Systems and methods for multiplexed proteomics

Abstract

The present disclosure provides methods and systems for detecting proteins, for example, from bulk and single cell lysates. Particularly the present disclosure includes methods including contacting immobilized proteins from a sample with a plurality of detection agents; analyzing target barcode oligonucleotides from barcoded detection agents bound to the immobilized proteins; and identifying proteins of interest in the sample.

Description

Atty. Docket No. JHU -43664.601P18336-03SYSTEMS AND METHODS FOR MULTIPLEXED PROTEOMICS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 748,100, filed January 22, 2025, and U.S. Provisional Patent Application No. 63 / 829,953, filed June 25, 2025, each of which is incorporated herein by reference.FIELD

[0002] The present disclosure provides methods and systems for detecting and analyzing proteins, for example, from bulk and single cell lysates.BACKGROUND

[0003] Comprehensive analysis of cellular proteins, their spatial distribution, and dynamic interactions is crucial for unraveling the complexity of cellular processes and understanding fundamental biological phenomena. However, cellular heterogeneity presents a challenge, as each cell exhibits a unique pattern of protein expression and modification, and this proteomic spectrum on the single cell level can have important implications on the system as a whole. The advent of singlecell RNA-seq (scRNA-seq) has transformed the study of heterogenous cell systems, providing more insights than ever observed before about the transcriptional landscape across individual cells.Techniques such as CITE-seq, REAP-seq, TEA-seq, and Abseq, to name a few, have further empowered our understanding of different cell types and their functional states by coupling scRNA-seq with the detection of surface markers in single cells through leveraging the multiplexing power of DNA-barcoded antibodies. This type of multimodal analysis has enabled additional complexity in the analysis and classification of cellular phenotypes that could not be previously achieved through transcriptomic information alone.

[0004] While surface protein profiling has provided valuable insights, it represents only a fraction of the vast protein landscape within cells, leaving the intracellular protein realm largely unexplored. Several groups have begun to employ barcoded antibodies to analyze intracellular protein expression, including nuclear protein expression, albeit on a small scale. The limited scale of these studies is likely due to the challenges posed by background noise from non-specific interactions between the negatively charged DNA barcode on the antibody and the cell nucleus. Several strategies have been devised to circumvent this issue, such as blocking cells with ssDNA or negatively charged polymers or blocking the antibody barcode with the ssDNA binding proteinAtty. Docket No. JHU -43664.601P18336-03 EcoSSB as in the NEAT-seq method. Despite the availability of these strategies, intracellular methods have mainly been employed on isolated nuclei rather than whole cells, thereby excluding a vast array of non-nuclear proteins from analysis.SUMMARY10005] Disclosed herein are methods for detecting proteins.10006] In some embodiments, the technology provides methods for detecting the identity of proteins in a sample. For example, in some embodiments, methods comprise (a) contacting the sample comprising proteins with magnetic beads, wherein the magnetic beads and proteins in the sample form bead-protein complexes; (b) contacting the bead-protein complexes with a plurality of detection agents, wherein each detection agent comprises: (i) an antibody or antibody fragment that binds to a protein within the bead-protein complexes; and (ii) a barcode oligonucleotide comprising: (1) at least one primer region; (2) a unique molecule identifier (UMI); and (3) a barcode sequence, wherein the barcode sequence is associated with the identity of a protein in the sample, wherein the antibody or the antibody fragment is covalently linked to the barcode oligonucleotide; (c) removing antibody or antibody fragments that are not bound to a protein; (d) contacting the sample with an enzyme solution, wherein the enzyme solution cleaves each antibody from each barcode oligonucleotide of the detection agents and removes the proteins from the sample; (c) isolating a plurality of barcode oligonucleotides from the sample; (f) amplifying the plurality of barcode oligonucleotides to form a plurality of amplified oligonucleotides; (g) for each of the oligonucleotides of the plurality of oligonucleotides, hybridizing an identification oligonucleotide to the barcode oligonucleotide and elongating the barcode oligonucleotide to incorporate an index sequence from the identification oligonucleotide into the barcode oligonucleotide to form elongated oligonucleotides, wherein the identification oligonucleotide comprises: (i) an adapter region that hybridizes to the at least one primer region; and (ii) an index sequence, wherein the index sequence is associated with a number for the sample; (h) sequencing the elongated oligonucleotides; and (i) analyzing the barcode sequences and identifying the associated identity of the proteins in the sample for each barcode sequence, thereby detecting the identity of proteins in the sample.

[0007] In some embodiments, the sample is a biological sample, a cell lysate of a single cell, a bulk cell lysate from a population of cells, or a bulk cell lysate from a tissue or organ. In some embodiments, the sample is not partitioned. In some embodiments, removing the antibody or the antibody fragments that are not bound to a protein comprises a blocking step. In some embodiments, the blocking step comprises contacting the bead-protein complexes in the sample with bovine serum albumin. In some embodiments, the enzyme solution comprises proteinase K. In some embodiments,Atty. Docket No. JHU -43664.601P18336-03 the method further comprises exposing the enzyme solution to heat to inactivate enzymes in the solution. In some embodiments, the amplifying comprises a polymerase chain reaction (PCR). In some embodiments, the amplifying is repeated one or more times.

[0008] In some embodiments, the index sequence comprises 2 to 20 base pairs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs). In some embodiments, the index sequence comprises 5 to 10 base pairs (e.g., 5, 6, 7, 8, 9, or 10 base pairs). In some embodiments, the index sequence comprises about 8 base pairs. In some embodiments, the identification sequence comprises an additional index sequence. In some embodiments, the barcode oligonucleotide further comprises a spacer between the UMI and the barcode sequence. In some embodiments, the plurality of detection agents is directed to 100 or more proteins of interest.

[0009] In some embodiments, the antibody or the fragment thereof comprises a monoclonal antibody or fragment thereof.

[0010] In some embodiments, the barcode oligonucleotide comprises a DNA, an RNA, or a hybrid of DNA and RNA. In some embodiments, the barcode oligonucleotide comprises a double stranded DNA. In some embodiments, the barcode oligonucleotide comprises a length of about 30 to about 150 base pairs (e.g., 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs).

[0011] In some embodiments, the technology described herein relates to a method for detecting proteins in a sample. For example, in some embodiments, methods comprise contacting immobilized proteins from the sample with a plurality of detection agents, wherein each detection agent comprises a binding agent specific to a protein of interest covalently attached to a barcode oligonucleotide; isolating target barcode oligonucleotides from barcoded detection agents bound to the immobilized proteins; analyzing the target barcode oligonucleotides; and identifying proteins of interest in the sample. In some embodiments, the plurality of detection agents is directed to 5 or more proteins of interest. In some embodiments, the plurality of detection agents is directed to 100 or more proteins of interest. In some embodiments, the binding agent comprises a protein or peptide. In some embodiments, the binding agent is an antibody or fragment thereof. In some embodiments, the binding agent is a monoclonal antibody.

[0012] In some embodiments, methods further comprise immobilizing proteins from the sample on a solid support. In some embodiments, the solid support is a bead or particle. In some embodiments, the immobilized proteins are in a suspension. In some embodiments, the contacting comprises incubating the immobilized proteins with the plurality of detection agents for at least 10 minutes.

[0013] In some embodiments, methods further comprise removing detection agents not bound to an immobilized protein. In some embodiments, the barcode oligonucleotide comprises DNA. In some embodiments, the barcode oligonucleotide comprises double-stranded DNA. In some embodiments,Atty. Docket No. JHU -43664.601P18336-03 each barcode oligonucleotide comprises a barcode and a unique molecular identifier (UMI) flanked by primer binding sites. In some embodiments, the barcode oligonucleotide has a length of about 30 to about 150 base pairs (e.g., 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs). In some embodiments, analyzing the target barcode oligonucleotides comprises amplifying and / or sequencing the barcode and UMI. In some embodiments, analyzing comprises introducing a sample identification tag to the barcode and amplifying and / or sequencing the sample identification tag, barcode, and UMI. In some embodiments, methods further comprise determining expression levels of the proteins of interest based on a comparison of barcode sequence numbers analyzed for each detection agent.

[0014] In some embodiments, methods further comprise determining transcript levels for the proteins of interest. In some embodiments, determining comprises capturing mRNA from the sample and generating an mRNA-seq library from the sample.

[0015] In some embodiments, methods further comprise determining one or more characteristics of the sample based on proteins of interest present in the sample, expression levels of the proteins of interest in the sample, transcript levels of the proteins of interest in the sample, or a combination thereof. In some embodiments, the sample is a biological sample. In some embodiments, the sample is a cell lysate of a single cell. In some embodiments, the sample is a bulk lysate of cells from a single cell type or tissue. In some embodiments, the sample is a bulk lysate of disease-associated cells.

[0016] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-1E show multiplexed detection of proteins at the bulk cell level. FIG. 1A shows a schematic of monoclonal antibodies with a barcode for BEND-Westem. FIG. IB shows the analysis of barcoding efficiency with Coomassie (left) and SYBR-gold (right) staining on a PAGE gel. FIG. 1C is a schematic for the BEND-Wcstcrn pipeline. FIG. ID shows a schematic (top) of the positive and negative controls alongside the cell lysate experimental sample and the corresponding results of the qPCR and Western analysis (bottom). FIG. IE shows graphs of the expression of select protein in brain and liver, as indicated, following BEND-Westem.

[0018] FIGS. 2A-2E show BEND-Westem to detect proteins at the single cell level. FIG. 2A shows the quantification of GAPDH from qPCR as compared to cell lysate dilution as indicated by cell number. FIGS. 2B-2D show traditional Western blot analysis performed utilizing approximately 250,000 cells per assay (left) and analysis of cell lysates from 4000 cells down to the single cell levelAtty. Docket No. JHU -43664.601P18336-03 using BEND- Western in which protein abundance (Cyclin A-FIG. 2A, Cyclin B-FIG. 2C, and Cyclin E -FIG. 2D) was analyzed using qPCR with barcode-specific primers at three different cell dilutions, as indicated (right three panels). FIG. 2E is a heatmap of the average protein expression across the cell cycle with known cell cycle peak expression for each protein denoted to the left of the heatmap.10019] FIGS. 3A-3F show single-cell differential protein expression across the cell cycle as determined by BEND-Westem. FIG. 3A is a pie chart of the selected 36 barcoded mAbs for singlecell analysis. FIG. 3B shows the counts for both the positive and negative controls, showing relative consistency across cell cycle. FIGS. 3C-3F show the cluster map of protein expression based on cell cycle stage.

[0020] FIGS. 4A-4F show BEND-Westem analysis of transcription factors (TFs) across cardiomyocyte differentiation at the single cell level with a schematic of the developmental stages of iPSC-derived cardiomyocytes, showing timepoints at which point cell lysates were collected for analysis (FIG. 4A) and a pie chart of the selected 36 barcoded mAbs for single-cell analysis of cardiomyocytc differentiation (FIG. 4B).DETAILED DESCRIPTION

[0021] Highly multiplexed detection of intracellular proteins has been a long-standing challenge in the field of cell biology, with previous studies being limited in multiplexing capabilities or largely focusing on surface protein detection. Provided herein are methods, systems, and kits for detecting and assessing the expression of hundreds to thousands of intracellular protein targets either in bulk samples or with single-cell sensitivity.

[0022] The disclosed methods immobilize proteins in a sample, e.g., a cellular lysate, which are then incubated with a cocktail of barcoded antibodies and washed thoroughly prior to release of the barcodes. Analysis of the resulting barcodes can be in a bulk cell fomrat using qPCR or at the single cell level through deep sequencing. The immobilization reduces background signal significantly by allowing for thorough washing of non-bound sample constituents, a step which is generally less efficient in typical antibody staining protocols. Thus, these methods can be especially useful for precious samples, such as needle biopsies, as very few cells are required to obtain expression data. The disclosed methods are also highly adaptable to diverse cell types, including complex tissues. These methods can be easily modified to simultaneously generate mRNA-seq libraries from the same single cells, allowing a comprehensive view of the expression and transcriptional landscape of thousands of single cells in a single experiment. These methods can also be applied to analyze paraffin-embedded tissues sections (FFPE), fresh-frozen tissue sections (OCT), and tissue lysates. These methods can also be for samples isolated by laser capture microdissection (ECM).Atty. Docket No. JHU -43664.601P18336-03

[0023] In other proteomic methods that utilize a cell staining based approach with antibodies or barcoded antibodies, there is a limit to the number of antibodies that can be utilized per experiment because at a certain number of antibodies a crowding effect will begin to be observed due to the inability of more antibody to enter the cell. The disclosed methods remove this crowding effect with more flexible access to cellular proteins without having to cross membranes or fit inside the cell.

[0024] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0025] The terms “comprise(s)”, “include(s)”, “having”, “has", “can”, “contain(s)”, and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. However, two or more copies are also contemplated. The singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising”, “consisting of”, and “consisting essentially of’, the embodiments or elements presented herein, whether explicitly set forth or not.

[0026] For the recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0027] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Fikewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C: A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0028] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0029] “Antibody” and “antibodies” as used herein refers to monoclonal antibodies, monospecific antibodies (e.g., which can either be monoclonal, or may also be produced by other means than producing them from a common germ cell), multi-specific antibodies, human antibodies, humanized antibodies (fully or partially humanized), animal antibodies such as, but not limited to, a bird (forAtty. Docket No. JHU -43664.601P18336-03 example, a duck or a goose), a shark, a whale, and a mammal, including a non-primate (for example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, etc.) or a non-human primate (for example, a monkey, a chimpanzee, etc.), recombinant antibodies, chimeric antibodies, single-chain Fvs (“scFv”), single chain antibodies, single domain antibodies, Fab fragments, F(ab’) fragments, F(ab’)2 fragments, disulfidc-linkcd Fvs (“sdFv”), and anti-idiotypic (“anti-Id”) antibodies, dual-domain antibodies, dual variable domain (DVD) or triple variable domain (FVD) antibodies (dual- variable domain immunoglobulins and methods for making them are described in Wu, C., et al., Nature Biotechnology, 25(11): 1290— 1297 (2007) and PCT International Application WO 2001 / 058956, the contents of each of which are herein incorporated by reference), or domain antibodies (dAbs) (e.g., such as described in Holt et al., Trends in Biotechnology 21:484-490 (2014)), and including single domain antibodies sdAbs that are naturally occurring, e.g., as in cartilaginous fishes and camelid, or which are synthetic, e.g., nanobodies, VHH, or other domain structure), and functionally active epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, namely, molecules that contain an analyte-binding site. Immunoglobulin molecules can be of any type (for example, IgG, IgE. IgM, IgD, IgA, and IgY), class (for example, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass. For simplicity’s sake, an antibody against an analyte is frequently referred to herein as being either an “anti-analyte antibody” or merely an “analyte antibody.”

[0030] “Antibody fragment” as used herein refers to a portion of an intact antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 25(9): 1126— 1129 (2005)) (e.g., comprises the antigen-binding site or variable region). Any antigen-binding fragment of the antibody described herein is within the scope of the present disclosure. The antibody may not include the constant heavy chain domains (e.g., CH2, CH3, or CH4, depending on the antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab’ fragments, Fab’-SH fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, diabodies, single-chain Fv (scFv) molecules, single-chain polypeptides containing only one light chain variable domain, single-chain polypeptides containing the three CDRs of the light-chain variable domain, single-chain polypeptides containing only one heavy chain variable region, and single-chain polypeptides containing the three CDRs of the heavy chain variable region.

[0031] Typically, an immunoglobulin or antibody is a protein that comprises at least one complementarity determining region (CDR). The CDRs form the “hypervariable region” of an antibody, which is responsible for antigen binding (discussed further below). A whole antibodyAtty. Docket No. JHU -43664.601P18336-03 typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2, and CH?) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The light chains of antibodies can be assigned to one of two distinct types, cither kappa (K) or lambda (A), based upon the amino acid sequences of their constant domains. In a typical antibody, each light chain is linked to a heavy chain by disulfide bonds, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other.

[0032] The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have the same general structure, with each region comprising four framework (FW or FR) regions. The term “framework region”, as used herein, refers to the relatively conserved amino acid sequences within the variable region which arc located between the CDRs. There are four framework regions in each variable domain, which are designated FR1, FR2, FR3, and FR4. The framework regions form the sheets that provide the structural framework of the variable region (see, e.g., C. A. Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y. (2001)).

[0033] The term “contacting" as used herein refers to bring or put in contact, to be in or come into contact. The term “contact” as used herein refers to a state or condition of touching or of immediate or local proximity.

[0034] The term “detecting” as used herein generally refers to any form of measurement and includes determining whether an element is present or not. This term includes quantitative and / or qualitative determinations.[0035| The terms “nucleic acid”, “polynucleotide”, and “oligonucleotide” are used herein to describe a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. As used herein in the context of a polynucleotide sequence, the term “bases” (or “base”) is synonymous with “nucleotides” (or “nucleotide”), the monomer subunit of a polynucleotide. The terms “nucleoside” and “nucleotide” are intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. InAtty. Docket No. JHU -43664.601P18336-03 addition, the terms “nucleoside” and “nucleotide” include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like. “Analogues” refer to molecules having structural features that arc recognized in the literature as being mimetics, derivatives, having analogous structures, or other like terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2’ -modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide that has added substituent groups, such as protecting groups or linking moieties.

[0036] The term “complementary” refers to specific binding between polynucleotides based on the sequences of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in a hybridization assay.Portions of polynucleotides arc complementary to each other if they follow conventional basepairing rules, e.g., A pairs with T (or U) and G pairs with C, although regions (e.g., less than 5 nucleotides) of mismatch, insertion, or deleted sequence may be present.

[0037] As used herein, the tern “primer” refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis of an extension product that is a complementary strand of nucleic acid (all types of DNA or RNA) when placed under suitable amplification conditions (e.g., buffer, salt, temperature and pH) in the presence of nucleotides and an agent for nucleic acid polymerization (e.g., a DNA-dependent or RNA-dependent polymerase). The term “amplification conditions”, as used herein, refers to conditions that promote annealing and / or extension of the amplification oligonucleotides. Such conditions are well-known in the art and depend on the amplification method selected. Amplification conditions encompass all reaction conditions including, but not limited to, temperature and / or temperature cycling, buffer, salt, ionic strength, pH, and the like.

[0038] As used herein, the tern “sample” is used in its broadest sense. In one sense, it is meant to include a specimen obtained from any source, including biological samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Such examples are not however to be construed as limiting the sample types. Preferably, a sample is a fluid sample such as a liquid sample. Examples of liquid samples that may be assayed include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, pleural effusions, ascites, thin needle aspirates and spinal fluid). Viscous liquid, semisolid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples. For example, throat or genital swabs may be suspended in a liquid solution to make aAtty. Docket No. JHU -43664.601P18336-03 sample. Samples can comprise biological materials, such as cells, microbes, organelles, and biochemical complexes. Liquid samples can be made from solid, semisolid, or highly viscous materials, such as fecal matter, tissues, organs, biological fluids, or other samples that are not fluid in nature. For example, solid or semisolid samples can be mixed with an appropriate solution, such as a buffer, a diluent, and / or extraction buffer. The sample can be macerated, frozen and thawed, or otherwise extracted to form a fluid sample. Residual particulates may be removed or reduced using conventional methods, such as filtration or centrifugation.

[0039] “Biological sample”, “sample from a subject”, “test sample”, and “patient or subject sample” as used interchangeably herein may be a sample of blood, such as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), tissue, urine, serum, plasma, amniotic fluid, an anal sample (such as an anal swab specimen), lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage, nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0040] A variety of cell types, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, disease samples (e.g., tumor samples), oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, frozen sections taken for histologic purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, red blood cells, platelets, an anal sample (such as an anal swab specimen), interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues may also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration. A tissue or cell type may be provided by removing a sample of cells from a human and a non-human animal but can also be provided by using previously isolated cells (e.g., isolated by another person, at another time, and / or for another puipose). Archival tissues or preserved tissues, such as those having treatment or outcome history, may also be used. The term “tissue” is used herein to denote any structure of cells, which may optionally be, for example, a structure, an organ, or part of a structure of organ. The tissue sample may be derived from a human or a non-human animal or plant tissue.

[0041] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse models, prokaryotic models (e.g., bacteria), archea, and single-celled eukaryotes (e.g., yeast). Likewise, subject may include either adults or juveniles (e.g., children). Moreover, patient may mean any livingAtty. Docket No. JHU -43664.601P18336-03 organism, preferably a mammal (e.g., humans and non-humans) that may benefit from the uses of compositions and methods contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the subject is a human.

[0042] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.2. Methods of Detection Proteins in a Sample

[0043] In one aspect, provided herein are methods for detecting proteins in a sample. The methods comprise contacting immobilized proteins from the sample with a plurality detection agents, wherein each detection agent comprises a binding agent specific to a protein of interest covalently attached to a barcode oligonucleotide; removing target barcode oligonucleotides from barcoded detection agents bound to the immobilized proteins; analyzing the target barcode oligonucleotides; and identifying proteins of interest in the sample.

[0044] The methods disclosed herein are suitable for use in detecting any number of proteins of interest, e.g., one or more proteins of interest. For example, the methods may be used to detect or identify a large number of proteins of interest simultaneously in a sample. The plurality of detection agents can include individual detection agent directed to any number of proteins of interest. For example, the plurality of detection agents may include detection agents individually directed to 5 or more proteins of interest, 10 or more proteins of interest, 50 or more proteins of interest, or 100 or more proteins of interest. Thus, the totality of the plurality of detection agents can target any number of different proteins of interest. The plurality of detection agents may further comprise more than one copy of any one detection agent directed to an individual protein of interest, allowing a measure of the quantity or relative quantity of the protein of interest. Accordingly, the methods allow for multiplexed detection of proteins in a sample. The disclosed methods can be used to detect and identify proteins of interest in bulk samples or single cell samples.

[0045] The methods are not limited by the proteins of interest. The proteins of interest may include cell cycle proteins, signal-pathway related proteins, disease-related proteins, transcriptionAtty. Docket No. JHU -43664.601P18336-03 factors, and the like. The proteins of interest may be cancer-related proteins. For example, the proteins of interest may include transcription factors with known roles in cancer, e.g., BACH 1 / 2, BRD9, CEBP family members, CREB5, ERG, FOS / JUN and related proteins, GATA4, FOX family members, KLFs, MYC and MYC-associated factors, SMAD family members, SOX family members, STAT family members, and multiple zinc fingers, as well as HDACs and other chromatin-associated proteins.

[0046] The methods may differentiate between modified and unmodified (e.g., phosphorylated, glycosylated, ubiquitinated, nitrosylated, methylated, acetylated, lipidated) versions of the proteins of the interest. For example, phosphorylation specific antibodies can be used for a protein of interest as applicable in addition or in alternative to a binding agent which detects any form of the protein of interest.

[0047] The proteins from the sample are immobilized on a solid support or surface. In some embodiments, the methods further comprise immobilizing proteins from the sample on the solid support or surface. Immobilizing the proteins from the sample allows the remainder of the sample to be used in additional analyses, e.g., to isolate, identify, and / or analyze other components such as nucleic acids in the sample, as described elsewhere herein.

[0048] The proteins may be immobilized using a variety of methods depending on the nature of the protein and the specific solid support or surface being utilized. The proteins may be covalently attached to the solid support or surface. For example, the surface may be chemically modified to introduce functional groups that form covalent bonds with the protein. For example, the solid support or surface may be functionalized with N-hydroxysuccinimide (NHS) ester groups, which react with primary amines on the protein to form stable amide bonds. Other covalent immobilization techniques may use carbodiimides to activate carboxyl groups on the solid surface or support, aldehydes to crosslink amino groups on the proteins to amino groups on the solid surface or support, and maleimide-functionalized surfaces to react with any free protein thiols. Other immobilization methods include physical adsorption due to electrostatic or hydrophobic interactions or affinity tag-based immobilizations, encapsulation in polymers or hydrogels.

[0049] In some embodiments, the solid support or surface is a bead or particle. By the terms “bead” or “particle” as used herein is meant any solid particle, of virtually any size and shape, for example, microbead, microparticle, microsphere, nanoparticle, nanobead, that is configured for immobilization of the proteins of interest. In some embodiments of the present invention, microbeads may be irregularly shaped, or substantially spherical, cubic, rhomboid, tetragonal, dodecahedral, ovoid, cylindrical, etc. Microbeads may have any surface texture. In some embodiments of theAtty. Docket No. JHU -43664.601P18336-03 present invention, microbeads may have surfaces that are substantially smooth, dimpled, wrinkled, angled, etc.

[0050] The beads or particles may be composed of any of a wide variety of materials, for example, polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, membranes, hydrogels, or any combinations thereof. For example, the beads may be fully or partially composed of agarose, sepharose, silica, or polystyrene. The beads or particles may be treated, coated, modified, printed or derivatized using polymers, chemicals to impart desired properties or functionalities to the array support surface. The beads or particles may be compatible with the range of conditions encountered during the methods including salt concentrations and solvents, and be stable under the application of magnetic fields. The bead or particle may be magnetic, e.g., magnetic beads, magnetic microbeads, paramagnetic particles, magnetically attractable particles, magnetic spheres, and magnetically responsive particles, which are capable of being manipulated in a liquid with the application of a magnetic field.

[0051] The beads or particles having proteins from the sample immobilized thereon may be provided in a suspension comprising beads or particles and diluent, where the beads or particles are evenly dispersed throughout the suspension or can be evenly dispersed upon mixing.

[0052] Contacting immobilized proteins with a plurality detection agents may comprise adding the plurality of detections agents to the suspension of beads or particles having proteins immobilized thereon. Alternatively, the suspension of beads or particles having proteins immobilized thereon can be added to a solution a plurality of detection agents. The sample and the immobilized proteins may be separated such that the contacting for a single sample may be completed over a series of different reaction mixtures or aliquots. Alternatively, the proteins from the entire sample may be incubated with the plurality of detection agents in a single mixture.

[0053] The detection agents comprise a binding agent specific to a protein of interest covalently attached to a barcode oligonucleotide. The binding agents include any moiety (e.g., nucleic acid, peptide, protein, or small molecule compound) which specifically binds to an individual protein of interest. In some embodiments, the binding agent is a peptide- or protein-based agent. In select embodiments, the binding agent is an antibody or fragment thereof directed to the protein of interest. Any suitable antibody or fragment thereof can be used in detection agents. Preferably, the antibodies or fragments thereof are validated with high specificity and selectivity. In some embodiments, the antibody or a fragment thereof binds directly to the protein of interest.

[0054] In some embodiments, each binding agent is selected from a monoclonal antibody, a multispecific antibody, a bispecific antibody, a trispecific antibody, a tetravalent antibody, a singledomain antibody, a chimeric antibody, and a polyclonal antibody mixture. In some embodiments, theAtty. Docket No. JHU -43664.601P18336-03 binding agent(s) are selected from a Fab, a scFv, a Fv, a scFv-Fc, a Fab', a Fab'-SH, a F(ab')2, a diabody, a minibody, and a tribody. In select embodiments, the binding agent is a monoclonal antibody which specifically binds to an individual protein of interest.

[0055] The binding agents are covalently attached to a barcode oligonucleotide. The respective barcode oligonucleotide for each of the binding agents provides a sequence which can be used to identify the protein of interest, the sample source (e.g., cell type, tissue, etc.) or both.

[0056] The barcode oligonucleotides comprise a barcode and a unique molecular identifier (UMI) flanked by primer binding sites. Each of the sequences for the barcode, UMI, and the primer binding sites can be separated by spacer base pairs or can be immediately adjacent to each other.

[0057] The barcode sequence may be any length or sequence. The barcode sequence can be used to specify the protein of interest to which the binding agent, and thereby the detection agent, is configured to bind. The UMI may be any suitable sequence of nucleic acids of any suitable length. The UMI may correlate with a specific protein of interest, but can also be used to account for PCR and sequencing artifacts in subsequent sequencing analysis, e.g., as a control for measuring read counts for any matched pair of barcode / UMI.

[0058] fhe barcode oligonucleotides can vary in length based on the size of each of the described components. For example, the barcode oligonucleotides can have a length of about 20 to about 150 base pairs. In some embodiments, the barcode about 5 to about 30 base pairs in length. In some embodiments, the UMI is about 5 to about 20 base pairs in length. In some embodiments, the primer binding sites can be about 10 to about 40 base pairs in length.

[0059] The barcode oligonucleotides can comprise DNA, RNA, or a combination thereof. In some embodiments, the barcode oligonucleotides are DNA. The barcode oligonucleotides can be fully or partially double stranded. In some embodiments, the barcode oligonucleotides are double stranded DNA.[0060| The barcode oligonucleotide is covalently attached to the binding agent (e.g., antibody or fragment thereof). In some embodiments, the covalent attachment is via a direct bond between the binding agent (e.g., antibody or fragment thereof) and the barcode oligonucleotide. In some embodiments, the barcode oligonucleotide is covalently attached via a linker.

[0061] General methods of conjugating oligonucleotides to proteins and peptides (e.g., antibodies and fragments thereof) are known to those skilled in the art. For example, a typical conjugation method includes use of a linker compound that includes two distinct reactive moieties, which react with different types of functional groups (e.g., one group that reacts with an amine, such as an activated ester group, and one group that reacts with a thiol, such as a maleimide group). Such reactive moieties used in conjugation reactions are well-known to those skilled in the art, and includeAtty. Docket No. JHU -43664.601P18336-03 activated esters such as succinimidyl and sulfosuccinimidyl esters and pentafluorophenyl esters, maleimides, azides, alkynes, hydrazines, isocyanates, isothiocyanates, haloacetamides, and the like. Methods of installing such reactive groups are well-known to those skilled in the art. As one nonlimiting example, an amino group can be installed at the 5 ’-end of an oligonucleotide via phosphoramiditc chemistry.

[0062] In some embodiments, the linker can include one or more nucleotides. For example, the linker can comprise an oligonucleotide sequence. Such a sequence may be considered separate from the barcode sequence.

[0063] The linker can include additional atoms or groups; for example, if the binding agent is reacted with 2-iminothiolane, it is understood that the linker will further include atoms derived from such reaction. In some embodiments, the linker further comprises one or more additional groups selected from -CH2-, -O-, -NH-, -S-, -C(=O)-, -C(=NH)-, and any combination thereof (e.g., combinations of such moieties could include ester groups (-C(=O)O-), amide groups (-C(=O)NH-), carbamate groups (-NHC(=O)O-), ethylene glycol groups (-CH2CH2O-), and the like.

[0064] In some embodiments, the linker comprises an antibody -binding domain. In some embodiments, an antibody binding domain (AbBD) comprises Protein A, Protein G, Protein L, CD4, or a fragment thereof. In some embodiments, the antibody -binding domain is an engineered antibody-binding domain, such as to include a non-natural amino acid, a photoreactive group, or a crosslinker. In some embodiments, the antibody binding domain is operably linked to a photoreactive amino acid group, for example, benzoylphenylalanine (BPA), resulting in a photoreactive antibody binding domain (pAbBD). In some embodiments, the antibody-binding domain (AbBD) is operably linked to a photoreactive amino acid which is operably linked to an antibody or a fragment thereof.

[0065] In some embodiments, a binding agent is first reacted with the linker compound to provide a functionalized binding agent, which is subsequently reacted with the barcode oligonucleotide to provide the detection agent. In other embodiments, a barcode oligonucleotide is first reacted with the linker compound to provide a functionalized barcode oligonucleotide, which is subsequently reacted with a binding agent to provide the detection agent.

[0066] In some embodiments, a binding agent is first conjugated with anchor oligonucleotide. The anchor oligonucleotide comprises a nucleic acid sequence which hybridizes to at least a portion of a single-stranded barcode oligonucleotide. To form a double-stranded barcode oligonucleotide the anchor oligonucleotide can be extended using known DNA synthetic enzymes and methods.

[0067] The contacting may be completed for any length of time or at any temperature which facilitates interactions between the binding agent and the proteins of interest. In some embodiments, the contacting comprises an incubation of the detection agent with the immobilized proteins ofAtty. Docket No. JHU -43664.601P18336-03 interest for at least 10 minutes (e.g., about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hours, or more). In some embodiments, the contacting comprises an incubation at about 4 °C to about 50 °C (e.g., about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C, about 11 °C, about 12 °C, about 13 °C, about 14 °C, about 15 °C, about 16 °C, about 17 °C, about 18 °C, about 1 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C, about 32 °C, about 33 °C, about 34 °C, about 35 °C, about 36 °C, about 37 °C, about 38 °C, about 39 °C, about 40 °C, about 41 °C, about 42 °C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, about 50 °C).[0068| In some embodiments, the contacting comprises mixing the reaction comprising the detection agents with the immobilized proteins of interest. In some embodiments, the reaction may be mixed during the incubation by stirring, shaking, rotating, swirling, vortexing, or other appropriate means based on the vessel.

[0069] In some embodiments, the methods further comprise separating or isolating detection agents and binding partners from the sample. In some embodiments, separating or isolating the detection agents and their respective binding partners also facilitates removal of unbound detection agents. In some embodiments, the methods comprise removing the unbound detection agents and washing the detection agent-protein of interest complexes to reduce background signals from unbound detection agents being retained for downstream analysis. Preferred wash solutions and conditions are those that do not change the configuration of the binding agent or protein of interest and do not dismpt the binding interactions in the detection agent-protein of interest complexes. Additionally, if non-covalent immobilization methods were used, the wash solution used will retain the non-covalent immobilization of the protein of interest to the solid support or surface.

[0070] The methods further comprise isolating target barcode oligonucleotides from barcoded detection agents bound to the immobilized proteins. Any methods known in the art for purifying or separating nucleic acid can be used to target barcode oligonucleotides. In some embodiments, the method comprises treating the immobilized detection agent-protein of interest complexes with a protease followed by DNA extraction. In some embodiments, the protease is selected from trypsin, proteinase K, pepsin, pronase, endoproteinase cAspN, and endoproteinase GluC.

[0071] Following isolation the target barcode oligonucleotides are analyzed. In some embodiments, analyzing the target barcode oligonucleotides comprises amplifying and / or sequencing the barcode and UMI. In some embodiments, the amplifying and / or sequencing utilize a corresponding barcode specific primer for each detection agent. Any suitable amplification method known in the art allowing for sensitive detection of DNA may be used, including by not limited toAtty. Docket No. JHU -43664.601P18336-03 polymerase chain reaction (PCR), preferably real time PCR. Sequencing can be accomplished using high-throughput systems, some of which allow detection of a sequenced nucleotide immediately after or upon its incorporation into a growing strand, e.g., detection of sequence in real time or substantially real time. In some embodiments, the sequencing is next-generation sequencing.

[0072] In some embodiments, the methods further comprise introducing a sample identification tag to the barcode prior to sequencing. For example, the sample identification tag may be a nucleic acid sequence used to tag or identify samples from certain cells, certain cell types, certain tissues or organs, and the like. Thus, the sample identification tag can, along with the barcode, indicate the protein of interest and its origin. The sample identification tag can be added by a ligation reaction or a short round of amplification to the barcode sequence using primers comprising the sample identification tag. As a result, a new set of primers may be added to the barcode having the sample identification tag which can then be used for subsequent rounds of amplification and / or sequencing.

[0073] In some embodiments, the methods further comprise identifying the protein(s) of interest based on the barcode and / or unique molecular identifier (UMI) sequences. The analysis can comprise extracting the barcodes, primer sequences, UMI, sample identification tag, and / or linkers from the detection agents, sequencing the oligonucleotides, and comparing them to the sequence files created during the analysis to identify those proteins of interest which associate to the sequences and samples. Details for the subsequent analysis is provided in Examples below. The analysis may be completed using computer implemented methods, also provided in the present disclosure.

[0074] The methods may further comprise determining the expression level of the proteins of interest based on a comparison of barcode sequence numbers analyzed for each detection agent. As described in the examples below, utilizing positive and negative control detection agents and quantifying the reads for any individual detection agent-protein of interest complex can provide a measure of the level of expression of the protein of interest in the sample.[0075| The methods may further comprise one or more analyses of the sample. In some embodiments, the methods further comprise determining the transcription level of the proteins of interest. The transcription level may be determined by analysis of the mRNA in the sample. The methods may further comprise capturing mRNA from the sample and generating an mRNA-seq library from the sample. In some embodiments, the mRNA is captured from the sample remaining after the proteins are immobilized on the solid support or surface.

[0076] In some embodiments, the sample is a biological sample. The biological sample can be derived from various sources. In some embodiments, the biological sample is derived from a subject or patient. In some embodiments, the biological sample is a tissue specimen or is derived from a tissue specimen (e.g., a tissue biopsy sample). In some embodiments, the biological sample is aAtty. Docket No. JHU -43664.601P18336-03 blood sample or is derived from a blood sample. In some embodiments, the biological sample is a cytological sample or is derived from a cytological sample. In some embodiments, biological sample is cultured cell(s).

[0077] In some embodiments, the sample is a cell lysate. The cell lysate may be derived from a single cell. Alternatively, the cell lysate may be a bulk cell lysate from a single cell type, a single tissue, or a subset of disease-associated cells.

[0078] In some embodiments, the methods comprise generating a cell lysate from a bulk or single cell sample. A cell or tissue sample may be lysed using chemical or physical technique to release the intracellular contents, including the intracellular proteins for immobilization and detection. The protein portion of the lysed sample may be isolated from the remaining contents by centrifugation, density gradients, or precipitation-based methods prior to immobilization.

[0079] In some embodiments, the sample is derived from paraffin-embedded tissues sections (FFPE), fresh-frozen tissue sections (OCT), or a tissue lysate. In some embodiments, the sample is isolated from tissues by laser capture microdissection (LCM).

[0080] In some embodiments, the methods further comprise determining one or more characteristics about the sample based on the identified proteins of interest, the expression level of the proteins of interest, the transcription level of the proteins of interest, status of posttranslational modification of the proteins of interest, mutation of the proteins of intertest, or a combination thereof. For example, the analysis of the proteins of interest may aid in diagnosing of a disease or disorder, determining the functional stage of a tissue or cell (e.g., cell cycle stage), distinguishing specific tissue / cell types of the sample, or other characteristics of the sample.3. Kits

[0081] In another aspect, the disclosure provides kits comprising a plurality of detection agents, as described herein. In some embodiments, the kits comprise one or more components to generate detection agents, as described herein. For example, the kit may include one or more antibodies, functionalized with a linker or an anchor oligonucleotide, a single stranded barcode oligonucleotide, functionalization reagents, DNA synthesis reagents, and the like.

[0082] fhe kits can also comprise other components necessary for carrying out the disclosed methods, including, but not limited to, primers, proteases, DNA purification reagents, and lysis reagents. Examples of additional suitable reagents for inclusion in the kit include conventional reagents employed in nucleic acid amplification reactions, such as, for example, one or more enzymes having polymerase activity, enzyme cofactors (such as magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, deoxyribonucleotide, or ribonucleotide triphosphatesAtty. Docket No. JHU -43664.601P18336-03 (dNTPs / rNTPs; for example, deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate) blocking agents, labeling agents, and the like.

[0083] The kits can also comprise instructions for using the components of the kit. The instructions arc relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instractions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0084] It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may further contain containers or devices for use with the methods or compositions disclosed herein. The kits optionally may provide additional components such as buffers and disposable single-use equipment (c.g., pipettes, tubes, flasks).

[0085] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.4. ExamplesMaterials and Methods

[0086] Labeling Monoclonal Antibodies with DNA Barcodes Highly specific monoclonal antibodies were conjugated to oligonucleotides that contain a protein specific barcode and UMI sequence. In the conjugation method, SMCC, a bifunctional crosslinker, was utilized to selectively link the amines on each antibody to a reduced thiol moiety on the anchor oligo DNA sequence using NHS and maleimide chemistry, respectively. The antibody was incubated with 7.5 and 20-fold excess of SMCC and reduced thiol DNA, respectively, overnight at 4C. After incubation, the reaction was quenched with Tris-Cl pH 8 to a final concentration of 50 mM on ice for 3 hours. After incubation on ice, the single stranded anchor oligonucleotide was made double stranded through second strand synthesis utilizing Klenow 3-5’ exo- with barcode DNA that has a complementary 3’ sequence to the anchor oligo. Unbound oligos were removed from the antibodies via centrifugal filtration with a 50 kDa cutoff molecular weight (Millipore, USA). Labeling of antibodies was verified via SDS-PAGE stained with Coomassie and SYBR Gold. Antibodies were stored at 4°C in Tris buffer until use.Atty. Docket No. JHU -43664.601P18336-03

[0087] Cell Culture HEK293t cells were maintained in DMEM media supplemented with 10% fetal bovine serum (FBS, Thermo Fisher, USA) at 37°C with 5% CO2. To generate cell cycle time points, cells were first arrested in G1 via serum starvation for 24 hours, then released from serum starvation. Time points were taken over 24 hours, which corresponds with the doubling time of HEK293t cells. For single cell experiments, single cells were isolated into hypotonic lysis buffer via FACS sorting at the JHMI Bloomberg School of Public Health Flow Cytometry and Immunology Core Facility.

[0088] Cell Lysate Preparation and Conjugation to NHS beads Cell lysates were generated utilizing hypotonic lysis as described by Abmayr et. al. For bulk cell experiments, BCA assays were employed to determine the total protein content of each lysate to ensure equal amounts are used for lysate bead generation. Eysate beads were generated as described in the protocol for Pierce NHS-Activated Magnetic beads (Thermo Fisher, USA). Immediately after lysis, lysates were added to unblocked NHS beads (10 pF for single cell experiments, bulk cell experiment volume needs to be determined from lysate protein content) and incubated at room temperature for 1 hour with mixing. After incubation with lysate, the beads were incubated with excess BS A for 30 minutes at room temperature. Next, beads were incubated with 3M ethanolamine pH = 9.0 for 2 hours at room temperature with mixing. Beads were washed one time with DI water, followed by twice with reaction buffer.

[0089] Incubation of Barcoded Antibodies with Lysate Beads Prior to each experiment, barcoded antibodies were pooled together. Amounts of each antibody in the cocktail started at 100 pg of each antibody per cell. Pooled antibodies were divided among each desired reaction and incubated with lysate beads suspended in reaction buffer (0.1 M Tris + 1% BSA) in DNA EoBind tubes or plates (Eppendorf, USA) for 30 minutes at room temperature. Each reaction was mixed by hand every 10 minutes to ensure beads remain in suspension. After incubation, beads were washed three times with High Salt Wash Buffer (IX TBST + 0.1 % BSA + 0.5 M NaCl). The beads were then resuspended in PBS buffer, treated with 2 uL Proteinase K (NEB, USA), and incubated at 37°C for 10 minutes to separate the DNA from the beads. After heat inactivation of the enzyme at 95°C for 10 minutes, the supernatant is suitable for downstream analysis with qPCR or NGS.

[0090] qPCR Analysis of Antibody Barcodes For each antibody included in the cocktail of the experiment, there is a corresponding barcode specific primer. Prior to qPCR, the total DNA concentration of the sample was measured using NanoDrop to ensure an appropriate amount of DNA was utilized. A total amount of 5-15 ng of input DNA is desired. The sample was aliquoted for each primer pair to be analyzed. Reactions were designed according to the instructions for GoTaq qPCRAtty. Docket No. JHU -43664.601P18336-03 Master Mix (Promega #A6001). To a full tube of the master mix, 20 uL of CXR dye was first added to act as a reference dye for the instrument. Because of this addition, for a 20 uL reaction, 10.2 uL of the 2X master mix was utilized instead of lOuL. Forward and barcode specific primers were added to a final concentration of 0.5 uM. Finally, 1 uL of the template at the desired total DNA reaction was added to the mix. Samples in the 96-wcll plate were centrifuged to remove the presence of any bubbles, and then samples were immediately placed in the sample tray and subjected to qPCR amplification. Typically, only 40 cycles according to the following parameters facilitate observation of sample signal.Hot Start: 95°C for 2 minutes40 cycles:Denature: 95°C for 15 secondsAnnealing: 60°C for 1 minute

[0091] Analysis of qPCR data Ct values were obtained from the instrument for each primer pair tested. First, Ct values were converted to "Relative Amounts" by performing the following calculation: 2CT. Background samples (IgG2b negative control, blank samples) were subtracted from unknown sample values. These values were normalized to positive controls (GAPDH, Beta Actin, etc.) through division. Values were plotted as a bar graph to compare normalized relative amounts for each sample for each antibody.

[0092] Sequencing Library Preparation from Antibody Barcodes Sequencing samples were prepared through two rounds of PCR with adapter and indexing primers. The first round of PCR was to add adapter sequences using GoTaq PCR Master Mix according to the manufacturer’s instructions for 10 cycles (Promega, USA). PCR reactions were then cleaned up using Ampure SPRI beads at a 1:1.6 ratio of sample to beads according to manufacturer’s instructions (Beckman, USA). The second round of PCR was to add unique indexes for cell identification and to add the P5 and P7 primers (Sequences as described by Mezger et al). This was completed using Q5 PCR Master Mix (NEB, USA) for 10 cycles according to manufacturer’s instructions. Finally, the PCR reactions were cleaned once more using Ampure SPRI beads at a ratio of 1 : 1.4 of sample to beads. DNA amounts for each cell were quantified using the Quanti-iT PicoGreen dsDNA Assay Kit (Thermo Fisher, USA) and all samples were pooled in an equimolar ratio. Samples were sequenced using iSeq at around 10,000 reads per cell.

[0093] Analysis of Sequencing Data Antibody tags and cell barcodes were matched to individual cells by direct extraction from the reads contained in the demultiplexed FASTQ files. Sequences were counted using a Hamming distance of less than 2. Reads containing the same cell barcode,Atty. Docket No. JHU -43664.601P18336-03 antibody barcode, and UMI were only counted once. Blank bead signal for each antibody was subtracted from the antibody read counts. For cell cycle experiments, read counts were then normalized to GAPDH control antibody reads to account for any bead loss during experiments. For the cardiomyocyte experiment, read counts were normalized to the total cell read count. Normalized read count matrices were generated using logNormCounts from the scRNAseq package scuttle.

[0094] Simultaneous rnRNA Capture and Sequencing Library Preparation For simultaneous capture of mRNA in addition to the proteins in cell lysate, agarose poly(dT) beads (MCLAB Products) are included with the NHS beads. After incubation of the beads with the lysate for 30 minutes at room temperature, the NHS and poly(dT) beads can be separated under the influence of a magnet and processed separately. The NHS beads undergo the previously described BEND-Westem workflow. The mRNA undergoes tagmentation with Illumina read primers, then samples were indexed as described above to introduce a cell barcode to each sample. mRNA can also be directly analyzed using qPCR with target specific primers.Example 1Multiplexed detection of proteins at the bulk cell level |0095] Monoclonal antibodies (mAbs) were barcoded with dsDNA sequences. Each barcode sequence contains a 20-nt priming sequence at its 5 ’-end, followed by an 11-nt barcode sequence with a Hamming distance of 2, a single nucleotide spacer, an 8-nt UMI, and another primer sequence at the 3’ -end (FIG. 1A). To reduce cost while maintaining consistency, an anchor DNA oligo of 20-nt with a thiol moiety attached to its 5 ’-end was first covalently conjugated to an mAb using a bivalent crosslinker SMCC. After quenching, a specific barcode oligo was annealed to the anchor oligo and converted to dsDNA using Klenow enzyme. Barcoding efficiency was examined with Coomassie and SYBR-gold staining on a PAGE gel (FIG. IB). Only those that had >60% barcoded species were allowed to pass this quality control step. Free, unbound DNA was removed with ammonium sulfate precipitation and spin columns.

[0096] Purified GAPDH and BSA were employed as positive and negative controls, respectively, and cell lysates from HEK293t cells were utilized as the experimental sample. Cell lysate, GAPDH, and BSA were immobilized separately on NHS-magnetic beads, as illustrated in FIG. ID. Each sample was then treated with a constant amount of barcoded anti-GAPDH mAb, washed thoroughly to remove any unbound antibody, and treated with Proteinase K to release the barcode sequence from the beads. Relative amounts of the GAPDH barcode DNA in each sample compared to the input antibody were determined utilizing GAPDH barcode specific primers in qPCR. Half of each sample was also amplified using traditional PCR and analyzed through gel electrophoresis. As expected, qPCR data demonstrated that immobilized GAPDH protein produced the strongest signal, as theAtty. Docket No. JHU -43664.601P18336-03 GAPDH protein immobilized met the capacity of the beads, hollowing this was the cell lysate, because the cellular GAPDH was a small fraction of the total lysate proteins. As expected, the signal obtained from immobilized BSA was the lowest (FIG. ID). Using gel electrophoresis (FIG. ID, bottom), these results in terms of relative amounts of barcode and the expected amplicon size were confirmed, indicating the signal from qPCR was indeed from the barcode DNA attached to the GAPDH mAb. This experiment provided evidence that BEND-Westem could determine differential protein levels across samples including cell lysate.

[0097] A panel of eight barcoded antibodies were employed to evaluate target protein expression in human brain and liver organoids derived from pluripotent stem cells in a bulk cell format. mAbs targeting six protein markers that are known to have differential expression profiles between the brain and liver were specifically selected. Anti-GAPDH and IgG2b were included as positive and negative controls, respectively. The total protein amount in the brain and liver organoids was quantified using a BCA assay and adjusted to contain the amount of protein present in -4000 cells. A blank, quenched NHS-bcad sample was also included as a negative control to account for any potential non-specific binding to the beads. Each cell organoid lysate was then separately immobilized on the beads. The mixture of barcoded mAbs was added to each sample at a final amount of 10 ng per mAb, equivalent to approximately 4 x 108mAb molecules. Assuming a median protein copy number of 18,000 molecules per cell (PMID: 22068331), the amount of each mAb added was in >22-fold excess. Barcodes were analyzed using qPCR with barcode specific primers. Every tested protein target showed elevated expression in the expected organoids where it is considered a marker or known to be more highly expressed (FIG. IE). For example, according to the Human Protein Atlas, actin, HLF, and HNF4a have higher expression levels in the liver than the brain, while LHX2, Myc, and NFIB have increased expression in the brain compared to the liver. These results demonstrate that BEND-Westem can reliably detect differentially expressed proteins in a multiplex fashion from different human organoids.Example 2Establishing BEND-Western to detect proteins at the single cell level

[0098] BEND-Western was performed on a dilution series of cell lysate, down to approximately one cell’s worth of lysate. A dilution series of cell lysates was prepared starting from 4000 cells and decreasing to approximately one cell, followed by immobilization on NHS magnetic beads. The beads were blocked with BSA, washed, and incubated with a mixture of anti-GAPDH mAb as a positive control and IgG2b mAb as a negative control. After removing the unbound mAbs, each sample was analyzed using qPCR with barcode-specific primers for the GAPDH and IgG2b barcodes separately. As illustrated in FIG. 2A, the amount of GAPDH quantified with the qPCR shows aAtty. Docket No. JHU -43664.601P18336-03 linear relationship with the number of cells. Conversely, barcodes from the IgG2b negative control antibody remained relatively low across all samples, as anticipated. Even down to approximately one cell, the amount of barcode detected for GAPDH was significantly higher (7-fold) than the amount detected for IgG2b negative control. This indicates that it was possible to detect intracellular proteins down to the single cell level with minimal background.

[0099] BEND Western was evaluated for its ability to accurately capture and quantify differential protein expression patterns throughout the mitotic cell cycle, from bulk cell to the single cell levels. The mitotic cell cycle is a model system with well-established temporal changes in protein abundance, featuring various proteins with known expression profiles linked to the cell cycle, notably the cyclins. These profiles have been thoroughly documented by the Human Protein Atlas database as well as the CycleBase database. Using these databases as a guide, a panel of 12 mAbs was designed to target proteins known to express differentially across the cell cycle. Also included were several positive controls, such as GAPDH and beta- Actin, which should be expressed at similar levels across the cell cycle, and negative controls like IgG2b that arc not expressed in any of the cells across the cell cycle.

[0100] HEK293t cells were synchronized at different stages of the cell cycle via serum starvation and release, and cells were collected at different time points corresponding to distinct phases of the cell cycle based on a 24-hour doubling time. Traditional Western blot analysis was performed utilizing approximately 250,000 cells per assay and the expected expression profiles of all proteins were confirmed at different cell cycle stages (FIGS. 2B-2D, left). Next, cell lysates from 4000 cells down to the single cell level were analyzed using BEND-Westem, and protein abundance was analyzed using qPCR with barcode-specific primers. Ct values obtained through qPCR were normalized to the values obtained for the positive controls to account for potential bead loss throughout the experiment. For each protein tested, similar cell cycle-specific profiles were observed in BEND-Westem assays with lysate from 4,000 cells down approximately a single cell, and importantly (FIGS. 2B-2D, three right-hand panels), they all match the profiles obtained with the traditional Western blot analysis. For example, enrichment of Cyclin A and Cyclin B was observed in the G2 / M phase of the cell cycle, while Cyclin E showed peak expression in the S phase population of cells, closely matching known data. Thus, BEND-Western can detect differential protein expression across cells down to the single cell level in a multiplexed fashion.Example 3Analysis of single-cell differential protein expression across the cell cycle

[0101] A total of 4,608 single cells (1152 per cell cycle stage) were subjected to the BEND-Western protocol utilizing a pool of 36 barcoded mAbs (FIG. 3A). 23 of the 36 antibodies target aAtty. Docket No. JHU -43664.601P18336-03 selection of proteins with known expression profiles across the cell cycle, three are phosphositespecific, as well as five positive controls (including anti-GAPDH and anti-b-actin) and five negative controls (including anti-rat IgG2a, IgG2b and IgGl, and anti-mouse ITGB1). The mixture of barcoded mAbs was added to each single cell at a final amount of 100 pg per mAb, equivalent to approximately 4 x 106mAb molecules, representing a 222-fold excess over the median protein copy number per cell. Blank bead samples containing no cell lysate were also included to examine nonspecific binding of the different antibodies with the beads themselves. Illumina compatible sequencing libraries were prepared, pooled, and sequenced using iSeq (4 million reads). The resulting barcode reads were normalized by the UMI to generate raw expression count matrices for each protein across the cell cycle. Background counts from blanks were subtracted, counts in each well were scaled to positive controls, and finally log-normalization was used to generate final normalized counts across wells. From the raw read counts of this experiment, a significant difference was observed between the positive control antibodies and the negative controls (FIG. 3B). As expected, the positive controls showed relatively consistent reads across each cell cycle stage, while the negative and blank bead controls produced little to no reads, indicating a high signal-to-noise ratio.

[0102] On the pseudo-bulk scale after normalization, the average protein expression across the cell cycle matched the known trends in the literature, as illustrated in FIG. 2E, with known peak expression denoted to the left of the heatmap. When cells were clustered by antibody read counts, clustering based on cell cycle stage was observed, demonstrating identification of distinct cell states using BEND-Westem intracellular protein expression data (FIG. 30). Interestingly, the clustered cells form a circular shape, representing the fluid and cyclic nature of protein expression across the cell cycle.

[0103] When examining the expression levels of different proteins in the clusters, known trends of protein expression were observed for each target protein tested. For example, Cyclin B is known to show peak protein expression towards the end of the cell cycle, with reduced signal in the earlier Gl / S phases. In the BEND-Western data, the same trend was observed (FIG. 3D). On the other hand, the protein TK1 shows significantly higher expression in the early stages of the cell cycle, particularly the G1 phase, and this trend of expression was observed in the BEND-Westem data as well (FIG. 3E). Most interestingly, cell cycle stage-specific phosphorylation events were also observed using phosphosite-specific antibodies. As an example, a Ser / Thr Polo Like Kinase 1 (PLK1), when activated by phosphorylation at the G2 / M phase boundary, promotes mitotic entry. The expression of PLK1 was observed throughout most of the cell cycle, with slightly reduced level in Gl, and notable elevated expression of pPLKl at the G2 / M phase boundary (FIG. 3F). BEND-Atty. Docket No. JHU -43664.601P18336-03 Western can detect differential protein expression across single cells and observe cell stage-specific signaling events, such as phosphorylation using phosphosite-specific antibodies.Example 4Analysis of hundreds unique TFs across cardiomyocyte differentiation at the single cell level 10104] 183 random anti-human transcription factor (TF) mAbs generated with funding of the NIH Protein Capture Reagent Program (PCRP) were selected to ensure the reproducibility and quantity of the single cell data. All the PCRP mAbs are mono-specific because they were rigorously tested for their target specificity on the human proteome (HuProt) arrays followed by cell-based Western blot and immunoprecipitation analyses. TFs are key regulators of differentiation, yet extensive proteomic expression profiling across the stages of differentiation has not yet been performed, presumably due to their low abundance. Also included were the 20 mAbs targeting cell cycle proteins and 24 positive and negative control mAbs, resulting in a mixture of 227 barcoded mAbs.

[0105] BEND Western technology was applied to examine the developmental stages of iPSC-dcrivcd cardiomyocytcs because it is a well-defined system with distinguishable cell morphology changes at different time points. The cardiomyocytes were sorted into single cells at Day 1 (1485 cells), Day 2 (1508 cells), Day 3 (1496 cells), and Day 4 (1485 cells) of differentiation. In addition to these 5974 wells containing single cells, 172 blank wells were processed as cell-free controls for non-specific binding of mAbs to the substrate, yielding 6146 total wells. After immobilizing the total proteins of each single cell on beads, a pool of 227 barcoded mAbs was added to each well (FIG. 4B). After incubation and stringent washes, single cell libraries were made, indexed, and pooled. The libraries were sequenced using MiSeq technology at a sequencing depth consistent with the sequencing depth used for CITE-seq ADT libraries.

[0106] After normalization with UMI, a total of 108,074,792 reads were successfully mapped to expected barcode sequences. The overall average read count per well was 17,600, with an average of 18,100 reads for non-blank wells and an average of 122 reads for blank wells. The anti-rat antibodies IgG, IgG2a, and IgG2b were used as negative controls. In non-blank wells, the average read count of the negative control antibodies was 60, and the average read count of the remaining antibodies was 80. Of the 183 TF and 20 cell cycle mAbs tested (230 mAbs), 62 produced average read counts at least 2x larger than the negative control antibodies. Each cell had at least 1 count for each antibody, without any dropouts.

[0107] Next, the UMAP analysis grouped the single cells into four major clusters that almost coincided with the four time points when they were collected (FIG. 4A). In addition, the clusters generated through this experiment largely resemble those reported previously utilizing scRNA-seq datasets. Protein enrichment analysis was performed for each of the different clusters, and theAtty. Docket No. JHU -43664.601P18336-03 identified enriched proteins in the iPSC-associated versus the cardiomyocyte-associated cluster were analyzed using STRING to identify known protein-protein associations and corresponding GO terms for each cluster (FIG. 4). In the case of both the iPSC-associated cluster and the cardiomyocyte enriched cluster, the enriched proteins were identified to have highly significant PPI enrichment p-valucs (p- value < 1.95 x 107and 1.95 x 10s, respectively). This demonstrated that those enriched proteins at the same time points have significantly more interactions among themselves than would be expected from a random list of proteins, suggesting that these proteins are working together with somewhat related biology as a group. Interestingly, the list of enriched proteins in the iPSC cluster showed enriched GO terms highly related to processes involved in the cell cycle. This was not unexpected since stem cell self-renewal relies on the processes of the cell cycle and cell proliferation. On the other hand, the GO terms identified for proteins enriched in the cardiomyocyte cluster suggested more specific relevancy to the heart and cardiomyocyte-relevant functions, such as smooth or cardiac muscle generation, heart structure development, and even cardiocyte differentiation (FIG.4). The highly expressed proteins identified in these clusters arc likely to perform functions associated with each cell type respectively, reflective of known expression in these cell types.

[0108] fhe known abundances of mRNA for each of the proteins tested was compared to the abundance observed for protein expression in this experiment across each of the time points studied. Using the existing RNA-seq data obtained at the same timepoints across cardiomyocyte differentiation, RNA heat maps were generated to show the relative expression profiles and compared with a pseudo-bulk protein dataset across the developmental process. Many proteins show similar or identical trends in their corresponding mRNA (FIG. 4). For example, CDK6 both showed peak expression of protein and mRNA in the final time point, with low expression of both species in the earlier time points (FIG. 4). Many proteins showed expression at the same time points as mRNA, but different relative expression across the time points when compared to mRNA (FIG. 4). For example, both ELF3 and its mRNA were found to be expressed in the first and last time points, but Elf3 shows peak expression in the first time point, while the protein shows peak expression in the last time point. This is an important piece of information that can be gained through the BEND-Western pipeline, as it solidifies the idea that high mRNA expression does not always correlate to high protein expression. Additionally, a subset of the proteins demonstrated delay in expression from the known RNA expression time points (FIG. 4). This is not an unexpected phenomenon, as it has been noted in the literature that there is often temporal delay between mRNA and protein expression. Using this dataset in combination with the RNA-seq data, protein dynamics such as degradation that are not possible to observe using RNA-seq data sets alone can be observed using the BEND-Western pipeline (FIG. 4).Atty. Docket No. JHU -43664.601P18336-03

[0109] Taken together, the observed discrepancy between protein levels and their corresponding messenger RNAs underscores the critical importance of advancing single-cell proteomics technologies. This discrepancy highlights the complex relationship between transcription and translation in cellular systems. While transcriptomics has provided valuable insights into gene expression patterns, it docs not always accurately reflect the functional protcomc of a cell.

[0110] Proteins are the ultimate effectors of cellular functions, serving as the molecular machinery that carries out a wide array of critical processes, including signal transduction, housekeeping, metabolism, survival, and cell-specific functions, to name a few. By focusing on single-cell proteomics, researchers can gain a more accurate understanding of cellular heterogeneity, post-translational modifications, and protein-protein interactions. The BEND-Westem pipeline can reveal insights into cellular function, development, and disease mechanisms that may not be apparent from genomic or transcriptomic data alone, facilitating unraveling the complexities of cellular biology and developing more effective therapeutic strategies.

[0111] It is understood that the foregoing detailed description and accompanying examples arc merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

[0112] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.

Claims

Atty. Docket No. JHU -43664.601P18336-03 CL IMSWe claim:

1. A method for detecting the identity of proteins in a sample, the method comprising:(a) contacting the sample comprising proteins with magnetic beads, wherein the magnetic beads and proteins in the sample form bead-protein complexes;(b) contacting the bead-protein complexes with a plurality of detection agents, wherein each detection agent comprises:(i) an antibody or antibody fragment that binds to a protein within the beadprotein complexes; and(ii) a barcode oligonucleotide comprising:(1 ) at least one primer region;(2) a unique molecule identifier (UMI); and(3) a barcode sequence, wherein the barcode sequence is associated with the identity of a protein in the sample,wherein the antibody or the antibody fragment is covalently linked to the barcode oligonucleotide;(c) removing antibody or antibody fragments that are not bound to a protein;(d) contacting the sample with an enzyme solution, wherein the enzyme solution cleaves each antibody from each barcode oligonucleotide of the detection agents and removes the proteins from the sample;(e) isolating a plurality of barcode oligonucleotides from the sample;(f) amplifying the plurality of barcode oligonucleotides to form a plurality of amplified oligonucleotides;(g) for each of the oligonucleotides of the plurality of oligonucleotides, hybridizing an identification oligonucleotide to the barcode oligonucleotide and elongating the barcode oligonucleotide to incorporate an index sequence from the identification oligonucleotide into the barcode oligonucleotide to form elongated oligonucleotides, wherein the identification oligonucleotide comprises:(i) an adapter region that hybridizes to the at least one primer region; and (ii) an index sequence, wherein the index sequence is associated with a number for the sample;(h) sequencing the elongated oligonucleotides; andAtty. Docket No. JHU -43664.601P18336-03 (i) analyzing the barcode sequences and identifying the associated identity of the proteins in the sample for each barcode sequence, thereby detecting the identity of proteins in the sample.

2. The method of claim 1, wherein the sample is a biological sample, a cell lysate of a single cell, a bulk cell lysate from a population of cells, or a bulk cell lysate from a tissue or organ.

3. The method of claim 1 or claim 2, wherein the sample is not partitioned.

4. The method of any one of claims 1 to 3, wherein (c) removing the antibody or the antibody fragments that are not bound to a protein comprises a blocking step, wherein the blocking step comprises contacting the bead-protein complexes in the sample with bovine serum albumin.

5. The method of any one of claims 1 to 4, wherein the enzyme solution comprises proteinase K.

6. The method of any one of claims 1 to 5, wherein the method further comprises exposing the enzyme solution to heat to inactivate enzymes in the solution.

7. The method of any one of claims 1 to 6, wherein the amplifying comprises a polymerase chain reaction (PCR).

8. The method of any one of claims 1 to 7, wherein the amplifying is repeated one or more times.

9. The method of any one of claims 1 to 8, wherein the index sequence comprises 2 to 20 base pairs.

10. The method of any one of claims 1 to 9, wherein the index sequence comprises 5 to 10 base pairs.

11. The method of any one of claims 1 to 10, wherein the index sequence comprises about 8 base pairs.Atty. Docket No. JHU -43664.601P18336-0312. The method of any one of claims 1 to 11, wherein the identification sequence comprises an additional index sequence.

13. The method of any one of claims 1 to 12, wherein the barcode oligonucleotide further comprises a spacer between the UMI and the barcode sequence.

14. The method of any one of claims 1 to 1 , wherein the plurality of detection agents are directed to 100 or more proteins of interest.

15. The method of any one of claims 1 to 14, wherein the antibody or the fragment thereof comprises a monoclonal antibody.

16. The method of any one of claims 1 to 15, wherein the barcode oligonucleotide comprises a DNA, an RNA, or a hybrid of DNA and RNA.

17. The method of any one of claims 1 to 16, wherein the barcode oligonucleotide comprises a double stranded DNA.

18. The method of any one of claims 1 to 17, wherein the barcode oligonucleotide comprises a length of about 30 to about 150 base pairs.

19. A method for detecting proteins in a sample, the method comprising:contacting immobilized proteins from the sample with a plurality of detection agents, wherein each detection agent comprises a binding agent specific to a protein of interest covalently attached to a barcode oligonucleotide;isolating target barcode oligonucleotides from barcoded detection agents bound to the immobilized proteins;analyzing the target barcode oligonucleotides; andidentifying proteins of interest in the sample.

20. The method of claim 1 , wherein the plurality of detection agents is directed to 5 or more proteins of interest.Atty. Docket No. JHU -43664.601P18336-03 21. The method of claim 19 or claim 20, wherein the plurality of detection agents is directed to 100 or more proteins of interest.

22. The method of any one of claims 19-21, wherein the binding agent comprises a protein or peptide.

23. The method of any one of claims 19-22, wherein the binding agent is an antibody or fragment thereof.

24. The method of any one of claims 19-23, wherein the binding agent is a monoclonal antibody.

25. The method of any of one claims 19-24, further comprising immobilizing proteins from the sample on a solid support.

26. The method of claim 25, wherein the solid support is a bead or particle.

27. The method of any one of claims 19-26, wherein the immobilized proteins are in a suspension.

28. The method of any one of claims 19-27, wherein the contacting comprises incubating the immobilized proteins with the plurality of detection agents for at least 10 minutes.

29. The method of any one of claims 19-28, further comprising removing detection agents not bound to an immobilized protein.

30. The method of any one of claims 19-29, wherein the barcode oligonucleotide comprises DNA.

31. The method of any one of claims 19-30, wherein the barcode oligonucleotide comprises double-stranded DNA.

32. The method of any one of claims 19-31, wherein each barcode oligonucleotide comprises a barcode and a unique molecular identifier (UMI) flanked by primer binding sites.Atty. Docket No. JHU -43664.601P18336-03 33. The method of any one of claims 19-32, wherein the barcode oligonucleotide has a length of about 30 to about 150 base pairs.

34. The method of claim 32 or claim 33, wherein analyzing the target barcode oligonucleotides comprises amplifying and / or sequencing the barcode and UMI.

35. The method of any one of claims 32-34, wherein the analyzing comprises introducing a sample identification tag to the barcode and amplifying and / or sequencing the sample identification tag, barcode, and UMI.

36. The method of any one of claims 19-35, further comprising determining expression levels of the proteins of interest based on a comparison of barcode sequence numbers analyzed for each detection agent.

37. The method of any one of claims 19-36, further comprising determining transcript levels for the proteins of interest.

38. The method of claim 37, wherein the determining comprises capturing mRNA from the sample and generating an mRNA-seq library from the sample.

39. The method of any one of claims 19-38, wherein the method further comprises determining one or more characteristics of the sample based on proteins of interest present in the sample, expression levels of the proteins of interest in the sample, transcript levels of the proteins of interest in the sample, or a combination thereof.

40. The method of any one of claims 19-39, wherein the sample is a biological sample.

41. The method of any one of claims 19-40, wherein the sample is a cell lysate of a single cell.

42. The method of any one of claims 19-41, wherein the sample is a bulk lysate of cells from a single cell type or tissue.

43. The method of any one of claims 19-42, wherein the sample is a bulk lysate of disease- associated cells.

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