more detectable label reagents comprise a metal-labeled antibody panel.

[0060] Embodiment 36: The method of any one of Embodiments 18 to 31, comprising performing on the recipient cells a technique selected from mass cytometry, mass cytometry imaging, and transmission electron microscopy, or another mass spectrometry-based single cell and / or imaging technique.

[0061] Embodiment 37: recipient cells produced by the method of Embodiment 18.

[0062] Embodiment 38: A method for detecting the EV of Embodiment 1 or Embodiment 16 and / or the recipient cells of Embodiment 37, comprising performing a technique selected from mass cytometry, mass cytometry imaging, and transmission electron microscopy, or another mass spectrometry-based single cell and / or imaging technique.

[0063] Embodiment 39: A kit for carrying out the method of Embodiment 6, comprising one or more mass-tagged components that can be taken up by production cells.

[0064] Embodiment 40: The EV of Embodiment 1 or Embodiment 16, a plurality of EVs of any one of Embodiments 2 - 5, a method of any one of Embodiments 7 - 13, 18 - 36, or 38, the recipient cells of Embodiment 37, or the kit of Embodiment 39, wherein the mass-tagged component comprises an amino acid or an analog thereof.

[0065] Embodiment 41: The EV, method, or kit of Embodiment 40, wherein the amino acid is phenylalanine or an analog thereof.

[0066] Embodiment 42: The EV, method, or kit of Embodiment 40 or Embodiment 41, wherein the EV, virus particle, or protein component of the cell or EV proteome or secretome is labeled with at least one mass tag.

[0067] Embodiment 43: The EV of Embodiment 1 or Embodiment 16, a plurality of EVs of any one of Embodiments 2 - 5, a method of any one of Embodiments 7 - 13, 18 - 36, or 38, the recipient cells of Embodiment 37, the EV or method of any one of Embodiments 40 - 42, or the kit of Embodiment 39, wherein the mass tag comprises an organic tellurophene tag.

[0068] Embodiment 44: The EV, method, or kit of Embodiment 43, wherein the organic tellurophene tag comprises L-2-tellurinylalanine (TePhe) or TeMal.

[0069] Embodiment 45: The EV, method, or kit of Embodiment 44, wherein a plurality of mass tags selected from isotopic substituents of TePhe or TeMal are provided or used to facilitate multiplex analysis.

[0070] Embodiment 46: The EV or method of Embodiment 43 or Embodiment 44, wherein the mass-tagged EV is substantially identical to an unlabeled EV produced from the same cell type under the same conditions as the labeled EV.

[0071] Embodiment 47: The EV or method of Embodiment 46, wherein the mass-tagged EV and the unlabeled EV have substantially the same effect on recipient cells.

[0072] Embodiment 48: The EV or method of Embodiment 47, wherein the effects of the mass-tagged EV and the unlabeled EV differ by no more than ±15, ±14, ±13, ±12, ±11, ±10, ±9, ±8, ±7, ±6, ±5, ±4, ±3, ±2, ±1, ±0.5 percent.

[0073] Embodiment 49: The EV or method of Embodiment 46, wherein the mass-tagged EV and the unlabeled EV have substantially the same MISEV2018 characteristics for one or more or all of the MISEV2018 characteristics.

[0074] Embodiment 50: The EV or method of Embodiment 47, wherein the characteristics of the mass-tagged EV and the unlabeled EV differ by no more than ±15, ±14, ±13, ±12, ±11, ±10, ±9, ±8, ±7, ±6, ±5, ±4, ±3, ±2, ±1, ±0.5% percent.

[0075] Embodiment 51: The use of a mass tag, characterized in that the mass tag is used to label a cell secretome and the mass-tagged components of the cell secretome are purified.

[0076] Embodiment 52: The use according to Embodiment 51, characterized in that the cell secretome is labeled by metabolic labeling.

[0077] Embodiment 53: The use according to Embodiment 51 or Embodiment 52, characterized in that the mass-tagged components of the cell secretome include one or more EVs.

[0078] Embodiment 54: The use according to any one of Embodiments 51 to 53, characterized in that the mass-tagged components of the cell components are used in research together with one or more other detectably labeled components.

[0079] Embodiment 55: The use according to Embodiment 54, characterized in that the research includes multiplex analysis.

[0080] Embodiment 56: A method for mass-tag labeling of a cell secretome, including metabolically labeling a secretome-producing cell containing extracellular vesicles, soluble proteins and peptides, and viruses with a mass-tagged amino acid or analog to simultaneously and uniformly label various components of the cell secretome.

[0081] Embodiment 57: The method according to Embodiment 56, wherein the mass-tag labeling is compatible with single-cell and imaging techniques based on mass spectrometry, imaging mass spectrometry, electron microscopy, and other mass spectrometry.

[0082] Embodiment 58: The method according to Embodiment 56, further including analysis of the labeled cell secretome components by single-cell and imaging techniques based on mass spectrometry, imaging mass spectrometry, electron microscopy, or other mass spectrometry.

[0083] Embodiment 59: The method according to Embodiment 56, wherein the mass-tag labeling does not require electroporation of extracellular vesicles or any other modification prior to metabolic labeling in the production cells.

[0084] Embodiment 60: The method of Embodiment 56, wherein the mass tag label provides increased sensitivity and specificity for the detection of low protein-containing components such as extracellular vesicles and viruses.

[0085] Embodiment 61: A mass tag-labeled cell secretome, extracellular vesicle, soluble protein, or virus produced by the method of Embodiment 56.

[0086] Embodiment 62: The mass tag-labeled cell secretome of Embodiment 61 for use in disease diagnosis, prognosis, monitoring of treatment response, identification of drug targets, in vivo distribution analysis, and biomarker discovery for basic research.

[0087] Embodiment 63: A kit for mass tag labeling of a cell secretome, comprising a reagent for metabolic labeling of secretome-producing cells containing a mass-tagged amino acid or analog, and instructions for subsequent analysis of the mass tag labeling of the cell secretome and the labeled cell secretome components.

[0088] Embodiment 64: A method for research on biomarker, drug target identification or cell communication using the mass tag-labeled cell secretome of Embodiment 61.

[0089] Embodiment 65: The mass tag-labeled cell secretome of Embodiment 61 for use in proteomics research to quantify the abundance of proteins, evaluate protein-protein interactions, and investigate post-translational modifications.

[0090] Embodiment 66: A mass tag-labeled cell secretome receptor cell analysis system, comprising a mass spectrometer, an imaging mass spectrometer, an electron microscope, or other mass spectrometry-based single cell and imaging devices, and a mass tag-labeled cell secretome prepared according to the method of Embodiment 56.

[0091] Embodiment 67: The system of Embodiment 66, further comprising a software package for the analysis and visualization of mass-tagged cell secretome receptor cell data.

[0092] Embodiment 68: A method for normalizing and comparing mass-tagged components of a cell secretome, in which the proteins in the secretome are uniformly labeled and normalized based on the Gaussian distribution of mass tag incorporation, enabling more accurate quantification and comparison of secreted peptides and proteins.

[0093] Embodiment 69: A study of the effects of mass-tagged cell secretome components on the phenotype and function of recipient cells, including the internalization of mass-tagged components such as proteins of extracellular vesicles or viruses or soluble proteins by recipient cells, and subsequent analysis using mass spectrometry, imaging mass spectrometry, electron microscopy, or other mass spectrometry-based single-cell and imaging techniques.

[0094] Embodiment 70: The method of Embodiment 69, in which the internalization of mass-tagged components of the cell secretome by recipient cells is not hindered by the incorporation of mass tags and the protein binding and phenotype remain unchanged.

[0095] Embodiment 71: A computer-readable medium containing a non-transitory program for analyzing data of mass-tagged cell secretomes generated by single-cell and imaging techniques based on mass spectrometry, imaging mass spectrometry, electron microscopy, or other mass spectrometry methods, the program including means for processing, analyzing, and visualizing the interaction between mass-tagged cell secretome components and recipient cells.

[0096] Embodiment 72: A method for purifying mass tag-labeled extracellular vesicles, soluble proteins, and viruses from a cell secretome, including a combination of density gradient centrifugation, size exclusion chromatography, or affinity purification techniques, with minimal contamination of other cellular components to isolate the mass tag-labeled components.

[0097] Embodiment 73: The method of Embodiment 72, wherein the purification of mass tag-labeled cell secretome components facilitates the identification and quantification of specific subpopulations of extracellular vesicles, soluble proteins, and viruses, enhancing the understanding of their roles in cell communication.

[0098] Embodiment 74: A method for multiplex analysis of mass tag-labeled cell secretome components of multiple cell types or treatment conditions, enabling simultaneous comparison of different experimental groups to identify changes in the secretome composition.

[0099] Embodiment 75: The method of Embodiment 74, wherein the multiplex analysis can be used to identify specific cell secretome components that change in response to different stimuli or in various disease states.

[0100] Embodiment 76: A method for validating the function of mass tag-labeled cell secretome components, including functional assays to evaluate the effects of these components on the receptor cell signaling pathways, gene expression, and overall cell function.

[0101] Embodiment 77: The method of Embodiment 76, wherein the function verification assay can be used to determine the biological relevance of mass tag-labeled cell secretome components in the context of cell communication and disease etiology.

[0102] Embodiment 78: A method for evaluating the effects of pharmacological agents on the composition and function of mass tag-labeled cell secretomes, facilitating the identification of drug targets and the evaluation of drug efficacy and safety.

[0103] Embodiment 79: A method for utilizing a high-throughput screening approach to identify novel mass-tagged cell secretome components that can be applied to therapy and to test the effects of these components on various disease models.

[0104] Embodiment 80: The mass-tagged cell secretome of Embodiment 61 for use in the development of diagnostic means for monitoring disease progression or treatment response by measuring changes in the composition and function of the cell secretome.

[0105] Embodiment 81: A method for creating a mass-tagged cell secretome library for high-throughput screening to enable the identification of novel secretome components that can be applied to therapy or diagnosis.

[0106] Embodiment 82: A method for optimizing a mass-tagging method that includes a systematic evaluation of labeling efficiency, signal-to-noise ratio, and the effect on cell secretome component function to ensure the reliability and reproducibility of the labeling method.

[0107] Embodiment 83: A method for integrating data of mass-tagged cell secretomes with other omics data such as transcriptomics, genomics, and metabolomics to provide a comprehensive understanding of cell communication and the molecular mechanisms underlying the etiology of diseases.

[0108] Embodiment 84: The method of Embodiment 56, wherein mass-tagging is adaptable to various cell types and organisms and facilitates the study of cell secretomes in diverse biological systems.

[0109] Embodiment 85: A method for tracking the in vivo distribution and localization of mass-tagged cellular secretome components in vivo using imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), or single photon emission computed tomography (SPECT) to monitor mass-tagged secretome components in a living body and enable evaluation of their biological roles and potential therapeutic applications.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0111] Definition The terms used in the claims and the specification are defined as follows, unless otherwise specified.

[0112] As used herein, the term "soluble component" means any component that is released or secreted from a cell into the extracellular space.

[0113] As used herein, the term "extracellular vesicle" ("EV") is used to mean a membrane vesicle that is released or discharged by a cell into the extracellular environment. Examples include, but are not limited to, microvesicles, exosomes, ectosomes, argosomes, and apoptotic bodies. These can be classified into different subtypes based on their biosynthesis, size, and composition. EVs contain a variety of biological molecules such as proteins, lipids, and nucleic acids that can be transferred between cells to mediate intercellular communication, control the immune response, and be involved in disease progression. Since they are involved in various pathological processes, the potential of EVs as diagnostic biomarkers and therapeutic targets is being explored.

[0114] As used herein, the term "mass tag" means a molecule containing at least one specific elemental isotope composition that is useful for distinguishing, using mass spectrometry, a tagged molecule or optionally the tag itself from other molecules containing different elemental isotope compositions. In some embodiments, the mass tag includes at least one elemental isotope and a support structure for at least one elemental isotope.

[0115] As used herein, the term "proteome" is used to mean the entire complement of proteins expressed in an organism, tissue, cell, or intracellular component (e.g., EVs, etc.). The structure of the proteome can vary depending on specific times and conditions. Thus, protein expression levels and modifications are dynamic entities as they can vary in response to environmental factors, developmental stages, and disease states. Proteomics, which is the large-scale study of the proteome, aims to identify, quantify, and characterize proteins in order to better understand their functions, interactions, and roles in cellular processes and disease mechanisms. The components of the cellular proteome include both the cellular secretome and all intracellular proteins that are not secreted (the intracellular proteome).

[0116] As used herein, the term "secretome" is used to mean all the elements secreted into the extracellular space by cells or their intracellular components (e.g., EVs, etc.). These elements include proteins, and the term "cellular secretome" is often used in the art to represent the aggregate of proteins secreted by cells into their surrounding extracellular environment. These proteins can include growth factors, cytokines, hormones, other signaling molecules, and extracellular vesicle proteins (the extracellular vesicle proteome). The secretome plays an important role in cell-cell communication, control of cellular processes, and maintenance of tissue homeostasis.

[0117] "Secreted soluble proteins" are a subset of the proteins within the secretome that are released into the extracellular environment in a soluble form. They can diffuse freely through the extracellular space and interact with other cells, receptors, or molecules to exert their functions. Examples of secreted soluble proteins include cytokines, chemokines, growth factors, antibodies, and hormones that play essential roles in the control of the immune response, cell growth and differentiation, and tissue repair and remodeling.

[0118] As used herein, the term "purify" means separating a desired component from at least one other component found together therewith. This term encompasses all degrees of purification, including purification to a level where the desired component is present at a level of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent in the purified composition.

[0119] As used herein, the term "producing cell" is used to mean a cell that produces EVs, viruses, and a secretome such as soluble proteins and peptides or components thereof.

[0120] As used herein, the term "receiving cell" is used to mean a cell that takes up one or more components of an EV, a virus, and / or a secretome such as soluble proteins and / or peptides.

[0121] As used herein, the term "detectable label" or "label" means any atom or molecule that can be used to provide a detectable and / or quantifiable signal. In particular, a label can be directly or indirectly attached to a nucleic acid or protein. Suitable labels include, but are not limited to, radioisotopes, fluorophores, chromophores, mass labels, high electron density particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0122] As used herein, the term "barcoding" is used to mean labeling an entity (e.g., a cell) with a detectable label or a combination of detectable labels that encode information. For example, a barcode can identify the source or type of a biological entity such as a cell. A barcode can be used, for example, to distinguish different samples, different cells, different treatments, different time points, etc.

[0123] When used in connection with an assay, the term "multiplex" means a situation in which a number of individual assays are run together within a single "pool" that is placed under a single set of conditions. For example, multiplex analysis enables a single study to be performed on a number of samples, each of which can be distinguished from all the others. This ability to distinguish between samples allows the study results to be deconvoluted so that a particular result can be assigned to a particular sample. Multiplex analysis has the advantage over singleplex analysis that it can eliminate artificial differences in the results that arise from running singleplexes in parallel (e.g., due to unavoidable differences in samples or conditions in individual singleplex assays).

[0124] As used herein, the term "antibody" shall mean monoclonal antibodies, polyclonal antibodies, and chimeric antibodies and their binding fragments, as well as antibodies of noncanonical structure from species such as camel and shark (e.g., nanobodies). Antibodies may be from recombinant sources and / or produced in transgenic animals. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be made by treating an antibody with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bonds to generate Fab' fragments. Papain digestion can cause the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized by recombinant techniques. As used herein, an antibody fragment means a fragment that binds to a target antigen.

[0125] As used herein, the term "amino acid residue" means a natural, synthetic, or modified amino acid. Various amino acid analogs include, but are not limited to, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine (beta-aminopropionic acid), 2-aminobutyric acid, 4-aminobutyric acid, pipecolic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, n-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n-methyllysine, n-methylvaline, norvaline, norleucine, ornithine, L-2-amino-3-guanidinopropionic acid (AGP), L-alpha,gamma-diaminobutyric acid (DAB), L-alpha,beta-diaminopropionic acid (DAP), L-alpha-t-butylglycine, and the like. These modified amino acid residues are merely illustrative and not limiting.

[0126] As used herein, the term "terulene" means a compound of the formula:

[0127]

Chemical formula

[0128] wherein the numbers are used for the naming of various substituents on the terulene ring.

[0129] The term "organic terulene" means a terulene substituted with at least one carbon-containing group.

[0130] As used herein, "organic tellurophene tag" means, in some embodiments, any tellurophene-containing compound that includes a tellurophene moiety and a linker that can conjugate, for example, to another molecule such as a small molecule, a biosensor, a polymer backbone, a biologically active substance, and / or an amino acid, and includes, for example, the organic tellurophene compounds described in International Application Publication No. WO 2016 / 0206046, which is incorporated herein by reference for the purposes of this description and / or for the purposes of this description. For example, an organic tellurophene tag can include a tellurophene moiety and a chemical bond or linker that conjugates that moiety directly or indirectly to another entity. In some embodiments, the entity can be tagged with an organic tellurophene tag by substituting a tellurophene group for another moiety in the entity, as shown below, when the phenyl group is substituted with an organic tellurophene analog. For example, phenylalanine substituted with an organic tellurophene can be synthesized as the L-enantiomer (>95%) in four steps from N-Boc-L-propargylglycine: (bromoethynyl)triisopropylsilane is added to yield the intermediates (S)-2-((tert-butoxycarbonyl)amino)-7-(triisopropylsilyl)hepta-4,6-dienoic acid and (S)-2-((tert-butoxycarbonyl)amino)-3-(tellurophen-2-yl)propanoic acid, which are ultimately converted to TePhe (L-2-telluranylalanine). (See Bassan, J. et al., (2019) Proc. Natl. Acad. Sci. USA 116(17):8155-8160, which is incorporated herein by reference for the purposes of the description of the synthesis and use of phenylalanine substituted with an organic tellurophene; and Vurgun, N. and Nitz, M. (2019) Chem. Europe 21(8): 1136-1139, which is incorporated herein by reference for the purposes of the description of the synthesis and use of phenylalanine substituted with an organic tellurophene). Organic tellurophene tags can include distinct tellurium isotopes to label different entities, for example, in multiplex assays, enabling distinct detection of each.

[0131] As used herein, L-2-tellurinylalanine, also referred to as TePhe, is a phenylalanine substituted with an organic tellurophene and has the chemical structure shown in Formula 1:

[0132]

Chemical formula

[0133] As used herein, N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(tellurophen-2-yl)propenamide, also referred to as TeMal, is a tellurophene having a thiol-reactive maleimide functional group and has the chemical structure shown in Formula 2:

[0134]

Chemical formula

[0135] As used herein, the term "distinct tellurium isotope" means a Te atom in a compound having one or more atoms of a single tellurium isotope. For example, a series of mass-tagged entities each having a different distinct tellurium isotope can be used in an assay such that each compound containing a distinct tellurium isotope can be distinguished from the other compounds.

[0136] As used herein, an "isotopic molecular species" is a chemical substance different from its parent chemical substance in which at least one atom has a different number of neutrons.

[0137] As used herein, the term "distinct mass" indicates that a compound has a unique combination of one or more atoms of a single tellurium isotope or tellurium isotope alone (e.g., a distinct tellurium mass) or a combination with other mass tags. One example is a series of compounds, optionally including polymers, each having a different level of a different distinct tellurium isotope alone or in combination with other mass tags, optionally for use in a barcoding embodiment.

[0138] As used herein, the term "metabolic label" means the incorporation of a labeled molecule into another molecule, such as a macromolecule, for example, in the incorporation of an amino acid into a protein or a monosaccharide into a polysaccharide or glycoprotein. Metabolic labeling is typically carried out by a cell or its metabolically active components that are exposed to the labeled molecule. Metabolic labeling is a technique that can be used to study intracellular cell turnover and transport. In some embodiments, it involves the incorporation of labeled amino acids (e.g., with isotopes, biotin, or fluorescent tags) into newly synthesized proteins over a defined period. By tracking the labeled amino acids, researchers can monitor protein synthesis, degradation, localization, and interactions in living cells or organisms. Metabolic labeling is widely used in proteomics research for the quantification of abundant proteins, the assessment of protein-protein interactions, and the investigation of post-translational modifications.

[0139] As used herein, the term "functional group" means a group of atoms or a single atom that reacts with another group of atoms or a single atom (the so-called "complementary functional group") to form a chemical interaction, typically a chemical bond between two groups or atoms. Moieties such as mass tags or other labels can be functionalized for conjugation or binding to molecules such as components that can be taken up by cells (e.g., for metabolic labeling) or antibodies that bind to cell surface antigens for labeling the cell surface.

[0140] As used herein, the phrase "effect on recipient cells" of an EV or other soluble component of the cell secretome means any effect on any function of a cell that is observed after the EV or soluble component has contacted and / or been taken up by the cell, including, but not limited to, cell functions (e.g., signaling) that can be measured using mass cytometry or mass cytometry imaging, or any change to one or more, or all, of the MISEV2018 characteristics.

[0141] As used herein, the term "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "include" and "includes"), or "containing" (and any form of "containing" such as "contain" and "contains") is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0142] As used herein, the term "consisting" and its derivatives are intended to be closed-ended terms that specify the presence of the recited features, elements, components, groups, integers, and / or steps, and also exclude the presence of other features, elements, components, groups, integers, and / or steps not recited.

[0143] As used herein, the term "consisting essentially of" is intended to specify the presence of the recited characteristics, elements, components, groups, integers, and / or steps, and also the presence of those that do not substantially affect the basic novel characteristics of these characteristics, elements, components, groups, integers, and / or steps. The basic novel characteristics of the mass-tagged EVs are the presence of a mass tag that does not differ substantially from the characteristics of EVs without the same type of mass tag, in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more characteristics.

[0144] As used herein, the terms "about," "substantially," and "approximately" mean a reasonable amount of deviation of a term so modified such that the end result does not vary appreciably. These terms of degree are considered to include at least a ±20% deviation of the modified term if the deviation does not negate the meaning of the phrase that the term modifies. Thus, for example, "substantially similar effect" or "substantially the same effect" on a recipient cell means one or more effects on the recipient cell typically caused by EVs or other components of the cell secretome that differ by ±20% or less. In some embodiments, for example, a high degree of similarity of at least ±15, ±14, ±13, ±12, ±11, ±10, ±9, ±8, ±7, ±6, ±5, ±4, ±3, ±2, ±1, ±0.5%, etc., or within any range limited by any of these values, for example, a similarity degree between ±5 and ±0.5 percent, is observed. As used with respect to mass-tagged EVs, more generally, "substantially similar" and "substantially the same," as compared to non-tagged EVs, mean similarity of the mass-tagged EVs as compared to non-tagged, as determined by methods acceptable in the art, such as described in the MISEV2018 guidelines, including morphology, physical and chemical properties, protein concentration, EV concentration, cell type specificity of uptake, size distribution, purification behavior, distribution of EV components (proteins, RNAs, lipids, polysaccharides, and glycans, etc.), and other general properties / behaviors described in the MISEV2018 guidelines. The degree of similarity described above with respect to "effects" is equally applicable to more general aspects of EVs such as those described in the MISEV2018 guidelines.

[0145] Mass Tagging of Cell Secretomes The present disclosure provides a method for mass tagging one or more soluble components of a cell secretome, which can include, for example, extracellular vesicles (EVs), virus particles, cell secretomes, EV proteomes or secretomes, or components of any of these. In certain embodiments, the method requires exposing cells, referred to as "producer cells," to mass-tagged components. In some embodiments, the components are those that can be taken up by the producer cells and incorporated by the cells into molecules produced by the cells, by a method referred to as "metabolic labeling." In some embodiments, the molecules produced by the cells are secreted, either by themselves or as part of a more complex structure such as an EV, or released in some other way, and in either case, the producer cells produce the mass-tagged soluble components. In some embodiments, the method requires purifying the mass-tagged soluble components. In other embodiments, the mass-tagged soluble components are produced by directly tagging the soluble components. However, the mass-tagged soluble components are produced and then can be used to study their binding, uptake, and / or biological effects in vitro or in vivo in a biological system of interest that can include tissues, and organs, or cells. The mass-tagged soluble components can be used in combination with other labeling strategies (including distinct mass tags) to, for example, characterize the biological effects of the soluble components and the tissues, organs, or cells affected. These studies can be performed at the single cell level.

[0146] To effectively compare the various components of the cell secretome, in some embodiments, it is essential to use an essentially normalized labeling technique. The mass tag labeling method for the cell secretome achieves this by randomly substituting Phe with TePhe in all proteins and peptides within the secretome (metabolic labeling of the cell secretome). This method follows a Gaussian distribution, ensuring consistent and uniform labeling throughout the cell secretome. In contrast, previous labeling methods lack these characteristics and are not suitable for facilitating direct comparison between different secretome components.

[0147] The novel mass tag labeling approach for the cell secretome has a wide impact on various fields of biotechnology and biomedical research, including but not limited to:

[0148] Biomarker discovery: This method can be used to identify novel biomarkers for disease diagnosis, prognosis, and monitoring of treatment response.

[0149] Drug target identification and biodistribution: Our method can facilitate the discovery of new cell drug targets and provide insights into the biodistribution of drugs for a wide range of diseases by enabling a more comprehensive understanding of the cell secretome.

[0150] Basic research: The proposed mass tag labeling technique can provide useful insights into the molecular mechanisms of cell communication and function, and deepen the understanding of the underlying biological communication methods.

[0151] Mass tag Mass cytometry reagents are tagged with a defined mass of metal isotopes that act as labels. The metals can be detected using inductively coupled plasma-time of flight mass spectrometry (ICP-TOF-MS). Many different types of mass tag reagents have been developed. These reagents include polymer-based mass tag reagents, non-polymer-based mass tag reagents, and inorganic nanoparticles. Metal chelate polymers (MCPs) are widely used to profile and quantify cell biomarkers. Biocompatible materials such as polystyrene and inorganic nanoparticles are also used in mass cytometry. Polystyrene allows for the incorporation of a wide variety of metals, while inorganic nanoparticles with high metal content provide an excellent opportunity to enhance the metal signal and detect low-concentration biomarkers. Non-polymer-based mass tag reagents can be used in a number of applications: cell detection, determination of cell cycle characteristics, biomarker detection, and mass tag cell barcoding (MCB). Recent developments have been achieved in live cell barcoding by targeting proteins (CD45, b2m, and CD298), using small non-polar probes, or ratio measurement barcoding. For mass cytometry reagents, see Delgado-Gonzalez and Sanchez-Martin (2021) Anal. Chem. 93(2):657-664, which is incorporated herein by reference for this description.

[0152] Mass tags useful in the methods and compositions described herein, in some embodiments, have one or more, or preferably all, of the following characteristics: being available for high-yield synthesis suitable for isotope incorporation, being stable under biologically relevant conditions, and having low toxicity. A mass tag probe (Telox) has been constructed for measuring cellular hypoxia (described in U.S. Patent Application No. 62 / 039762). This probe uses 2-nitroimidazole as an active group for hypoxia-specific labeling and methyl telluroether functionality as a tag unit for MC detection. Tellurium is known to form stable bonds with carbon and has eight naturally occurring isotopes that can be obtained to generate a series of unique identifiable biologically distinguishable mass spectrometry (MC) probes, so it was selected as the element for detection. In Telox, the stability of the telluroether functionality is moderate and the metabolic LD50 value is close to the required assay concentration. The synthesis, aqueous / aerobic stability, and in vitro toxicity of a series of alkyl telluroether and tellurophene functional groups, and their use, are described in detail in International Application Publication No. 2016026046, which is incorporated herein by reference for this description. Based on the guidelines therein, one of ordinary skill in the art can label components with organic tellurophene tags.

[0153] The mass tag can be incorporated directly into the biological component of interest, for example, by substituting the phenyl portion in the component with tellurophene and / or adding it to the component via a linker. The biological component can also be labeled directly or indirectly using a binding moiety functionalized with a mass tag such as an antibody, streptavidin-biotin, etc. A wide variety of labeling strategies are available and can be readily adapted to the use of the mass tag as a detectable label.

[0154] Mass tagging method Tagging by metabolic labeling One skilled in the art of cell biology understands that a wide variety of components are taken up by cells and incorporated into other molecules during cell metabolism. Examples include, but are not limited to, amino acids, sugars, salts, lipids, and elements. In some embodiments, the selection of a particular component can be determined by the purpose of mass-tagging a particular aspect of the cell. For example, if one desires to mass-tag the cell proteome, amino acids can be conveniently used to introduce the mass tag into the cell's proteins. Similarly, the selection of a particular amino acid can be influenced by the structure of the mass tag. Organic tellurophene tags can be readily incorporated into amino acids by substitution of the phenyl moiety. This approach is illustrated in the following examples using a mass-tagged form of phenylalanine L-2-tellurinylalanine (TePhe).

[0155] For metabolic tagging, the cells are exposed to the mass-tagged component under conditions such that the cells can take up a sufficient amount of the mass-tagged component to facilitate the desired labeling. This may require reducing the tendency of the mass-tagged component to degrade, aggregate, or bind to other components in the cellular environment that may inhibit uptake of the mass-tagged component, and / or providing conditions that promote uptake of the mass-tagged component by the cells. In the case of mass-tagged amino acids, in some embodiments, it may be advantageous to expose the cells to the mass-tagged amino acids under serum-free conditions, as shown in the following examples. One skilled in the art knows or can experimentally determine the appropriate conditions for a particular mass-tagged component, cell, and cellular aspect to be mass-tagged.

[0156] Any cell that can take up and incorporate mass-tagged components can be used in this metabolic labeling approach for mass tagging. In the case of TePhe, for example, the cell preferably has several means of taking up Phe, such as a Phe transporter. Suitable cells include bacteria, protozoa, fungi, and higher organisms such as plants or animals, particularly mammals, more particularly human cells. Metabolic labeling with mass tags can be performed on cell lines (e.g., HEK293T, HeLa, OSU-CLL, and PANC-1), primary cells (e.g., chronic lymphocytic leukemia cells), and in vivo. The only requirement is that the cells are viable and can incorporate the mass-tagged components.

[0157] Direct tagging In some embodiments, for example, a soluble component of interest, such as an extracellular vesicle (EV) or a viral particle, can be directly tagged by contacting the soluble component with a mass tag functionalized to bind to a characteristic of the soluble component. Such binding can be of any type, e.g., covalent or non-covalent, direct or indirect. In some embodiments, the soluble component (e.g., EV) is purified from a biological fluid or tissue prior to tagging.

[0158] For example, mass tag labeling of EVs derived from solid tissues or biological fluids can be performed directly with TeMal isotopologues after size-exclusion chromatography-based separation and ultrafiltration-based concentration of the EV fraction. See, for example, Willis, L.M. et al., (2018) “Tellurium-based mass cytometry barcode for live and fixed cells” Cytometry, https: / / doi.org / 10.1002 / cyto.a.23495 (incorporated herein by reference for the description of direct tagging with TeMal). In an exemplary embodiment, TeMal at a total concentration of 6 μM can be added to the concentrate of the EV-containing fraction (200 μL) after 10 kDa MWCO ultrafiltration. Next, the EV concentrate is incubated with TeMal for 30 minutes at room temperature in an ultrafiltration unit and then washed twice (4 °C, 20 minutes, 3000 RCF) with 14.8 mL and 15.0 mL of PBS, respectively. After the final wash step, the mass-tagged sample is ready for use in mass cytometry and imaging mass cytometry assays.

[0159] Other labeling strategies that can be used in combination with the methods described herein are Qin, W. et al., (2021) “Deciphering molecular interactions by proximity labeling” Nat. Methods 18:133 - 143 (incorporated herein by reference for this description) and Sufi, J. et al., (2021) “Multiplexed single-cell analysis of organoid signaling networks” Nat. Protoc. 16(10):4897 - 4918 (incorporated herein by reference for this description).

[0160] Nucleic acid tagging In some embodiments, the cellular aspect to be tagged is the nucleic acid of the cell. Suitable components for mass tags for labeling nucleic acids are known to those skilled in the art and include, for example, nucleic acid binders or DNA intercalators such as cisplatin, iridium, or rhodium. These components can be introduced into cells or EVs by standard techniques such as transfection or electroporation. See, for example, Wang, J. et al., (2020) “Loading of metal isotope-containing intercalators for mass cytometry-based high-throughput quantitation of exosome uptake at the single-cell level” Biomaterials 255:120152 (incorporated herein by reference for this description). One thing to note in relation to this approach is that the nucleic acid cargo of EVs can vary quite significantly at the single EV level and not all EVs have been proven to carry nucleic acids. Also, transfection and electroporation can change the membrane structure and thus, for example, EV function. Metabolic labeling circumvents both of these problems.

[0161] Purification of Mass-Tagged Secretome Components Mass-tagged secretome components can generally be purified using essentially the same techniques as those used for their tagless counterparts, and depending on the nature of the soluble components, appropriate purification procedures can be devised by those skilled in the art.

[0162] Various purification techniques that can be used include filtration, membrane crossflow ultrafiltration or microfiltration, tangential flow ultrafiltration or microfiltration, or dead-end filtration, centrifugation (e.g., density gradient centrifugation), decantation. Further steps can include one or more of the following: nanofiltration, concentration, extraction, crystallization, precipitation, adsorption, and chromatography (e.g., size exclusion or ion exchange).

[0163] In certain embodiments shown in the following examples, mass-tagged EVs were purified by a combination of filtration, ultrafiltration, and size-exclusion chromatography. Specifically, it was 0.2 μM filtration followed by 10 kDa ultrafiltration and then qEV / 35 nm chromatography.

[0164] Mass Tagging of Extracellular Vesicles Extracellular vesicles (「EV」) are membrane-bound vesicles that are released or discharged by cells into the extracellular environment. Examples include microvesicles, exosomes, ectosomes, argosomes, and apoptotic bodies. Typically, EVs are vesicles derived from endosomes and / or the plasma membrane. These EVs can represent a mode of intercellular communication, particularly by functioning as a medium for the transport of membrane and cytoplasmic proteins, lipids, and RNA between cells. The term exosome was initially used for vesicles in the range of 40 to 1,000 nm released by various cultured cells (see, for example, Trams et al., (1981) Biochim. Biophys. Acta. 645: 63-70), but the intracellular origin of these vesicles remained unknown. Subsequently, this nomenclature was adopted for 40-100 nm vesicles released during reticulocyte differentiation as a result of the fusion of multivesicular endosomes (MVE) with the plasma membrane (see, for example, Harding et al., (1984) Eur. J. Cell Biol. 35: 256-263; Pan et al., (1985) J. Cell Biol. 101: 942-948). Subsequently, exosomes were discovered to be released by B lymphocytes and dendritic cells through a similar pathway (see, for example, Zitvogel et al., (1998) Nat. Med. 4: 594-600; Raposo et al., (1996) J. Exp. Med. 183: 1161-1172). Additional cell types of both hematopoietic and non-hematopoietic origin, including but not limited to cytotoxic T cells, platelets, mast cells, neurons, oligodendrocytes, Schwann cells, and intestinal epithelial cells, have also been shown to release exosomes by MVE fusion with the cell surface (see, for example, Simons & Raposo (2009) Curr. Opin. Cell Biol. 21: 575-581; Thery E (2009) Nat. Rev. Immunol. 9: 581-593).

[0165] EVs are increasingly being used as diagnostic tools and, in their purified form, are therapeutically used in a variety of diseases. Ongoing clinical trials aim to test EV-based therapeutics for the treatment of metastatic pancreatic cancer, colon cancer, and non-small cell lung cancer. The methods and compositions described herein will facilitate further development of such applications by providing analytical means for characterizing EVs and their recipient cells. In particular, the methods and compositions described herein will help answer questions regarding where EVs go when administered (e.g., organs, spatial tissue distribution, and recipient cell type), as well as the biological effects of therapeutically administered EVs (e.g., proliferation, apoptosis, and intracellular signaling).

[0166] Mass-tagged EVs can be produced from any cell that produces a secretome.

[0167] In some embodiments, EVs can be tagged with different detectable labels, such as distinct mass tags to distinguish one set of EVs from another. For example, EVs prepared from different producer cells and / or different samples can be mass-tagged and then combined into a single pool of EVs, after which further studies can be performed, with the advantage that the EVs are all exposed to the same study conditions. In certain embodiments, different sets of EVs can be distinguished by different combinations of detectable labels. For example, a set of mass-tagged EVs can have different combinations of mass tags and / or can have common mass tags, but can also have another different detectable label of a different type (e.g., fluorescence) that allows for the unique identification of EVs from a particular set.

[0168] These mass tag / labeling strategies described for EVs are equally applicable to any mass-tagged soluble secretome component of interest, including viruses and proteins.

[0169] The procedures described in the following examples demonstrate that mass tagging of EVs, particularly of the EV proteome, does not substantially alter important EV characteristics such as size, morphology, or composition. Thus, surprisingly, mass-tagged EVs are not substantially different from non-labeled EVs produced from the same cell type under the same conditions as the mass-tagged EVs. In particular, it was not reasonably warranted or anticipated prior to the operations of the present invention that mass-tagged EVs would be secreted by producer cells, have substantially similar morphology, physical and biochemical properties, be taken up by recipient cells in substantially the same manner as non-tagged EVs, and have substantially similar effects on one or more cellular functions. In particular, the procedures described herein demonstrate for the first time that mass-tagged EVs and non-labeled EVs have substantially the same MISEV2018 characteristics.

[0170] Methods of using mass-tagged EVs and / or other mass-tagged components of the cell secretome Mass-tagged components of the cell secretome are individually useful in the functional evaluation of producer cells. For example, as described herein, antibody-producing hybridoma cells can be contacted with TePhe to produce mass-tag-labeled monoclonal antibodies, which can be purified and their biodistribution and functional effects on specific cell types analyzed by mass spectrometry and imaging mass spectrometry. In another example, mass-tagged components of the cell secretome can be used in combination with magnetic resonance imaging to further elucidate the state.

[0171] Mass-tagged EVs are useful, in particular, in studies to characterize EV distribution in tissues or whole organisms, recipient cells that take up EVs, and EV-mediated changes in cell behavior. Mass-tagged EVs can readily contact potential recipient cells, and in some embodiments, uptake can be detected by any means capable of detecting the mass tag.

[0172] Particularly of interest is the study of EV uptake in heterogeneous cell populations. Using available means to identify specific cell types, such as labeled antibodies that bind to antigens or combinations of antigens characteristic of a particular cell type, it is possible to determine which cells in a heterogeneous cell population take up specifically mass-tagged EVs. When a population of EVs derived from a combination of different sets of EVs with different labels (e.g., mass tags) or combinations of labels is brought into contact with a heterogeneous population of cells, it is possible to determine which types of EVs are taken up by the recipient cell type, enabling multidimensional analysis that has hitherto been impossible with conventional labeling methods.

[0173] For example, Figure 11 shows a panel of mass-tagging reagents that enable the identification of EV uptake in more than 30 different cell types (Standard BioTools (formerly Fluidigm Corporation) MaxPar® Direct Immune Profiling Assay (MDIPA)). Standard BioTools supports mass spectrometry studies of over 50 metal-tagging reagents, including antibodies, nucleic acid intercalators and analogs, and other biochemical ligands. Each reagent is detected and quantified by cytometry by time-of-flight mass spectrometry on Standard BioTools' fully automated CyTOF® system. The high purity and selection of metal isotopes ensure minimal signal overlap or background noise from endogenous cell components. Standard BioTools' catalog contains over 800 antibodies that detect over 400 unique human or mouse targets. If the desired antibody is not included, Standard BioTools enables the user to create it themselves using Standard BioTools' metal-labeling kits. Metal-labeled antibodies can also be custom ordered from Standard BioTools.

[0174] Figure 12 schematically shows a CD45 live cell barcoding (select 3 out of 7 combinations) approach that generates 35 unique barcodes that can be used to distinguish different types of cells. Modifications to these, and other approaches known or developed, can be used in combination with the mass-tagged EVs described herein. For example, a novel barcoding technique that utilizes 10 different tags, 7 cadmium (Cd) tags, and 3 Pd tags that have excellent signal strength that does not interfere with lanthanide detection, enables improved storage of samples under a number of experimental conditions, and significantly increases sample throughput. See Muftuoglu et al., (2021) "Extended live-cell barcoding approach for multiplexed mass cytometry" Scientific Reports 11, 12388 (incorporated herein by reference for the purposes of this description).

[0175] The label can also be used to identify one or more changes in cell function after EV uptake by using mass-tagged EVs in combination with other labeling components such as antibodies or other binding partners for antigens / ligands associated with changes in cell function. For example, the reagent panel shown in Figure 11 can detect markers of DNA damage response, chronic lymphocytic leukemia (CLL) cell biology, tyrosine kinase signaling, cell cycle, apoptosis, and checkpoints. Optionally, in combination with a mass-tagging reagent that differentiates cell types, such reagents that are also labeled with mass tags enable simultaneous detection of one or more of the following parameters: EV source or type, changes in markers of cell function, and recipient cell source or type. Changes in cell function can be detected, for example, as differences in the levels of one or more biomarkers at a time point before EV uptake and at a time point after EV uptake, or as differences in biomarker "fingerprints" (presence or relative levels of two or more biomarkers). Such changes can indicate changes in almost all cell functions, including but not limited to apoptosis, DNA damage response, migration, proliferation, and tyrosine kinase signaling.

[0176] Mass tags can be detected by any means known in the art, such as mass spectrometry, mass spectrometry imaging, and transmission electron microscopy, as well as single cell and imaging techniques based on other mass spectrometry methods. Detection of other types of labels can be performed by any available method appropriate for the specific label.

[0177] These mass tag / labeling strategies described for EVs are equally applicable to any mass-tagged soluble secretome components of interest, such as viruses and proteins.

[0178] Kit The kit according to the present invention can include one or more reagents useful for performing one or more methods described herein. The kit generally includes one or more individual compositions, or optionally, as a mixture to the extent the reagents permit, one or more containers that house the reagents (e.g., mass-tagged components such as functionalized mass tags or mass-tagged amino acids) in a package. The kit can also include other materials that may be desired from the user's perspective, such as buffers, diluents, standards, etc., and / or any other materials useful for performing any other steps of sample processing, washing, or assay.

Example

[0179] Introduction For this example, a novel mass-tag labeling of the cell secretome (e.g., TeLEV, a labeling method based on proteomic incorporation of L-2-tellurienylalanine (TePhe), a tellurium-containing phenylalanine mimic that enables tracking of EV uptake in heterogeneous cell samples) was developed. By using mass-tag labeling of the cell secretome (e.g., TeLEV) with extracellular and intracellular / nuclear mass cytometry (MC) staining with 50 markers, changes mediated by EVs in cell functions such as apoptosis, DNA damage response, migration, proliferation, and tyrosine kinase signaling can be analyzed at the single-cell level in vitro and in vivo and directly correlated with EV uptake. To pool 35 samples in one tube, the mass-tag labeling of the cell secretome (e.g., TeLEV) can be used in combination with, for example, a cadmium-based live-cell barcoding approach (a combination of choosing 3 from 7) to eliminate staining variability and enable, in particular, sensitive EV dose-response studies and functional EV uptake kinetics.

[0180] According to MISEV2018, a wide range of multi-platform property evaluations including the bulk method and the single EV method were carried out, and it was demonstrated that Te-containing EVs are no different from those of unlabeled EVs. The cell secretomes of 8 cell lines (HEK293T, Hela, JVM-3, etc.) and primary cells (chronic lymphocytic leukemia cells, etc.) were mass-tag labeled (e.g., TeLEV) under serum-free conditions, and sEVs were separated by a combination of 0.2 μm filtration, 10 kDa ultrafiltration, and size exclusion chromatography (qEV / 35 nm).

[0181] Materials and Methods Cell Culture: Before mass-tag labeling, cell lines were cultured in RPMI1640 medium (Gibco) supplemented with 10% FCS (Gibco), 1% GlutaMax (Gibco), and 1% penicillin-streptomycin (Gibco). Immediately before mass-tag labeling, primary chronic lymphocytic leukemia (CLL) cells were isolated by negative selection of EDTA blood from CLL patients with a white blood cell count in the range of 50 - 100 white blood cells per nL by adding 50 μL of RosetteSep human B cell enrichment cocktail (STEMCELL Technologies) at RT for 20 minutes and then density gradient layering (Lymphopure, BioLegend). The viability of cell lines and primary cells (DAPI exclusion, DAPI - cells > 95%) was evaluated by flow cytometry analysis immediately before preparing cell lines / primary cells for mass-tag labeling.

[0182] Mass-Tag Labeling: Monoisotopic L-2-terranylalanine diluted with water (e.g., 130TePhe) was thawed and directly added at a concentration of 50 μM to all tested cell lines and primary cells in serum-free synthetic medium supplemented with 1% GlutaMax (Gibco) and 1% penicillin-streptomycin (Gibco). The cytotoxicity of monoisotopic TePhe was tested for all cells / cell lines to find the optimal concentration for mass-tag labeling of all components of the cell secretome.

[0183] The cell line or primary cells were washed twice with PBS (RT, 5 minutes, 350 RCF). Next, the cells / cell line were resuspended in TePhe-containing medium at a cell density of 1e6 / mL (depending on the cell type) for 48 hours (depending on the cell type) to generate cell / cell line conditioned medium (CCM) containing mass-tagged components of the cell secretome.

[0184] Separation / concentration of mass-tagged components of the cell secretome: First, the CCM was collected after 48 hours by centrifugation (RT, 5 minutes, 350 RCF). The resulting supernatant was used to purify mass-tagged extracellular vesicles and mass-tagged soluble proteins in the following steps. Next, the supernatant was centrifuged once at 2000 RCF (4°C, 10 minutes) and then twice at 3000 RCF (4°C, 10 minutes). Thereafter, the clarified supernatant was filtered through a 0.2 μm filter according to the remaining components of the cell secretome to be separated / concentrated.

[0185] 50 mL of the filtered CCM was centrifuged using a 10 kDa MWCO ultrafiltration unit (Vivaspin Turbo 15 RC, Sartorius) (4°C, 20 minutes, 3000 RCF) to obtain 500 μL of CCM concentrate. Next, this 500 μL was layered on top of a size exclusion chromatography column (qEVoriginal / 35nm, Izon), and EV purification was performed according to the manufacturer's instructions. To recover most of the mass-tagged EVs with high purity, 2.5 mL of the void volume was discarded and 400 μL fractions were collected. The first 5 fractions (2 mL) containing EVs were pooled and concentrated to 200 μL by 10 kDa MWCO ultrafiltration. All other elution fractions were also collected in 400 μL fractions containing mass-tagged soluble proteins (such as antibodies, cytokines, and hormones) of the cell secretome. After concentration / separation, the mass-tagged samples were prepared for mass spectrometry and imaging mass spectrometry analysis.

[0186] Transmission electron microscopy (TEM) imaging of EVs: 5 μl of the diluted sample was added on top of a copper grid (formvar coated, Science Services) and incubated for 20 min as described above (6). After fixation with 2% paraformaldehyde for 5 min, the sample was washed with PBS and refixed with 1% glutaraldehyde for 5 min. The grid was washed with Milli-Q water and contrasted for 4 min in the presence or absence of 1.5% uranyl acetate (or left uncontrasted). EVs were imaged using a Gatan OneView 4K camera mounted on a Jem-2100Plus (Jeol) operated at 200 kV.

[0187] Mass spectrometry analysis of EV recipient cells In 1 mL of freshly supplemented RPMI 1640 medium, 1e6 freshly purified peripheral blood mononuclear cells were cultured for 16 hours in the presence of mass-tagged EVs from different cells / cell lines. EVs were administered for 48 hours according to the cell equivalent of 2e6 cell synthetic medium. Next, the cells were washed, live cells were barcoded (anti-CD45-Cd conjugate), pooled, and counted. 10e6 barcoded cells were used for mass cytometry staining. Cells were sequentially stained with the Maxpar Direct Immune Profiling assay (MDIPA, Fluidigm) and other surface and intracellular / nuclear markers according to the manufacturer's instructions, then the cells were freshly fixed (1.6% FA in PBS) and incubated overnight with 125 nM of the Cell-ID Intercalator-Ir (Fluidigm). At least 1e6 cells at a concentration of 5e5 cells per mL were acquired by the Helios mass cytometer (Fluidigm) in the cell acquisition solution (Fluidigm) containing 0.1X EQ 4-element calibration beads (Fluidigm). Data were normalized with CyTOF software (Fluidigm), barcode removed (ParkerICI / premessa, FR package), and cleaned up and analyzed using FCS Express 7 (new software). The Opt-SNE and FlowSOM algorithms were used for dimensionality reduction and clustering. The analyzed data were visualized as tSNE plots and multi-dimensional heatmaps to evaluate EV uptake and phenotypic and functional changes in EV recipient cells.

[0188] Conclusion In conclusion, the present invention provides a novel mass tag labeling approach for studying the interactions and effects of cell secretome components, such as extracellular vesicles (EVs), soluble proteins, and viruses, on the phenotype and function of recipient cells. This approach overcomes the limitations of existing labeling methods, such as low efficiency, high cost, and incompatibility with high-dimensional single-cell analysis. The mass tag labeling approach described herein ensures uniform and normalized labeling of various secretome components, eliminates the need for electroporation or other modifications, and enables compatibility with mass spectrometry, imaging mass spectrometry, electron microscopy-based techniques, and other mass spectrometry-based single-cell and imaging technologies. This new method improves labeling efficiency, enhances multiplexing capabilities, simplifies procedures, reduces costs, provides adaptability, increases sensitivity and specificity, and is a widely applicable and powerful tool for researchers studying the cell secretome.

[0189] References [1] van Niel, G., D’Angelo, G., & Raposo, G. (2018). Shedding light on the cell biology of extracellular vesicles. Nature Reviews. Molecular Cell Biology, 19(4), 213-228. [2] Moller, A., & Lobb, R. J. (2020). The evolving translational potential of small extracellular vesicles in cancer. Nature Reviews. Cancer, 20(12), 697-709. [3] Herrmann, I. K., Wood, M. J. A., & Fuhrmann, G. (2021). Extracellular vesicles as a next-generation drug delivery platform. Nature Nanotechnology, 16(7), 748-759. [4] Colombo, M., Raposo, G., & Thery, C. (2014). Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annual Review of Cell and Developmental Biology, 30, 255-289. [5] van Niel, G., Carter, D. R. F., Clayton, A., Lambert, D. W., Raposo, G., & Vader, P. (2022). Challenges and directions in studying cell-cell communication by extracellular vesicles. Nature Reviews. Molecular Cell Biology, 1-14. [6] Bachurski, D., Schuldner, M., Nguyen, P.-H., Malz, A., Reiners, K. S., Grenzi, P. C., Babatz, F., Schauss, A. C., Hansen, H. P., Hallek, M., & Pogge von Strandmann, E. (2019). Extracellular vesicle measurements with nanoparticle tracking analysis - An accuracy and repeatability comparison between NanoSight NS300 and ZetaView. Journal of Extracellular Vesicles, 8(1), 1596016. [7] Verweij, F. J., Balaj, L., Boulanger, C. M., Carter, D. R. F., Compeer, E. B., D’angelo, G., El Andaloussi, S., Goetz, J. G., Gross, J. C., Hyenne, V., & Others. (2021). The power of imaging to understand extracellular vesicle biology in vivo. Nature Methods, 18(9), 1013-1026. [8] Ramirez, M. I., Amorim, M. G., Gadelha, C., Milic, I., Welsh, J. A., Freitas, V. M., Nawaz, M., Akbar, N., Couch, Y., Makin, L., Cooke, F., Vettore, A. L., Batista, P. X., Freezor, R., Pezuk, J. A., Rosa-Fernandes, L., Carreira, A. C. O., Devitt, A., Jacobs, L., … Dias-Neto, E. (2018). Technical challenges of working with extracellular vesicles. Nanoscale, 10(3), 881-906. [9] Simonsen, J. B. (2019). Pitfalls associated with lipophilic fluorophore staining of extracellular vesicles for uptake studies. Journal of Extracellular Vesicles, 8(1), 1582237.

[10] Thery, C., Witwer, K. W., Aikawa, E., Alcaraz, M. J., Anderson, J. D., Andriantsitohaina, R., Antoniou, A., Arab, T., Archer, F., Atkin-Smith, G. K., & Others. (2018). Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles, 7(1), 1535750.

[11] Adapted from “Extracellular Vesicle Separation by Density Gradient” by BioRender.com (2022). Retrieved from https: / / app.biorender.com / biorender-templates.

[12] S. H. Ranganath, O. Levy, M. S. Inamdar, and J. M. Karp, “Harnessing the Mesenchymal Stem Cell Secretome for the Treatment of Cardiovascular Disease,” Cell Stem Cell, vol. 10, no. 3, 2012, doi: 10.1016 / j.stem.2012.02.005.

[13] C. R. Harrell, C. Fellabaum, N. Jovicic, V. Djonov, N. Arsenijevic, and V. Volarevic, “Molecular Mechanisms Responsible for Therapeutic Potential of Mesenchymal Stem Cell-Derived Secretome,” Cells, vol. 8, no. 5, 2019, doi: 10.3390 / cells8050467.

[14] C. Giesen et al., “Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry,” Nat. Methods, vol. 11, no. 4, 2014, doi: 10.1038 / nmeth.2869.

[15] M. Le Rochais, P. Hemon, J.-O. Pers, and A. Uguen, “Application of High-Throughput Imaging Mass Cytometry Hyperion in Cancer Research,” Front. Immunol., vol. 13, 2022, Accessed: May 02, 2023. [Online]. Available: https: / / www.frontiersin.org / articles / 10.3389 / fimmu.2022.859414

[16] J. Bassan et al., “TePhe, a tellurium-containing phenylalanine mimic, allows monitoring of protein synthesis in vivo with mass cytometry,” Proc. Natl. Acad. Sci. U. S. A., vol. 116, no. 17, pp. 8155-8160, Apr. 2019, doi: 10.1073 / pnas.1821151116.

[17] J. Wang et al., “Loading of metal isotope-containing intercalators for mass cytometry-based high-throughput quantitation of exosome uptake at the single-cell level,” Biomaterials, vol. 255, p. 120152, Oct. 2020, doi: 10.1016 / j.biomaterials.2020.120152.

[18] J. Wang et al., “High-throughput single-cell analysis of exosome mediated dual drug delivery, in vivo fate and synergistic tumor therapy,” Nanoscale, vol. 12, no. 25, pp. 13742-13756, Jul. 2020, doi: 10.1039 / d0nr02344b.

[19] H. Liu, Y. Tian, C. Xue, Q. Niu, C. Chen, and X. Yan, “Analysis of extracellular vesicle DNA at the single-vesicle level by nano-flow cytometry,” J. Extracell. Vesicles, vol. 11, no. 4, p. e12206, 2022, doi: 10.1002 / jev2.12206.

[20] K. B. Johnsen et al., “Evaluation of electroporation-induced adverse effects on adipose-derived stem cell exosomes,” Cytotechnology, vol. 68, no. 5, pp. 2125-2138, Oct. 2016, doi: 10.1007 / s10616-016-9952-7.

[21] J. Shin et al., “Comparative analysis of differentially secreted proteins in serum-free and serum-containing media by using BONCAT and pulsed SILAC,” Sci. Rep., vol. 9, no. 1, 2019, doi: 10.1038 / s41598-019-39650-z.

Claims

1. A method for producing mass-tagged soluble components from production cells, Exposing at least one type of production cell to a mass-tagged component that can be taken up by the at least one type of production cell, and Purifying a mass-tagged soluble component produced by at least one of the aforementioned production cells, A method that includes this.

2. The method according to claim 1, wherein the mass-tagged soluble component is selected from extracellular vesicles (EVs), viral particles, cellular secretomes, EV proteomes, EV secretomes, or any of the above components.

3. (i) At least one of the production cells is exposed to the mass-tagged component under serum-free conditions; (ii) The at least one of the production cells is derived from a cell line; and / or (iii) At least one of the production cells is derived from primary cells, The method according to claim 1.

4. The method according to claim 2, wherein the mass-tagged soluble component is extracellular viable (EV), and the EV is purified by filtration, ultrafiltration, or size exclusion chromatography.

5. A method using extracellular vesicles (EVs), wherein the components of the EVs are labeled with at least one mass tag, A method comprising bringing the EV and the receptor cell into contact so that the receptor cell takes up the EV.

6. (i) an in vivo method; (ii) It is an in vitro method; or (iii) including studies on in vivo distribution, The method according to claim 5.

7. The method according to claim 5, further comprising measuring changes in cellular function of the receptor cell after taking up the EV compared to before the receptor cell took up the EV, wherein the changes in cellular function are selected from the group consisting of apoptosis, DNA damage response, migration, proliferation, and tyrosine kinase signaling.

8. The aforementioned receptor cells are labeled with at least one detectable label. The at least one detectable label indicates the characteristics of the receptor cell, and The aforementioned characteristics of the receptor cell, either alone or in combination with other characteristics, distinguish the cell type of the receptor cell from at least one other cell type and / or identify the cell type of the receptor cell. The method according to claim 5.

9. The method according to claim 8, wherein each of the at least one detectable markers includes a mass tag.

10. The method according to claim 8, wherein the receptor cells are subjected to CD45-based live cell barcoding or palladium-based fixed cell barcoding to identify cells from different samples and / or cells of different cell types.

11. The method according to claim 8, further comprising using detectably labeled receptor cells and / or one or more detectably labeled reagents to characterize EV uptake and / or EV-mediated effects, to identify receptor cells and / or in multiplex analysis, wherein one or more detectably labeled reagents comprise one or more antibodies.

12. The method according to claim 5, comprising performing a technique selected from the group consisting of mass cytometry, mass cytometry imaging, and transmission electron microscopy on receptor cells.

13. A kit for carrying out the method of claim 1, comprising one or more mass-tagged components that can be taken up by at least one type of production cell.

14. The method according to claim 1, wherein the mass-tagged soluble component comprises an amino acid or an analog thereof.

15. The method according to claim 1, wherein the mass tag of the mass-tagged component includes an organic tellophene tag.

16. The method according to claim 1, wherein the mass-tagged soluble component comprises mass-tagged extracellular vesicles (EVs), the mass-tagged extracellular vesicles (EVs) being substantially indistinguishable from unlabeled EVs produced from the same cell type under the same conditions as labeled EVs.

17. The method according to claim 16, wherein mass-tagged EVs and unlabeled EVs have substantially the same effect on receptor cells.

18. The method according to claim 17, wherein the effects of mass-tagged EVs and unlabeled EVs differ by only ±15, ±14, ±13, ±12, ±11, ±10, ±9, ±8, ±7, ±6, ±5, ±4, ±3, ±2, ±1, or ±0.5 percent.

19. The method according to claim 16, wherein mass-tagged EVs and unlabeled EVs have substantially the same MISEV2018 characteristics with respect to one or more minimal information for studies of extracellular vesicles 2018 (MISEV2018) characteristics.

20. The method according to claim 17, wherein the properties of the mass-tagged EV and the unlabeled EV differ by only ±15, ±14, ±13, ±12, ±11, ±10, ±9, ±8, ±7, ±6, ±5, ±4, ±3, ±2, ±1, or ±0.5 percent.

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