Methods and compositions for activity-dependent protein labeling

Abstract

Aspects herein relate, in part, to system and methods for evaluating a protein of interest. In particular, certain aspects use activity probes, which can be fused to recognition elements, to contact a protein of interest for further evaluation or analysis of the protein of interest.

Description

METHODS AND COMPOSITIONS FOR ACTIVITY-DEPENDENT PROTEINLABELINGBACKGROUNDI. Cross Reference to Related Applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 696,271, filed on September 18, 2024, the contents of which are hereby incorporated by reference in their entirety.

[0002] Further applicable details may be found in U.S. Provisional Application Serial No. 63 / 696,341 filed September 18, 2024 entitled “Reversible and Irreversible Kinome Probes and Uses Thereof,” U.S. Provisional Application Serial No. 63 / 696,346 filed September 18, 2024 entitled “Kinase Inhibitor Compositions and Therapeutic Uses Thereof,” and PCT Application No. PCT / US2018 / 062231 filed November 21, 2018 entitled “Chemical Probe-Dependent Evaluation of Protein Activity and Uses Thereof,” all of which are incorporated by reference in their entireties herein.II. Sequence Listing

[0003] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 18, 2025, is named ARCD_P0853WO_sequence_listing.xml and is 65,302 bytes in size.III. Field of the Invention

[0004] The current disclosure relates to the field of molecular biology, therapeutic methods, and diagnostics.IV. Description of Related Art

[0005] The study of protein function has traditionally been a reductionist endeavor, where proteins are expressed and purified from orthogonal hosts and then studied in isolation. However, the functional properties of a protein are imparted by the complexity of the surrounding environment, including participation in protein-protein complexes, spatial localization to distinct sub-cellular compartments, post-translational chemical modifications and even mechanical forces within or between cells. Despite an appreciation for these influences, traditional biophysical and biochemical techniques rarely capture the effects of these events. The field of proteomics aims to provide a comprehensive accounting of thecomplement of proteins in a biological sample. In the decade since orbitrap mass spectrometers and analysis algorithms have become commercially available, the field of proteomics has found mainstream applications in basic chemical, biological and clinical research. Despite the power of these technologies, standard proteomic platforms are typically limited to providing two pieces of information: whether a specific protein is present in a sample, and the relative abundance of a protein within a sample. While this information is important, it does not provide information on the functional state of the detected proteins. Activity-based proteomic technologies, on the other hand, integrate enzyme- or protein-family specific chemical probes with traditional mass spectrometry or gel-based profiling methods in order to detect and quantify protein activity, rather than abundance. These measurements can be made directly with complex samples such as lysate, tissues and biological fluids to measure changes in protein activity, often for entire families of proteins of a hundred or more, that result from endogenous biological signals or the action of exogenous molecules (e.g., therapeutics).

[0006] Activity-based profiling approaches and the mass spectrometry platforms upon which they rely have two major limitations. First, gel-based or mass spectrometry-based proteomic experiments impose significant limits on the amount of sample needed, which generally prevents the analysis of limited abundance samples (e.g., patient tissue) and single cell measurements. Even with ample input proteome, gel-based and data-dependent LC- MS / MS measurements are heavily biased toward high abundance proteins, often omitting a majority of the proteome in routine analyses. CyTOF and imaging mass spectrometry approaches can provide quantitative information on protein abundance with single-cell resolution, however these approaches require expensive mass spectrometry equipment and antibody conjugates, and do not report on protein function. Second, current proteomic methods require homogenization and manipulation of the biological sample, which results in the loss of spatial information about protein activity, both at intra- and intercellular levels. Expression of fluorescent protein-tagged proteins or the use of proximity ligation assays targeting complexes or modified forms of a protein of interest can provide information on sub-cellular localization, however these approaches often require genetic manipulation, availability of multiple proteoform-specific antibodies, and a priori information correlating functional state with specific proteoforms of a protein. Activity -based probes detect protein activity, but involve loss of spatial information and require significant input proteome. Small molecule “turn-on” probes typically lack the ability to provide precise spatial information due to signal diffusion, and sometimes do not reflect activity of a single protein but a protein family. Several recent studies299713335.1 - 2 -have applied iterative medicinal chemistry and screening to transform non-selective familywide probes into enzyme-specific reporter probes for lipid hydrolases and caspase-family cysteine proteases. Through the covalent tagging of active enzymes with a fluorescent reporter, these probes have enabled sub-cellular and intercellular visualization and quantification of active enzymes, in live cells and in vivo. While providing a step forward in chemical proteomics, like “turn-on” probes this approach is hardly general, as each enzyme requires de novo development of tailored chemical probes that exhibit extremely high target selectivity. Therefore, there is a need in the art for methods to study the activity profile of native proteins in their natural environment.SUMMARY OF THE DISCLOSURE

[0007] Aspects of the disclosure relate to systems, methods, and products for evaluating a protein of interest (POI) using modular activity-dependent proximity ligation (ADPL). Modular ADPL refers to Applicant’s system wherein the elements of the system can be replaced or “swappable” so that the system is customizable for a variety of uses. Elements can include:1) a POI2) an epitope tag fused to the protein (also referred to herein as an “protein tag”)3) an activity probe (also referred to herein as a “discovery probe” or a “chemical probe”) that includes:(a) a targeting group which binds to the POI (also referred to herein as a “warhead”)(b) a retrieval tag4) recognition elements such as:(a) an epitope tag recognition element which binds the epitope tag (also referred to as a “POI recognition oglio”); can include an epitope tag binding component and / or a barcode.(b) a retrieval tag recognition element which binds the retrieval tag (also referred to as “probe recognition oglio”); can include an epitope tag binding component and a barcode.(c) an epitope tag binding component which binds the epitope tag and an epitope-retrieval recognition element which binds the epitope tag binding component; and5) oligonucleotides (also referred to herein as “oligos”) with barcodes.299713335.1 - 3 -For such elements, the element can be replaced, exchanged, or swapped with another element. For example, the POI can be replaced with another POI, the epitope tag can be replaced with another epitope tag, and so forth. In certain aspects, it is contemplated that one or more of the elements are specifically excluded from a composition, system, method, or other aspect disclosed herein.

[0008] Aspects of the disclosure relate to a system for evaluating a protein of interest. In some aspects, the system can include at least or include at most 1, 2, 3, or 4 of the following: (i) a protein of interest fused to an epitope tag; (ii) an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises an epitope tag binding component and a first oligonucleotide; (iii) an activity probe, wherein the activity probe comprises a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; and / or (iv) a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval-binding tag comprises a second oligonucleotide.

[0009] In some aspects, the system can , include at least or include at most 1, 2, 3, 4, or 5 of the following:(i) a protein of interest fused to an epitope tag; (ii) an epitope tag binding component capable of binding the epitope tag; (iii) an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide; (iv) an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; and / or (v) a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval-binding tag comprises a second oligonucleotide.

[0010] Aspects of the disclosure relate to a method for evaluating a protein of interest. In some aspects, the method can include at least or include at most 1, 2, 3, 4, or 5 of the following: : (i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest (ii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises a epitope tag binding component and a first oligonucleotide; (iii)contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (iv) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second299713335.1 - 4 -oligonucleotides are in close proximity to each other; and / or (v) detecting ligated or annealed first and second oligonucleotide.

[0011] In some aspects, the method can , include at least or include at most 1, 2, 3, 4, 5, or 6 of the following: (i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; (ii) contacting the composition with an epitope tag binding component capable of binding the epitope tag; (iii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide; (iv) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (v) incubating the composition under conditions sufficient for ligation and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and / or (vi) detecting the ligated and / or annealed first and second oligonucleotide. In this aspect, steps (iii) and (iv) can be performed consecutively with (iii) before (iv); consecutively with (iv) before (iii); or concurrently.

[0012] In certain aspects, at least 1, 2, 3, 4, or 5 of the of the steps are specifically excluded.

[0013] In some aspects of the system or method, the first and / or second oligonucleotide single stranded. The first and / or second oligonucleotide can include a DNA barcode.

[0014] In some aspects of the system or method, the first and the second oligonucleotides are capable of being ligated and / or annealing when in sufficient proximity to each other. As used herein, “Sufficient proximity” or “close proximity” is defined as a physical proximity sufficient to cause ligation and / or annealing of the first and / or the second oligonucleotide when ligation reagents are added. Ligation reagents include a ligase, primers, dNTPs, etc.

[0015] In some aspects, the system further comprises one or more bridging oligonucleotides, wherein at least one of the one or more bridging oligonucleotides comprises complementary regions to both the first and second oligonucleotide.

[0016] In some aspects, the system further comprises one or more reagents for rolling circle amplification.

[0017] In some aspects, the system further comprises a recognition oligonucleotide conjugated to a label, wherein the recognition oligonucleotide is complementary to the first299713335.1 - 5 -oligonucleotide, the second oligonucleotide or at least one of the one or more bridging oligonucleotides. The label can include a fluorescent protein, a pull down tag or a split recognition system or the like.

[0018] In some aspects, the system further comprises a first labeled primer and / or second labeled primer, wherein the first labeled primer is specifically complementary to the first oligonucleotide and non-complementary to the second oligonucleotide and the second labeled primer is specifically complementary to the second oligonucleotide and non-complementary to the first oligonucleotide. In some aspects, the first labeled primer and / or second labeled primer is complementary to a barcode. In some aspects, the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

[0019] In some aspects, the first and the second oligonucleotides are capable of being ligated and / or and annealing when in sufficient proximity to each other in the presence of ligation reagents.

[0020] In some aspects of the system or method, the protein of interest is an enzyme, but any protein of interest can be used. The protein of interest can include, for example, a serine hydrolase, cysteine protease, kinase, metalloprotease, P-retaining glycosidase, tyrosine phosphatase, cytochrome P450, or the like. In some aspects, the protein of interest is a DNA repair enzyme. In some aspects, the protein of interest is a non-enzyme.

[0021] In some aspects of the system or method, the epitope tag includes a florescent protein, a pull down tag, or a split recognition system or protein tags for covalent labeling. In some aspects, the florescent protein comprises GFP, RFP, mCherry, and / or the like. In some aspects, the pull down tag comprises GST, a flag, MBP, His, HA, and / or the like. In some aspects, the split recognition system comprises a split gfp, spytag and catcher, and / or the like. In some aspects, the protein tags for covalent labeling comprise snap tag, halo tag, and / or the like.

[0022] In some aspects of the system or method, the epitope tag is incorporated into the protein of interest, by combining genetic material, for example in an expressed fusion construct. In some aspects of the system or method, the protein of interest with an epitope tag is a recombinant protein.

[0023] In some aspects of the system or method, the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, aptamer and / or the like.299713335.1 - 6 -

[0024] In some aspects of the system or method, the targeting group is any moiety that can bind to a POI. Examples include, but are not limited to, flourophosphonate, diphenylphosphonate, sulfonyl fluoride, acyloxymethyl ketone, phenoxymethylketone, vinyl sulfone, epoxide, halomethylketone, alpha-haloester, alpha-haloamide, a, P-unsaturated ester, a, P-unsaturated ketone, diazomethylketone, acyl phosphate, acylphosphonate, hydroxamate, carbamate, ester, thioester, 2-deoxy-2-fluoro glycoside, a-bromobenzylphosphonate, 2- ethynylnaphthalene, acrylamide, butyneamide, crotonamide, chloroacetamide, alphamethylchloroacetamide, and / or the like. The targeting group can be a small molecule. In certain aspects, the targeting group is a small molecule moiety linked in the activity probe. “Small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000 Da. In some embodiments, they are less than 5,000 Da, less than 1,000 Da, or less than 500 Da (and any range derivable therein). This class of modifiers includes chemically synthesized molecules, for example, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified from screening methods described herein. Methods for generating and obtaining small molecules are well known in the art (Schreiber, Science 2000; 151 : 1964-1969; Radmann et al., Science 2000; 151 : 1947-1948, which are hereby incorporated by reference).

[0025] In some aspects of the system or method, the retrieval tag and / or retrieval tag recognition element are one or more of the following retrieval tag and retrieval tag recognition element: biotin and streptavidin, biotin and avidin, biotin and anti-biotin, desthiobiotin and streptavidin, desthiobiotin and avidin, desthiobiotin and anti-biotin, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne , a tetrazine and a tran- cyclooctene, an epitope and an epitope-specific antibody, or the like.

[0026] In some aspects of the system or method, the targeting group is selected based on the protein family of interest. In some aspects of the system or method, the protein of interest is a serine hydrolase and the targeting group comprises

[0027] In some aspects of the system or method, the protein of interest is a kinase and thetargeting group comprises299713335.1

[0028] In some aspects of the system or method, the protein of interest is a cysteine proteaseand the targeting group comprises one of:

[0029] In some aspects of the system or method, the targeting group is linked to the retrieval tag with a linker. In some embodiments, the linker is an organic linker. In some embodiments, the linker comprises an aliphatic linker. In some embodiments, the linker comprises a hydrocarbon chain of 3-20 carbon atoms. In some embodiments, the linker comprises a hydrocarbon chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any derivable range therein) carbon atoms. Linker length can be optimized to enhance solubility and / or minimize background in assays.

[0030] In some aspects of the system or method, the retrieval tag comprises an orthogonal recognition element. In some aspects, the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, O6-benzylguanine, SNAP protein, an alkyne, an azide, a cyclooctyne a tetrazine, a trans-cyclooctene, a peptide epitope, a peptide epitope-specific antibody, and / or the like.

[0031] In some aspects of the system or method, the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form. The term “specified functional form” refers to a form or state of the protein that performs a particular function and / or is considered an active state. In some embodiments, the specified functional form is an active form of the protein. In some embodiments, the specified functional form is an active form of the protein that can / is capable of acting as a catalyst in an enzymatic reaction. In some aspects of the system or method, the specialized functional form is an active form.

[0032] In some aspects of the system or method, the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form. In some aspects of the system or method, the non-specialized functional form is an inactive form.

[0033] In some aspects of the system or method, the system further comprises a non-activity probe capable of binding the protein of interest in non-specialized functional form, or the method further comprises incubating the composition with a non-activity probe capable of binding the protein of interest in non-specialized functional form.299713335.1 - 8 -

[0034] Some aspects of the disclosure relate to a method for evaluating a target protein of interest comprising the any of the systems disclosed herein. The method can include evaluating activity and / or abundance of the protein of interest.

[0035] Some aspects of the disclose relate to any of the elements or combination of elements according to any of the systems or methods disclosed herein. The element can include one or more of POI, epitope tag, activity probe, epitope tag recognition element, retrieval tag recognition element, epitope retrieval recognition element, and / or the like, and / or any combination thereof. Some aspects of the disclose relate to an activity probe according to any of the systems or methods disclosed herein.

[0036] Some aspects of the disclosure relate to an epitope tag recognition element. The epitope tag recognition element can include an epitope binding component and a barcode, the epitope binding component can be interchangeable with at least another epitope binding component.

[0037] Some aspects of the disclose relate to an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest. In some aspects the activity probe is customizable. The term “customizable” refers to customizing a probe or element for a use by, for example, changing a feature of the probe or element. In some aspects, the targeting group is interchangeable with at least another targeting group. In some aspects, the targeting group is selected based on the protein of interest.

[0038] In some aspects of the method, the protein of interest is in a cell. In some aspects, wherein step (i) occurs in a live cell and steps (ii) and onward occur after fixing and / or lysing of the cell.

[0039] In some aspects of the disclosure, the evaluating comprises measuring activity and / or quantity, wherein the target protein of interest is in a specialized functional form.

[0040] In some aspects of the disclosure, the specialized functional form is an active form of the protein of interest.

[0041] In some aspects of the disclosure, the detecting the ligated or annealed oligonucleotides comprises imaging, qPCR, a sequencing based readout, and / or the like.

[0042] In some aspects of the disclosure, the method further comprising determining abundance of the target protein of interest. In some aspects of the disclosure, the abundance of299713335.1 - 9 -the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0043] In some aspects of the disclosure, determining the abundance of the target protein or interest comprises primer extension and / or PCR amplification of the ligated and / or annealed first and second oligonucleotides. For example, when the first, second, and / or bridging oligo are in close proximity, they may be constructed so that a proximity extension assay can be performed. The proximity of the oligos creates a real-time PCR amplicon in a proximitydependent manner enabled by the action of a DNA polymerase (eg. 3 'Exonuclease-capable polymerase).

[0044] In some aspects of the disclosure, the steps are performed in chronological order.

[0045] In some aspects of the disclosure, the method further includes contacting the composition with one or more bridging oligonucleotides, wherein at least one of the one or more bridging oligonucleotides comprises or are at least 80, 85, 90, 95, or 100% complementary regions (or any range derivable therein) (100% complementary is the same as “fully complementary”) to both the first and second oligonucleotide.

[0046] In some aspects of the disclosure, the method further comprises performing rolling circle amplification after the detecting the ligated and / or annealed first and second oligonucleotides.

[0047] In some aspects of the disclosure, the method further includes contacting the composition with one or more recognition oligonucleotides conjugated to a label, wherein the recognition oligonucleotide nucleotide is or is at least 80, 85, 90, 95, or 100% complementary (or any range derivable therein) (100% complementary is the same as “fully complementary”) to the first oligonucleotide, the second oligo nucleotide or at least one of the one or more bridging oligonucleotides. In some aspects, the label comprises a flourescent protein, a pull down tag or a split recognition system.

[0048] In some aspects of the disclosure, the method further includes contacting the composition with a first labeled primer and / or second labeled primer, wherein the first labeled primer is specifically complementary to the first oligonucleotide and non-complementary to the second oligonucleotide and the second labeled primer is specifically complementary to the second oligonucleotide and non-complementary to the first oligonucleotide. In some aspects, the first labeled primer and / or second labeled primer is complementary to a barcode. In some aspects, the first labeled primer and second labeled primer are labeled with differentially299713335.1 - 10 -detectable molecular labels. The term “differentially detectable” refers to two labels that can be quantitatively and / or qualitatively detected at the same time when the two labels are intermixed in the same composition and / or microscopically visualized within the same cells. The term “specifically complementary” as used herein refers to “at least 80, 85, 90, 95, or 100% complementary” and the term “non-complementary” as used herein refers to “less than 80, 85, 90, 95, or 100% complementary.”

[0049] In some aspects of the disclosure, wherein first and the second oligonucleotides are capable of being ligated and / or and annealing when in sufficient proximity to each other in the presence of ligation reagents.

[0050] In some aspects of the disclosure, the method or the system further includes spatially detecting the target protein of interest.

[0051] In some aspects, the activity probe covalently modifies, or is capable of covalently modifying the proteins of interest. For example, the probe can cause a covalent bond with the target protein and that enables downstream barcoding. For example, the probe can enable downstream recognition element (e.g., epitope tag recognition element) binding to the epitope POI and downstream ‘barcoding’ (ligation and amplification of oligo signal).

[0052] In some aspects of the disclosure, the composition comprises fewer than 5000 cells. In some embodiments, the composition comprises less than 1000000, 900000, 800000, 700000, 600000, 500000, 400000, 300000, 200000, 100000, 90000, 80000, 70000, 60000, 50000, 40000, 30000, 20000, 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 500, 400, 300, 200, or 100 cells (or any derivable range therein). In some embodiments, the composition comprises less than 1 pg of total protein. In some embodiments, the composition comprises less than 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0.4, 0.3, 0.2, 0.1, or 0.01 pg (or any derivable range therein) of total protein. In some aspects, the composition comprises less than 1 pg of total protein.

[0053] In some aspects of the disclosure, the method further comprises detecting a cellular marker. Such methods may include one or more of the following steps: addition of a markerspecific antibody, detection of binding of the marker-specific antibody, detection of the binding of the marker-specific antibody by immunofluorescence, isolating cells comprising the detected binding by cell sorting (e.g., fluorescence activated cell sorting, FACS), flow cytometry of the cells, and / or culturing of the isolated cells.299713335.1 - 11 -

[0054] In some aspects of the disclosure, the method further comprises determining the total amount of target protein of interest. In some aspects of the disclosure, the determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

[0055] In some aspects of the disclosure, determining the total amount of target protein of interest comprises directly or indirectly detecting the specialized functional form and nonspecialized functional forms of the protein.

[0056] In some aspects of the disclosure, the method excludes liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry.

[0057] Also disclosed are methods of competitive profiling comprising any of the methods herein, wherein step (i) further comprises contacting the composition with a non-activity probe capable of binding the protein of interest in non-specialized functional form.

[0058] Also disclosed are methods of evaluating two or more proteins of interest according to any of the methods disclosed herein wherein the label and / or the recognition oligonucleotides are different for each protein.

[0059] Also disclosed are methods of determining a half maximal inhibitory concentration (ICso) according to any of the methods disclosed herein. IC50 values of a compound of interest can be determined by performing one or more of the ADPL steps described herein using the compound of interest operatively linked to a retrieval tag, including any retrieval tag described herein. Differing concentrations of the composition of interest operatively linked to the retrieval tag can be introduced to a biological sample containing a protein of interest, tissue containing a protein of interest, a cell containing a protein of interest, or an isolated protein of interest. In some embodiments, the differing concentrations include concentrations of at least, at most, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nM, pM, or mM (or any range derivable therein). In some embodiments, determining the IC50 value includes starting the concentration of the compound of interest operatively linked to a retrieval tag at approximately 1, 10, or 100 nM, pM, or mM and increasing the concentration by 1, 10, or 100 nM, pM, or mM until a final desired concentration is reached. Other starting concentrations or299713335.1 - 12 -concentration increases may be performed. In some embodiments, the starting concentration and concentration increases are based on known IC50 values of the compound of interest (or similar compounds or analogs thereof) for the protein of interest (or similar proteins or homologs thereof). Detecting signal from the ADPL method can be used to determine the binding and / or inhibition of the compound of interest at the introduced concentration. In some embodiments, the compound of interest is a known drug, a drug candidate, a biological probe, experimental compound, or a compound for which IC50 values against a protein of interest are desired. In some embodiments, the compound of interest is a compound described herein. In some embodiments, the protein of interest is an oncogene, a drug target, or a protein implicated in a disease.

[0060] In some embodiments, the method further comprises obtaining a biological sample from a patient. In some embodiments, the biological sample comprises blood, serum, or tissue. In some embodiments, the biological sample comprises cancerous cells. In some embodiments, the cancerous cells comprise ovarian or prostate cancerous cells. In some embodiments, the cancerous cells are from a cancer described herein. In some embodiments, the method further comprises determining the level or abundance of the protein of interest in the specified functional form in the biological sample.

[0061] In some embodiments, the method further comprises comparing the level or abundance of a protein of interest in the specified functional form in the biological sample compared to a control. In some embodiments, the control comprises the level of the protein of interest in a specified functional form in a biological sample from a patient having non- aggressive cancer or a non-cancerous sample. In some embodiments, the control comprises the level of the protein of interest in the specified functional form in a biological sample from a patient having non-aggressive ovarian or prostate cancer or a non-cancerous sample. In some embodiments, the non-aggressive cancer comprises stage I or stage II cancer. In some embodiments, the non-aggressive ovarian or prostate cancer comprises stage I or stage II ovarian or prostate cancer. In some embodiments, diagnosing the patient based on the determined level of protein of interest in the specified functional form. In some embodiments, the method further includes diagnosing the patient with aggressive or non-aggressive cancer based on the level or abundance of the protein of interest in the specified functional form in the biological sample from the patient. In some embodiments, the method further includes diagnosing the patient with aggressive or non-aggressive prostate or ovarian cancer based on the level or abundance of the protein of interest in the specified functional form in the biological299713335.1 - 13 -sample from the patient. In some embodiments, the method further comprises treating the patient diagnosed with aggressive cancer with a stage 3 or 4 cancer therapeutic treatment regimen or treating the patient diagnosed with non-aggressive cancer with a stage 1 or 2 cancer therapeutic treatment regimen. In some embodiments, the method further comprises treating the patient diagnosed with aggressive ovarian or prostate cancer with a stage 3 or 4 ovarian or prostate cancer therapeutic treatment regimen or treating the patient diagnosed with non- aggressive ovarian or prostate cancer with a stage 1 or 2 ovarian or prostate cancer therapeutic treatment regimen.

[0062] In some embodiments, the retrieval tag comprises biotin, streptavidin, avidin, antibiotin, desthiobiotin, O6-benzylguanine, SNAP protein, alkyne, azide,tetrazine,, a peptide epitope or a peptide epitope-specific antibody. In some embodiments, the retrieval tag binding component comprises biotin, streptavidin, avidin, anti- biotin, desthiobiotin, O6-benzylguanine, SNAP protein, alkyne, azide,tetrazine,, a peptide epitope or a peptide epitope-specific antibody.

[0063] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0064] Aspect 1 includes a system for evaluating a protein of interest comprising: (i) a protein of interest fused to an epitope tag; (ii) an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises an epitopebinding component and a first oligonucleotide; (iii) an activity probe, wherein the activity probe comprises a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; and (iv) a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval-binding tag comprises a second oligonucleotide.299713335.1 - 14 -

[0065] Aspect 2 includes a system for evaluating a protein of interest comprising: (i) a protein of interest fused to an epitope tag; (ii) an epitope tag binding component capable of binding the epitope tag; (iii) an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide; (iv) an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; and (v) a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval-binding tag comprises a second oligonucleotide.

[0066] Aspect 3 depends upon any one of Aspects 1 to 2, wherein the first and / or second oligonucleotide is single stranded.

[0067] Aspect 4 depends upon any one of Aspects 1 to 3, wherein the first and / or second oligonucleotides comprise a DNA barcode.

[0068] Aspect 5 depends upon any one of Aspects 1 to 4, further comprising one or more bridging oligonucleotides, wherein at least one of the one or more bridging oligonucleotides comprises complementary regions to both the first and second oligonucleotide.

[0069] Aspect 6 depends upon any one of Aspects 1 to 5, wherein the system further comprises one or more reagents for rolling circle amplification.

[0070] Aspect 7 depends upon any one of Aspects 1 to 6, further comprising a recognition oligonucleotide conjugated to a label, wherein the recognition oligonucleotide is complementary to the first oligonucleotide, the second oligonucleotide or at least one of the one or more bridging oligonucleotides.

[0071] Aspect 8 depends upon Aspect 7, wherein the label comprises a flourescent protein, a pull down tag or a split recognition system.

[0072] Aspect 9 depends upon any one of Aspects 1 to 8, further comprising a first labeled primer and / or second labeled primer, wherein the first labeled primer is specifically complementary to the first oligonucleotide and non-complementary to the second oligonucleotide and the second labeled primer is specifically complementary to the second oligonucleotide and non-complementary to the first oligonucleotide.

[0073] Aspect 10 depends upon Aspect 9, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.299713335.1 - 15 -

[0074] Aspect 11 depends upon Aspect 9 or 10, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

[0075] Aspect 12 depends upon any one of Aspects 1 to 11, wherein the first and the second oligonucleotides are capable of being ligated and / or annealing when in sufficient proximity to each other.

[0076] Aspect 13 depends upon any one of Aspects 1 to 12, wherein the protein of interest is an enzyme.

[0077] Aspect 14 depends upon any one of Aspects 1 to 13, wherein the protein of interest comprises a serine hydrolase, cysteine protease, kinase, metalloprotease, P-retaining glycosidase, tyrosine phosphatase, or cytochrome P450.

[0078] Aspect 15 depends upon any one of Aspects 1 to 14, wherein the protein of interest is a DNA repair enzyme.

[0079] Aspect 16 depends upon any one of Aspects 1 to 15, wherein the protein of interest is a non-enzyme.

[0080] Aspect 17 depends upon any one of Aspects 1 to 16, wherein the epitope tag comprises a fluorescent protein, a pull down tag, a split recognition system, or a protein tag for covalent labeling, optionally wherein the protein tags for covalent labeling comprise snap tag or halo tag.

[0081] Aspect 18 depends upon Aspect 17, wherein the fluorescent protein comprises GFP, RFP, or mCherry.

[0082] Aspect 19 depends upon Aspect 17, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

[0083] Aspect 20 depends upon Aspect 17, wherein the split recognition system comprises split gfp or spytag and catcher.

[0084] Aspect 21 depends upon any one of Aspects 1 to 20, wherein the epitope tag is fused to the protein of interest by combining genetic material.

[0085] Aspect 22 depends upon any one of Aspects 1 to 21, wherein the protein of interest fused to the epitope tag is a recombinant protein.

[0086] Aspect 23 depends upon any one of Aspects 1 to 22, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.299713335.1 - 16 -

[0087] Aspect 24 depends upon any one of Aspects 1 to 23, wherein the targeting group comprises a flourophosphonate, diphenylphosphonate, sulfonyl fluoride, acyloxymethyl ketone, phenoxymethylketone, vinyl sulfone, epoxide, halomethylketone, alpha-haloester, alpha-haloamide, a, P-unsaturated ester, a, P-unsaturated ketone, diazomethylketone, acyl phosphate, acylphosphonate, hydroxamate, carbamate, ester, thioester, 2-deoxy-2-fluoro glycoside, a-bromobenzylphosphonate, 2-ethynylnaphthalene, or acrylamide, butyneamide, crotonamide, chloroacetamide, alpha-methylchloroacetamide.

[0088] Aspect 25 depends upon any one of Aspects 1 to 24, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are one or more of the following retrieval tag and retrieval tag binding component pairs: biotin and streptavidin, biotin and avidin, biotin and anti -biotin, desthiobiotin and streptavidin, desthiobiotin and avidin, desthiobiotin and anti-biotin, 06- benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a tran-cyclooctene, or an epitope and an epitope-specific antibody.

[0089] Aspect 26 depends upon any one of Aspects 1 to 25, wherein the targeting group is selected based on the protein family of interest.

[0090] Aspect 27 depends upon any one of Aspects 1 to 26, wherein the protein of interest is a serine hydrolase and the targeting group comprises .

[0091] Aspect 28 depends upon any one of Aspects 1 to 27, wherein the protein of interest is a kinase and the targeting group comprises .

[0092] Aspect 29 depends upon any one of Aspects 1 to 28, wherein the protein of interest is a cysteine protease and the targeting group comprises one of: .

[0093] Aspect 30 depends upon any one of Aspects 1 to 29, wherein the targeting group is linked to the retrieval tag with a linker.

[0094] Aspect 31 depends upon Aspect 30, wherein the linker is organic.

[0095] Aspect 32 depends upon Aspect 30 or 31, wherein the linker comprises an aliphatic linker.

[0096] Aspect 33 depends upon any one of Aspects 1 to 32, wherein the retrieval tag comprises an orthogonal recognition element.

[0097] Aspect 34 depends upon any one of Aspects 1 to 33, wherein the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, O6-benzylguanine, SNAP299713335.1 - 17 -protein, an alkyne, an azide, a cyclooctyne, a tetrazine, a trans-cyclooctene, a peptide epitope or a peptide epitope-specific antibody.

[0098] Aspect 35 depends upon any one of Aspects 1 to 34, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

[0099] Aspect 36 depends upon Aspect 35, wherein the specialized functional form is an active form.

[0100] Aspect 37 depends upon any one of Aspects 1 to 36, wherein the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form.

[0101] Aspect 38 depends upon Aspect 37, wherein the non-specialized functional form is an inactive form.

[0102] Aspect 39 depends upon any one of Aspects 1 to 38, wherein the system further comprises a non-activity probe capable of binding the protein of interest in non-specialized functional form.

[0103] Aspect 40 includes a method for evaluating a target protein of interest comprising the system of any one of Aspects 1 to 39.

[0104] Aspect 41 depends upon Aspect 40, wherein the evaluating comprises measuring activity and / or quantity.

[0105] Aspect 42 includes an epitope tag recognition element according to any one of Aspects 1 to 41.

[0106] Aspect 43 includes an epitope tag recognition element comprising an epitopebinding component and a barcode.

[0107] Aspect 44 depends upon Aspect 43, wherein the epitope-binding component is interchangeable with at least another epitope-binding component.

[0108] Aspect 45 includes an activity probe according to any one of Aspects 1 to 41.

[0109] Aspect 46 includes an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest.

[0110] Aspect 47 depends upon Aspect 46, wherein the targeting group is interchangeable with at least another targeting group.299713335.1 - 18 -

[0111] Aspect 48 depends upon Aspect 46 or 47, wherein the targeting group is selected based on the protein of interest.

[0112] Aspect 49 includes a method for evaluating a target protein of interest, the method comprising: (i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; (ii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises an epitope tag binding component and a first oligonucleotide; (iii) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (iv) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and (v) detecting the ligated or annealed first and second oligonucleotide.

[0113] Aspect 50 depends upon Aspect 49, wherein step (i) occurs in a live cell and steps (ii)-(v) occur after fixing and / or lysing of the cell.

[0114] Aspect 51 includes a method for evaluating a target protein of interest, the method comprising: (i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; (ii) contacting the composition with an epitope tag binding component capable of binding the epitope tag; (iii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide; (iv) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (v) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and (vi) detecting the ligated and / or annealed first and second oligonucleotides.

[0115] Aspect 52 depends upon Aspect 51, wherein steps (iii) and (iv) are performed: (a) consecutively with (iii) before (iv); (b) consecutively with (iv) before (iii); or (c) concurrently.299713335.1 - 19 -

[0116] Aspect 53 depends upon Aspect 51, wherein step (i) occurs in a live cell and steps (ii)-(vi) occur after fixing and / or lysing of the cell.

[0117] Aspect 54 depends upon any one of Aspects 49 to 52, wherein the protein of interest is in a cell.

[0118] Aspect 55 depends upon any one of Aspects 49 to 54, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialized functional form.

[0119] Aspect 56 depends upon Aspect 52, wherein the specialized functional form is an active form of the protein of interest.

[0120] Aspect 57 depends upon any one of Aspects 49 to 56, wherein detecting the ligated or annealed oligonucleotides comprises imaging, qPCR or a sequencing-based readout.

[0121] Aspect 58 depends upon any one of Aspects 49 to 57, wherein the method further comprises determining abundance of the target protein of interest.

[0122] Aspect 59 depends upon Aspect 58, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0123] Aspect 60 depends upon Aspect 58 or 59, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0124] Aspect 61 depends upon any one of Aspects 49 to 60, wherein the steps are performed in chronological order.

[0125] Aspect 62 depends upon any one of Aspects 49 to 61, further comprising contacting the composition with one or more bridging oligonucleotides, wherein at least one of the one or more bridging oligonucleotides comprises complementary regions to both the first and second oligonucleotide.

[0126] Aspect 63 depends upon any one of Aspects 49 to 62, wherein the method further comprises performing rolling circle amplification after detecting the ligated and / or annealed first and second oligonucleotides.

[0127] Aspect 64 depends upon any one of Aspects 49 to 63, further comprising contacting the composition with one or more recognition oligonucleotides conjugated to a label, wherein299713335.1 - 20 -the recognition oligonucleotide is complementary to the first oligonucleotide, the second oligonucleotide or at least one of the one or more bridging oligonucleotides.

[0128] Aspect 65 depends upon Aspect 64, wherein the label comprises a fluorescent protein, a pull down tag or a split recognition system.

[0129] Aspect 66 depends upon any one of Aspects 49 to 65, further comprising contacting the composition with a first labeled primer and / or second labeled primer, wherein the first labeled primer is specifically complementary to the first oligonucleotide and non- complementary to the second oligonucleotide and the second labeled primer is specifically complementary to the second oligonucleotide and non-complementary to the first oligonucleotide.

[0130] Aspect 67 depends upon Aspect 66, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

[0131] Aspect 68 depends upon Aspect 66 or 67, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

[0132] Aspect 69 depends upon any one of Aspects 49 to 68, wherein the first and the second oligonucleotides are capable of being ligated and / or annealing when in sufficient proximity to each other in the presence of ligation reagents.

[0133] Aspect 70 depends upon any one of Aspects 49 to 69, wherein the method further comprises spatially detecting the target protein of interest.

[0134] Aspect 71 depends upon any one of Aspects 49 to 70, wherein the activity probe covalently modifies, or is capable of covalently modifying the proteins of interest.

[0135] Aspect 72 depends upon any one of Aspects 49 to 71, wherein the composition comprises fewer than 5000 cells.

[0136] Aspect 73 depends upon any one of Aspects 49 to 72, wherein the composition comprises less than 1 pg of total protein.

[0137] Aspect 74 depends upon any one of Aspects 49 to 73, wherein the method further comprises detecting a cellular marker.

[0138] Aspect 75 depends upon any one of Aspects 49 to 74, wherein the method further comprises determining the total amount of target protein of interest.299713335.1 - 21 -

[0139] Aspect 76 depends upon Aspect 75, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

[0140] Aspect 77 depends upon Aspect 75 or 76, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the specialized functional form and non-specialized functional forms of the protein.

[0141] Aspect 78 depends upon any one of Aspects 49 to 77, wherein the method excludes performing liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and / or mass spectrometry.

[0142] Aspect 79 depends upon any one of Aspects 49 to 78, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are one or more of the following retrieval tag and retrieval tag binding component pairs: biotin and streptavidin, biotin and avidin, biotin and anti -biotin, desthiobiotin and streptavidin, desthiobiotin and avidin, desthiobiotin and anti-biotin, 06- benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a tran-cyclooctene, or an epitope and an epitope-specific antibody.

[0143] Aspect 80 depends upon any one of Aspects 49 to 79, wherein the first and / or second oligonucleotide is single-stranded.

[0144] Aspect 81 depends upon any one of Aspects 49 to 80, wherein the first and / or second oligonucleotide comprises a DNA barcode.

[0145] Aspect 82 depends upon any one of Aspects 49 to 81, wherein the protein of interest is an enzyme.

[0146] Aspect 83 depends upon any one of Aspects 49 to 82, wherein the protein of interest is a serine hydrolase, cysteine proteases, kinases, metalloproteases, P-retaining glycosidases, tyrosine phosphatases, or cytochrome P450s.

[0147] Aspect 84 depends upon any one of Aspects 49 to 83, wherein the protein of interest is a DNA repair enzyme.

[0148] Aspect 85 depends upon any one of Aspects 49 to 84, wherein the protein of interest is a non-enzyme.

[0149] Aspect 86 depends upon any one of Aspects 49 to 85, wherein the epitope tag comprises a fluorescent protein, a pull down tag, a split recognition system, or a protein tag for299713335.1 - 22 -covalent labeling, optionally wherein the protein tag for covalent labeling comprises snap tag or halo tag.

[0150] Aspect 87 depends upon Aspect 86, wherein the fluorescent protein comprises GFP, RFP, or mCherry.

[0151] Aspect 88 depends upon Aspect 86, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

[0152] Aspect 89 depends upon Aspect 86, wherein the split recognition system comprises split gfp or spytag and catcher.

[0153] Aspect 90 depends upon any one of Aspects 49 to 89, wherein the epitope tag is fused to the protein of interest by combining genetic material.

[0154] Aspect 91 depends upon any one of Aspects 49 to 90, wherein the protein of interest fused to the epitope tag is a recombinant protein.

[0155] Aspect 92 depends upon any one of Aspects 49 to 91, wherein the protein of interest is introduced into a cell by transfection.

[0156] Aspect 93 depends upon Aspect 92, wherein the cell is a live cell.

[0157] Aspect 94 depends upon any one of Aspects 49 to 93, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

[0158] Aspect 95 depends upon any one of Aspects 49 to 94, wherein the targeting group comprises a flourophosphonate, diphenylphosphonate, sulfonyl fluoride, acyloxymethyl ketone, phenoxymethylketone, vinyl sulfone, epoxide, halomethylketone, alpha-haloester, alpha-haloamide, a, P-unsaturated ester, a, P-unsaturated ketone, diazomethylketone, acyl phosphate, acylphosphonate, hydroxamate, carbamate, ester, thioester, 2-deoxy-2-fluoro glycoside, a-bromobenzylphosphonate, 2-ethynylnaphthalene, acrylamide, butyneamide, crotonamide, chloroacetamide, or alpha-methylchloroacetamide.

[0159] Aspect 96 depends upon any one of Aspects 49 to 95, wherein the targeting group is selected based on the protein family of interest.

[0160] Aspect 97 depends upon any one of Aspects 49 to 96, wherein the protein of interestO is a serine hydrolase and the targeting group comprises299713335.1 - 23 -

[0161] Aspect 98 depends upon any one of Aspects 49 to 96, wherein the protein of interestis a kinase and the targeting group comprises

[0162] Aspect 99 depends upon any one of Aspects 49 to 96, wherein the protein of interestis a cysteine protease and the targeting group comprises one of

[0163] Aspect 100 depends upon any one of Aspects 49 to 99, wherein the targeting group is linked to the retrieval tag with a linker.

[0164] Aspect 101 depends upon Aspect 100, wherein the linker is organic.

[0165] Aspect 102 depends upon Aspect 100 or 101, wherein the linker comprises an aliphatic linker.

[0166] Aspect 103 depends upon any one of Aspects 49 to 102, wherein the retrieval tag comprises an orthogonal recognition element.

[0167] Aspect 104 depends upon any one of Aspects 49 to 103, wherein the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, O6-benzylguanine, SNAP protein, an alkyne, an azide, a cyclooctyne, a tetrazine, a trans-cyclooctene, a peptide epitope or a peptide epitope-specific antibody.

[0168] Aspect 105 depends upon any one of Aspects 49 to 104, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

[0169] Aspect 106 depends upon Aspect 105, wherein the specialized functional form is an active form.

[0170] Aspect 107 depends upon any one of Aspects 49 to 106, wherein the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form.299713335.1 - 24 -

[0171] Aspect 108 depends upon Aspect 107, wherein the non-specialized functional form is an inactive form.

[0172] Aspect 109 depends upon any one of Aspects 49 to 108, wherein the system further comprises a non-activity probe capable of binding the protein of interest in non-specialized functional form.

[0173] Aspect 110 includes a method of competitive profiling comprising any one of Aspects 49 to 109, wherein step (i) further comprises contacting the composition with a nonactivity probe capable of binding the protein of interest in non-specialized functional form.

[0174] Aspect 111 includes a method of analyzing two or more proteins of interest using any one of Aspects 49 to 109, wherein the label and the recognition oligonucleotides are different for each protein.

[0175] Aspect 112 includes a method of determining a half maximal inhibitory concentration (IC50) value of a compound of interest against a protein of interest, the method comprising: (i) contacting a composition comprising the protein of interest fused to an epitope tag with an activity probe comprising the compound of interest operatively linked to a retrieval tag, wherein the compound of interest is capable of binding to the protein of interest; (ii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises an epitope tag binding component and a first oligonucleotide; (iii) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (iv) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and (v) detecting the ligated or annealed first and second oligonucleotide.

[0176] Aspect 113 depends upon Aspect 112, wherein step (i) occurs in a live cell and steps (ii)-(v) occur after fixing and / or lysing of the cell.

[0177] Aspect 114 includes a method of determining a half maximal inhibitory concentration (IC50) value of a compound of interest against a protein of interest, the method comprising: (i) contacting a composition comprising the protein of interest fused to an epitope tag with an activity probe comprising the compound of interest operatively linked to a retrieval tag, wherein the compound is capable of binding to the protein of interest; (ii) contacting the composition with an epitope tag binding component capable of binding the epitope tag; (iii)299713335.1 - 25 -contacting the composition with an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide; (iv) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (v) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and (vi) detecting the ligated and / or annealed first and second oligonucleotides.

[0178] Aspect 115 depends upon Aspect 114, wherein steps (iii) and (iv) are performed: (a) consecutively with (iii) before (iv); (b) consecutively with (iv) before (iii); or (c) concurrently.

[0179] Aspect 116 depends upon Aspect 114, wherein step (i) occurs in a live cell and steps (ii)-(vi) occur after fixing and / or lysing of the cell.

[0180] Aspect 117 depends upon any one of Aspects 112 to 115, wherein the protein of interest is in a cell.

[0181] Aspect 118 depends upon any one of Aspects 112 to 117, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialized functional form.

[0182] Aspect 119 depends upon Aspect 115, wherein the specialized functional form is an active form of the protein of interest.

[0183] Aspect 120 depends upon any one of Aspects 112 to 119, wherein detecting the ligated or annealed oligonucleotides comprises imaging, qPCR or a sequencing-based readout.

[0184] Aspect 121 depends upon any one of Aspects 112 to 120, wherein the method further comprises determining abundance of the target protein of interest.

[0185] Aspect 122 depends upon Aspect 121, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0186] Aspect 123 depends upon Aspect 121 or 122, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0187] Aspect 124 depends upon any one of Aspects 112 to 123, wherein the steps are performed in chronological order.299713335.1 - 26 -

[0188] Aspect 125 depends upon any one of Aspects 112 to 124, further comprising contacting the composition with one or more bridging oligonucleotides, wherein at least one of the one or more bridging oligonucleotides comprises complementary regions to both the first and second oligonucleotide.

[0189] Aspect 126 depends upon any one of Aspects 112 to 125, wherein the method further comprises performing rolling circle amplification after detecting the ligated and / or annealed first and second oligonucleotides.

[0190] Aspect 127 depends upon any one of Aspects 112 to 126, further comprising contacting the composition with one or more recognition oligonucleotides conjugated to a label, wherein the recognition oligonucleotide is complementary to the first oligonucleotide, the second oligonucleotide or at least one of the one or more bridging oligonucleotides.

[0191] Aspect 128 depends upon Aspect 127, wherein the label comprises a fluorescent protein, a pull down tag or a split recognition system.

[0192] Aspect 129 depends upon any one of Aspects 112 to 128, further comprising contacting the composition with a first labeled primer and / or second labeled primer, wherein the first labeled primer is specifically complementary to the first oligonucleotide and non- complementary to the second oligonucleotide and the second labeled primer is specifically complementary to the second oligonucleotide and non-complementary to the first oligonucleotide.

[0193] Aspect 130 depends upon Aspect 129, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

[0194] Aspect 131 depends upon Aspect 129 or 130, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

[0195] Aspect 132 depends upon any one of Aspects 112 to 131, wherein the first and the second oligonucleotides are capable of being ligated and / or annealing when in sufficient proximity to each other in the presence of ligation reagents.

[0196] Aspect 133 depends upon any one of Aspects 112 to 132, wherein the method further comprises spatially detecting the target protein of interest.

[0197] Aspect 134 depends upon any one of Aspects 112 to 133, wherein the compound of interest covalently modifies, or is capable of covalently modifying the proteins of interest.299713335.1 - 27 -

[0198] Aspect 135 depends upon any one of Aspects 112 to 134, wherein the composition comprises fewer than 5000 cells.

[0199] Aspect 136 depends upon any one of Aspects 112 to 135, wherein the composition comprises less than 1 pg of total protein.

[0200] Aspect 137 depends upon any one of Aspects 112 to 136, wherein the method further comprises detecting a cellular marker.

[0201] Aspect 138 depends upon any one of Aspects 112 to 137, wherein the method further comprises determining the total amount of target protein of interest.

[0202] Aspect 139 depends upon Aspect 138, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

[0203] Aspect 140 depends upon Aspect 138 or 139, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the specialized functional form and non-specialized functional forms of the protein.

[0204] Aspect 141 depends upon any one of Aspects 112 to 140, wherein the method excludes performing liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and / or mass spectrometry.

[0205] Aspect 142 depends upon any one of Aspects 112 to 141, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are one or more of the following retrieval tag and retrieval tag binding component pairs: biotin and streptavidin, biotin and avidin, biotin and anti -biotin, desthiobiotin and streptavidin, desthiobiotin and avidin, desthiobiotin and anti-biotin, 06- benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a tran-cyclooctene, or an epitope and an epitope-specific antibody.

[0206] Aspect 143 depends upon any one of Aspects 112 to 142, wherein the first and / or second oligonucleotide is single-stranded.

[0207] Aspect 144 depends upon any one of Aspects 112 to 143, wherein the first and / or second oligonucleotide comprises a DNA barcode.

[0208] Aspect 145 depends upon any one of Aspects 112 to 144, wherein the protein of interest is an enzyme.299713335.1 - 28 -

[0209] Aspect 146 depends upon any one of Aspects 112 to 145, wherein the protein of interest is a serine hydrolase, cysteine proteases, kinases, metalloproteases, P-retaining glycosidases, tyrosine phosphatases, or cytochrome P450s.

[0210] Aspect 147 depends upon any one of Aspects 112 to 146, wherein the protein of interest is a DNA repair enzyme.

[0211] Aspect 148 depends upon any one of Aspects 112 to 147, wherein the protein of interest is a non-enzyme.

[0212] Aspect 149 depends upon any one of Aspects 112 to 148, wherein the epitope tag comprises a fluorescent protein, a pull down tag, a split recognition system, or a protein tag for covalent labeling, optionally wherein the protein tag for covalent labeling comprises snap tag or halo tag.

[0213] Aspect 150 depends upon Aspect 149, wherein the fluorescent protein comprises GFP, RFP, or mCherry.

[0214] Aspect 151 depends upon Aspect 149, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

[0215] Aspect 152 depends upon Aspect 149, wherein the split recognition system comprises split gfp or spytag and catcher.

[0216] Aspect 153 depends upon any one of Aspects 112 to 152, wherein the epitope tag is fused to the protein of interest by combining genetic material.

[0217] Aspect 154 depends upon any one of Aspects 112 to 153, wherein the protein of interest fused to the epitope tag is a recombinant protein.

[0218] Aspect 155 depends upon any one of Aspects 112 to 154, wherein the protein of interest is introduced into a cell by transfection.

[0219] Aspect 156 depends upon Aspect 155, wherein the cell is a live cell.

[0220] Aspect 157 depends upon any one of Aspects 112 to 156, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

[0221] Aspect 158 depends upon any one of Aspects 112 to 157, wherein the targeting group comprises a flourophosphonate, diphenylphosphonate, sulfonyl fluoride, acyloxymethyl ketone, phenoxymethylketone, vinyl sulfone, epoxide, halomethylketone, alpha-haloester, alpha-haloamide, a, P-unsaturated ester, a, P-unsaturated ketone, diazomethylketone, acyl299713335.1 - 29 -phosphate, acylphosphonate, hydroxamate, carbamate, ester, thioester, 2-deoxy-2-fluoro glycoside, a-bromobenzylphosphonate, 2-ethynylnaphthalene, acrylamide, butyneamide, crotonamide, chloroacetamide, or alpha-methylchloroacetamide.

[0222] Aspect 159 depends upon any one of Aspects 112 to 158, wherein the targeting group is selected based on the protein family of interest.

[0223] Aspect 160 depends upon any one of Aspects 112 to 159, wherein the protein ofO interest is a serine hydrolase and the targeting group comprises

[0224] Aspect 161 depends upon any one of Aspects 112 to 159, wherein the protein ofinterest is a kinase and the targeting group comprises

[0225] Aspect 162 depends upon any one of Aspects 112 to 159, wherein the protein of interest is a cysteine protease and the targeting group comprises one of

[0226] Aspect 163 depends upon any one of Aspects 112 to 162, wherein the targeting group is linked to the retrieval tag with a linker.

[0227] Aspect 164 depends upon Aspect 163, wherein the linker is organic.

[0228] Aspect 165 depends upon Aspect 163 or 164, wherein the linker comprises an aliphatic linker.

[0229] Aspect 166 depends upon any one of Aspects 112 to 165, wherein the retrieval tag comprises an orthogonal recognition element.

[0230] Aspect 167 depends upon any one of Aspects 112 to 166, wherein the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, O6-benzylguanine, SNAP protein, an alkyne, an azide, a cyclooctyne, a tetrazine, a trans-cyclooctene, a peptide epitope or a peptide epitope-specific antibody.299713335.1 - 30 -

[0231] Aspect 168 depends upon any one of Aspects 112 to 167, wherein the compound of interest binds to the protein of interest when the protein of interest is in a specialized functional form.

[0232] Aspect 169 depends upon Aspect 168, wherein the specialized functional form is an active form.

[0233] Aspect 170 depends upon any one of Aspects 112 to 169, wherein the compound of interest does not bind to the protein of interest when the protein of interest is in non-specialized functional form.

[0234] Aspect 171 depends upon Aspect 170, wherein the non-specialized functional form is an inactive form.

[0235] Aspect 172 depends upon any one of Aspects 112 to 171, wherein the system further comprises a non-activity probe capable of binding the protein of interest in non-specialized functional form.

[0236] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0237] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Any term used in singular form also comprise plural form and vice versa.

[0238] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” “(x and z) or y,” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an aspect or aspect.

[0239] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.299713335.1 - 31 -

[0240] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0241] It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0242] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0243] Use of the one or more systems or compositions may be employed based on any of the methods described herein. Other aspects and embodiments are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0244] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of the Disclosure, Brief Description of the Drawings, Detailed Description, and / or Claims.

[0245] Other obj ects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and299713335.1 - 32 -scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0246] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0247] FIG. 1A-1C. Schematics of ADPL iterations. (A) Primary ADPL System: In this system, the recognition element directly binds to the protein of interest (POI) and, when in close proximity to the probe recognition oligonucleotide, triggers a ligation event, generating a signal. However, a significant drawback is the limited availability of suitable antibodies for some targets, which restricts the system's ability to expand its target scope. (B) Sandwich ADPL System: This system uses a secondary recognition element conjugated to an oligonucleotide that indirectly recognizes the POI. The secondary recognition element binds to a primary recognition element already attached to the POI. A key limitation of this system is that it hinders the ability to perform multiplexing. (C) Modular ADPL System: This method addresses the limitations of both the Primary and Sandwich ADPL systems by utilizing a modular tag fused to the POI for recognition. This system eliminates the need for antibody optimization for each specific target. The recognition element binds to a common epitope tag, while the probe recognition element attaches to the probe tag. Since the oligonucleotide is directly conjugated to the recognition element there is also room for multiplexing. Also, information on target protein localization and abundance can be detected simultaneously in the case of GFP or other fluorescent proteins. The systems and methods allow for better efficiency and specificity than previous methods. The systems and methods also allow for a modular approach where proteins of interest that do not have specific recognition abilities (e.g. the protein of interest lacks a specific antibody that recognizes the protein of interest) can be more easily and more efficiently assayed in specific environments.

[0248] FIG. 2. Mechanism of modular ADPL. How modular ADPL leads to a signal: On treating cells with a chemical probe (“activity probe”), the probe reacts with the active POI in cells. When recognition elements are added to the system, the protein recognition element (“epitope tag recognition element”) binds to the common epitope tag and the probe recognition299713335.1 - 33 -element (“retrieval tag recognition element”) binds to the probe tag (“retrieval tag”). When these two recognition elements include oligonucleotides and are in good proximity, one more more bridging oligo can binds to them and further ligation which leads to a robust ligated circular DNA. This DNA further undergoes rolling circular amplification and is detected by detection oligonucleotides (“recognition oligonucleotides”). If any one of the recognition elements is missing from the system there is no ligation product and there will be no signal.

[0249] FIG. 3. Modular ADPL platform overview. The figure depicts the system. The protein recognition side can be generally replaced by several specific epitope tags - for example, GFP-tagged proteins can be expressed in diverse cellular formats, then modular ADPL can be run using a GFP-recognition element (oligo-conjugated anti-GFP antibody) and then implemented into imaging, qPCR or sequencing based readouts. These quantitative ADPL measurements can be multiplexed with probes targeting various protein targets, including other expressed ET -proteins or endogenous proteins. This modular approach can be applied to diverse enzymes and activity probes, such as serine hydrolases, kinases, DNA repair enzymes, and non-enzymes with in-cell binding sites. Competitive profiling can be performed as well to find non-discovery probes (i.e., drug-like modulators and small molecules) can compete with discovery probe binding, which prevents the ability of ADPL proximity complex to be formed and DNA barcode to be generated. Thus, degree of labeling by discovery probe in the cell, blocked by small molecule interaction, is reflected in the amount of generated DNA barcode.

[0250] FIG. 4. Exemplary proteins and tags that can be adapted to the platform. This figure provides a non-exhaustive list of target POIs for which GFP-fusions can be used for ADPL. This list can be expanded to any protein within the cell and any family of proteins. The figure also provides a list of common tags that can be fused to the POI.

[0251] FIG. 5. Activity probe of the system. This figure depicts an activity probe. The chemical probes used in the study in the examples are serine hydrolase and kinase active site reactive family wide probes. These probes can be expanded to other families of proteins like cathepsins, DNA repair enzymes as well as non-enzymes. The modular elements in the chemical probes include 1) warhead (“targeting group”), 2) linker and 3) tag. A few variations of these elements are given in the figure but can be expanded. For example, this could be expanded to probes containing other electrophilic chemical groups like chloroacetamide, Alpha-methylchloroacetamide, bromoacetamide, iodoacetamide, acrylamide, crotonamide, butyneamide and others.299713335.1 - 34 -

[0252] FIG. 6. Recognition elements of the system. The figure depicts the recognition elements: (1) a protein of interest recognition element (“epitope tag recognition element”) and (2) a probe recognition element (“retrieval tag recognition element”). The recognition elements for ADPL bind to the common epitope tag fused to the protein of interest and the probe recognition elements bind to the tag on the chemical probe (“activity probe”). These recognition elements are also modular and can vary. Examples are provided in the figure.

[0253] FIG. 7. Exemplary oligonucleotides used for imaging. This figure depicts example oligonucleotides that can be used in the system for imaging of a single POI.

[0254] FIG. 8. Exemplary oligonucleotides used for multiplexed imaging. This figure depicts example oligonucleotides that can be used in the system for imaging multiple POIs.

[0255] FIG. 9. Exemplary barcoded oligonucleotides used for sequencing use: Nextera Addition. This figure depicts example oligonucleotides that can be used in the system for sequencing analysis.

[0256] FIG. 10. Exemplary barcoded oligonucleotides used for sequencing use: Nextera immediately compatible. This figure depicts example oligonucleotides that can be used in the system for sequencing analysis. Sequences are slightly different from the previous figure to be compatible with a different sequencing platform.

[0257] FIG. 11. Exemplary barcoded oligonucleotides used for sequencing use: TruSeq Immediately Compatible. This figure depicts example oligonucleotides that can be used in the system for sequencing analysis. Sequences are slightly different from the previous figure to be compatible with a different sequencing platform.

[0258] FIG. 12. Exemplary barcoded oligonucleotides used for sequencing use: TruSeq / Nextera Mixed. This figure depicts example oligonucleotides that can be used in the system for sequencing analysis. Sequences are slightly different from the previous figure to be compatible with a different sequencing platform.

[0259] FIG. 13. Exemplary kinase activity probes. The examples include family-wide kinase probes that covalently attach to active kinases in cells. They are compatible with modular or standard ADPL readout. Linker length and overall molecular properties are very important and non-obvious for proper function. This is demonstrated by X4K not generating any ADPL signal for a target kinase, but X8K and X12K yielding a productive signal.299713335.1 - 35 -

[0260] FIG. 14. Validation of Modular ADPL imaging. This figure depicts data from experiments with serine hydrolases and a snap tag. POI - Snaptag-GFP fusion, chemical probe - benzylguanine biotin GFP channel shows expression of SNAP protein, Modular ADPL signal is generated on addition of the chemical probe (red), and no signal is generated on addition of inhibitor or removal of one of the recognition elements. Similarly, results are seen on expansion to a protein targeted by a family wide probe. POI - PAFAH2, Chemical probe - FP-bio. Increasing signal on addition of increasing chemical probe and decreasing signal on addition of increasing concentration of inhibitor was observed.

[0261] FIG. 15A-15D. Modular ADPL detection of kinase family protein activity and ligand binding. This figure depicts data from experiments with CDK2 and LIMK2.. POI - CDK2 (FIG. 15 A) and LIMK2 (FIG. 15D), Chemical probe - X12K GFP channel shows expression of CDK2 and LIMK2 protein, Modular ADPL signal is generated on addition of the chemical probe, and no signal is generated on addition of inhibitor or removal of one of the recognition elements. (B) mean intensity of CDK2 signal normalized to GFP. (C) mean intensity of LIMK2 signal normalized to GFP.

[0262] FIG. 16. Validation of expression and probe labeling. Validation of the POI-gfp fusion cell lines. The figure shows images of the gfp expressing cells.

[0263] FIG. 17. Modular ADPL platform overview. Schematic representation of a Protein of interest (POIe, fused to an Epitope tag (ET) labeled with a discovery probe (DP) lead to ADPL signal due to proximity ligation. Other proteins are also labelled by the DP but the signal is generated only when the POI is fused to an ET. b) In the event that the protein in inactive due to structural changes or due to the binding of an inhibitor, no signal is generated since the DP no longer binds to the POI. c) The DP label any family member depending on the warhead and any of the mentioned tags can be used as a handle for readout, d) One can also easily “plug and play” the epitope tags and any one of the mentioned tags can be used for modular ADPL experiments, e) One can implement the use of multiple epitope binding reagents in modular ADPL studies.

[0264] FIG. 18A-18C. Modular ADPL signal on treating with ibrutinib. (A) Imaging of cells treated with DMSO (a), an X12K control (b), XO44+X12K (c), and ibrutinib+X12K (d). (B) Quantification of (A) showing the % modular ADPLsignal. (C) % modular ADPLsignal at differing concentrations of ibrutinib+X12K.299713335.1 - 36 -

[0265] FIG. 19A-19C. Kinetics of modular ADPL signal with ibrutinib. (A) Time course of ibrutinib+X12K showing the ICso at various time points. (B) Western blot analysis of ibrutinib+X12K at increasing concentrations. (C) Quantification of (B) to calculate the ICso value using Western blotting analysis.DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0266] The methods of the disclosure provide for a chemical proteomic platform to address several shortcomings that plague current proteomic profiling approaches. Chief among these were the inability to probe a wide dynamic range of sample abundance, provide information on the functional state of proteins, and the capacity to quantify this information with spatial resolution at the inter- and intracellular scale. Compared to existing activity-based proteomic approaches with gel- or LC-MS / MS as a readout, the incorporation of a specific and robust amplification scheme applied in native cell environments allows for significant expansion of the questions that can be addressed in biological, diagnostic, and therapeutic systems. First, the disclosed methods permit quantification of enzyme activity across a high dynamic range with respect to sample input as well as relative abundance within the proteome of a given cell. The examples of the disclosure demonstrate that the disclosed methods allow for single-cell resolution, as well as interrogation of low abundance or low activity protein targets, both of which represent important contexts in biology. Single-cell resolution and low sample requirements enable the detection and quantification of enzyme activity in heterogeneous cellular populations, including cellular co-culture and primary ovarian cancer spheroids. The fact that the methods of the disclosure do not require any genetic manipulation is also important to allow for direct compatibility with other types of primary tissues and fluids. Furthermore, readouts such as fluorescence imaging, qPCR, and sequencing can all be combined with the methods of the disclosure. Additionally, implementation of barcoded oligo-fluorophores or primers enable multiplexed readout of active enzymes within and between families, as well as integration with methods to simultaneously capture information on transcript and protein abundance. The use of cell permeable family-wide chemical probes permits tagging of active proteins in their native cellular context, which provides a better representation of their functional properties. The Examples of the application demonstrate the applicability of the disclosed methods to detect activity of a kinase, specifically of CDK2. Since this technique does not require sample homogenization it allows for retention of quantitative, activitydependent information at the inter- and intracellular scale. These aspects of the disclosed methods are useful for the study of the relationship between protein abundance, localization299713335.1 - 37 -and activity in a variety of biological contexts (e.g., cancer, inflammation, immune function, development).

[0267] The disclosed methods can be used to detect endogenous differences in enzyme activity among distinct cellular phenotypes, as well as to interrogate the action of small molecule inhibitors on enzyme function directly in live cells. Additionally, this approach can be used to verify target engagement in cells without relying on downstream peripheral biomarkers, an important capability in both basic and translational research. This modularity may be extended to other mechanism-driven or affinity-based probes, greatly expanding the information that can be captured on these proteins with spatial resolution, high dynamic range, and in native environments. This approach enables the interrogation of important basic and translational questions in biology and medicine and provides novel therapeutic and diagnostic approaches to treat and detect disease.

[0268] It has been previously disclosed and shown that one can quantitatively measure small molecule probe binding to endogenous proteins inside or outside of live cells, by performing probe-protein adduct (covalent adduct)-dependent ‘proximity barcoding’ to generate an oligonucleotide product for detection and quantification of the probe-protein amount or concentration. This general concept has been demonstrated for many target protein examples, after their binding and adduct formation with different ‘family-wide’ activity probes or other covalent chemical probes, in diverse cellular settings, including cell culture, isolated single cells, isolated tissues and biofluids. ADPL-generated oligonucleotide products can be subsequently detected and quantified by fluorescence imaging, digital droplet PCR, quantitative reverse transcription PCR (RT-qPCR) and next generation sequencing. In this iteration, the generation of an oligonucleotide signal from a protein of interest (POI)-probe adduct required detection of the specific POI through an antibody, aptamer, nanobody or one of many other POI specific binding reagents. These binding reagents (BRs) were conjugated to single stranded DNA for subsequent ADPL processing, as described previously. However, this approach is therefore limited to the availability, synthesis and suitable activity of POI- specific BR-oligo conjugates to detect new target proteins.

[0269] As described above, previous ADPL systems have drawbacks due to the limited availability of suitable antibodies for some targets, which restricts the system's ability to expand its target scope. Other systems are limited because they are hindered in their ability to perform multiplexing. The disclosed system addresses the limitations of previous ADPL systems by utilizing a modular tag fused to the POI for recognition. The novel system eliminates the need299713335.1 - 38 -for antibody optimization for each specific target. Since the oligonucleotide can be directly conjugated to a recognition element (“epitope binding probe”) there is also room for multiplexing. Also, information on target protein localization and abundance can be detected simultaneously in the case of GFP or other fluorescent proteins.

[0270] The disclosure relates to a ‘modular’ ADPL solution. In some aspects, a POI is expressed as an epitope-tagged protein of interest (E-POI), which can be recognized via an epitope-binding reagent (EBR-oligo) (“epitope-binding probe”) fused to a single stranded DNA barcode. Example epitopes (“epitope tags”) include GFP or other fluorescent proteins, MBP, FLAG-tag, SNAP -tag, Clip-Tag, halo-tag, Myc-tag, HA-tag, an azide group, an alkyne group, a ketone group or other specific epitopes expressed as a fusion on the N-terminus, C- terminus or interior of any given POI. The Examples demonstrate that the synthesis of complementary EBR-oligos, such as anti-GFP antibody-oligo conjugates or oligo-conjugates to a specific EBR for any of the above epitopes, can be utilized to generate chemical probedependent, and ADPL-dependent product oligonucleotides for detection and quantification.

[0271] EBR-oligo conjugates are described to recognize any E-POI being expressed either transiently or under stable expression in any cellular or tissue context in any cellular environment, however ADPL signal can only be produced if said protein has been chemically labeled by a small molecule chemical probe (activity probe or discovery probe, DP, wherein DPs all are organic small molecules that contain an orthogonal recognition element, such as biotin, desthiobiotin, alkyne, azide, alkylchloride, benzylguanine, tetrazine, TCO, DBCO or other). Small molecule chemical probes are also referred to as “activity probes” throughout this disclosure. Thus, the terms “discovery probe,” “DP” and “activity probe” are used interchangeably.

[0272] The DPs are capable of interacting with and chemically tagging target proteins within the context of whole proteome based on their structural state or activity state as they exist inside of cells, which is a unique and more biologically relevant environment relative and some of these small molecule binding sites may only exist in the context of cellular environments.

[0273] The modularity within the protein-recognition side (ET-protein), small molecule recognition side (both with complementary DNA barcodes) are brought in post small molecule tagging such that only if an ET-protein has been labeled by a discovery probe do the DNA299713335.1 - 39 -barcodes come into close proximity, enabling subsequent ligation or bridging primer binding to create a new DNA barcode that combines the protein- and probe-identity information.

[0274] Using this system, modular ADPL signals from numerous target POIs with numerous chemical probes in numerous cellular settings can be used. These data validate the concept of general ADPL detection of ligand binding for any protein containing a ‘modular’ epitope tag, by combining epitope-tag binding reagent-oligo (EBR-oligo) conjugate, where the oligo conjugates are between 7-40 nucleotides, and contain barcode sequences for ADPL- dependent ligation, primer binding, oligonucleotide amplification and subsequent readout, as described in demonstrative examples in subsequent descriptions and drawings.

[0275] The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a gene product or functional protein.

[0276] The terms “oligonucleotide” and “oligo” are used interchangeably and refer to short DNA or RNA molecules.

[0277] The terms “contacted” and “exposed,” when applied to a cell or composition, are used herein to describe the process by which an agent is delivered to a target cell or are placed in direct juxtaposition with the target cell, composition, or target molecule.

[0278] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein and that different embodiments may be combined.

[0279] Use of the one or more compositions may be employed based on methods described herein. Use of one or more compositions may be employed in the preparation of medicaments for treatments according to the methods described herein. Other embodiments are discussed throughout this application. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. The embodiments in the Example section are understood to be embodiments that are applicable to all aspects of the technology described herein.1. Assay ComponentsA. Molecular Construct

[0280] Embodiments of the disclosure relate to methods and compositions comprising a molecular construct comprising: a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest. The targeting group299713335.1 - 40 -specifically binds to the specified functional form of the proteins of interest. Exemplary embodiments of the molecular construct and fragments thereof are described throughout the application.

[0281] The concentration of the molecular construct in the compositions and methods of the disclosure may be at least, at most, or exactly IxlO'10, IxlO'9, IxlO'8, IxlO'7, IxlO'6, 1x10'5, IxlO’4, IxlO’3, IxlO’2, IxlO’1, 1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO'2, IxlO'1, or 1, IxlO10(or any derivable range therein) pM, nM, pM, mM, M, cM, or dM.

[0282] In some embodiments, the molecular construct is cell permeable.B. Targeting Groups (“warhead)

[0283] The targeting group is a moiety that specifically binds to a protein of interest, or family of proteins, or group of enzymes having a similar or the same enzymatic activity and / or active site structure. The protein of interest can be a protein that is in specified functional form. The term “specifically binds to active specified functional form of a protein” refers to a binding that includes the specified functional form and excludes the non-specified functional form or wherein the targeting group binds to less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1% (or any derivable range thereof) of the total non-specified functional form of the protein. In some embodiments, the interaction of the target protein with the specified or non-specified functional form of the protein of interest may be defined as being a Kd or Kmof at least, at most, or exactly IxlO'10, IxlO'9, IxlO'8, IxlO'7, IxlO'6, IxlO'5, IxlO'4, IxlO'3, IxlO'2, IxlO'1, 1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO'2, IxlO'1, or 1, IxlO10(or any derivable range therein) pM, nM, pM, mM, M, cM, or dM. The targeting group can be obtained from a commercial library.

[0284] FIG. 17 exemplifies family-wide targeting groups (warhead groups) that can be used in the methods of the disclosure. FIG. 17 further exemplifies which probes are useful for certain enzyme families. For example, fluorophosphonate, diphenylphosphonate, and sulfonyl fluoride are useful in methods wherein the protein of interest is a serine hydrolase / serine protease. Sulfonyl fluoride is also useful in methods wherein the protein of interest is a kinase. Acyloxymethyl ketone, phenoxymethylketone, vinyl sulfone, epoxide, halomethylketone, a, P- unsaturated ester, a, P-unsaturated ketone, and diazomethylketone are useful when the protein of interest is a cysteine protease. Nucleotide acyl phosphate is useful when the protein of interest is a kinase. Hydroxamate is useful when the protein of interest is a metalloprotease.299713335.1 - 41 -2-deoxy-2-fluoro glycoside is useful when the protein of interest is a P-retaining glycosidase, a-bromobenzylphosphonate is useful when the protein of interest is a tyrosine phosphatase, and 2-ethynylnaphthalene is useful when the protein of interest is a cytochrome P450.

[0285] In some embodiments, the activity prove comprises desthiobiotin-ADP or desthiobiotin-ADP, binding to active kinase results in acylation of conserved, proximal lysine epsilon-amine(s), tagging the protein with a biotin or desthiobiotin. All kinases have active site proximal lysines, and therefore all kinases and ATP -binding proteins can be profiled using this and similar probes. The treated cellular sample can be processed for ADPL as with other probe classes for in situ cellular imaging, or in lysate and homogenized digital quantitation by qPCR, digital PCR or next-generation sequencing readout. Other kinase probes with diverse electrophilic warheads including sulfonylfluoride-containing, acylphosphate,299713335.1 - 42 -acylphosphonate, N-hydroxysuccinimide, ester and others can be appended onto kinase- binding small molecule scaffolds and used for ADPL kinase profiling as well.

[0286] Further examples of family-wide activity probes useful in the methods and compositions of the disclosure include:299713335.1 - 43 -299713335.1 -44 -299713335.1 -45 -2. Retrieval Tags and Retrieval Tag Binding Component Pairs

[0287] Exemplary retrieval tags and retrieval tag binding component (partner) pairs are shown in FIG. 14.299713335.1 - 46 -

[0288] It is contemplated that the retrieval tag and retrieval tag binding component / partner may be interchangeable. For example, a compound identified herein as a retrieval tag may be used as a retrieval tag binding component / partner, and the compound described as the retrieval tag binding component / partner may be used as a retrieval tag.

[0289] Further exemplary retrieval tag and retrieval tag binding component pairs include:

[0290] In some embodiments, the retrieval tag and / or retrieval tag binding component has an atomic mass of at least, at most, or exactly 200, 175, 150, 125, 100, 75, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, or 0.01 kDa (or any range derivable therein). In some embodiments, the retrieval tag and / or retrieval tag binding component has a molar mass of at least, at most, or exactly 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 250, 225, 200, 175, 150, 125, 100, 75, 50, or 25 g / mol (or any derivable range therein).3. Linker

[0291] In order to conjugate two molecules, such as a retrieval tag to a targeting group (“warhead”) or an oligo to an antibody or to a retrieval tag binding component (“binder”), the following exemplary techniques and linking reagents can be applied.

[0292] Functional groups and reactive groups may be used to link two molecules together. The term “functional groups” as used herein is not restricted to reactive chemical groups299713335.1 - 47 -forming covalent bonds, but also includes chemical groups leading to an ionic interaction or hydrogen bonds with one or more components of the assay.

[0293] The functional groups or the linking molecules bearing them may be amino groups, carbonic acid groups, thiols, thioethers, disulfides, guanidino, hydroxyl groups, amine groups, vicinal dioles, aldehydes, alpha-haloacetyl groups, mercury organyles, ester groups, acid halide, acid thioester, acid anhydride, isocyanates, isothiocyanates, sulfonic acid halides, imidoesters, diazoacetates, diazonium salts, 1,2-diketones, phosphonic acids, phosphoric acid esters, sulfonic acids, azolides, imidazoles, indoles, N-maleimides, alpha-beta-unsaturated carbonyl compounds, arylhalogenides or their derivatives.

[0294] Non-limiting examples for other linking molecules with higher molecular weights are nucleic acid molecules, polymers, copolymers, polymerizable coupling agents, silica, proteins, and chain-like molecules having a surface with the opposed polarity with respect to the substrate or nanosphere. Nucleic acids can provide a link to affinity molecules containing themselves nucleic acid molecules, though with a complementary sequence with respect to the linking molecule.

[0295] As examples for polymerizable coupling agents, diacetylene, styrene butadiene, vinylacetate, acrylate, acrylamide, vinyl compounds, styrene, silicone oxide, boron oxide, phosphorous oxide, borates, pyrrole, polypyrrole and phosphates can be cited.

[0296] The molecule can be chemically modified, for instance by the binding of a phosphonic acid derivative having a functional reactive group. One example of these phosphonic acid or phosphonic acid ester derivates is imino-bis(methylenphosphono) carbonic acid which can be synthesized according to the "Mannich-Moedritzer" reaction. The phosphonic acid (ester) derivative, or linking molecules bound thereto, may display the same functional groups as given above.

[0297] For non-covalent linking techniques, chain-type molecules having a polarity or charge opposite to that of the substrate or nanosphere surface are particularly suitable. Examples for linking molecules which can be non-covalently linked to core / shell nanospheres involve anionic, cationic or zwitter-ionic surfactants, acid or basic proteins, polyamines, polyamides, polysulfone or polycarboxylic acid. The hydrophobic interaction between a molecule and amphiphilic reagent having a functional reactive group can generate the necessary link. In particular, chain-type molecules with amphiphilic character, such as299713335.1 - 48 -phospholipids or derivatised polysaccharides, which can be crosslinked with each other, are useful.

[0298] Protocols for coupling reactions of functional groups to biological molecules can be found in the literature, for instance in "Bioconjugate Techniques" (Greg T. Hermanson, Academic Press 1996). The biological molecule (e.g., MHC molecule or derivative thereof) can be coupled to the linking molecule, covalently or non-covalently, in line with standard procedures of organic chemistry such as oxidation, halogenation, alkylation, acylation, addition, substitution or amidation. These methods for coupling the covalently or non- covalently bound linking molecule can be applied prior to the coupling of the linking molecule to the substrate or nanosphere or thereafter. Further, it is possible, by means of incubation, to effect a direct binding of molecules to correspondingly pre-treated substrate or nanospheres (for instance by trimethyl silyl bromide), which display a modified surface due to this pretreatment (for instance a higher charge or polar surface).C. Oligos1. Nucleic Acids

[0299] The current disclosure includes embodiments of nucleic acids comprising one or more of a heterologous receptor gene and an inducible reporter. The terms “oligo,” "oligonucleotide,” “polynucleotide,” and “nucleic acid are used interchangeably and include linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, a-anomeric forms thereof, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to- monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Usually monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as "ATGCCTG," it will be understood that the nucleotides are in 5'— >3' order from left to right and that "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, unless otherwise noted. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoranilidate, phosphoramidate, and the like. It is clear to those skilled in the art when oligonucleotides having natural or non-natural nucleotides may be299713335.1 - 49 -employed, e.g., where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.

[0300] The nucleic acid may be an “unmodified oligonucleotide” or “unmodified nucleic acid,” which refers generally to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In some embodiments a nucleic acid molecule is an unmodified oligonucleotide. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside linkages. The term “oligonucleotide analog” refers to oligonucleotides that have one or more non-naturally occurring portions which function in a similar manner to oligonucleotides. Such non-naturally occurring oligonucleotides are often selected over naturally occurring forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases. The term “oligonucleotide” can be used to refer to unmodified oligonucleotides or oligonucleotide analogs.

[0301] Specific examples of nucleic acid molecules include nucleic acid molecules containing modified, i.e., non-naturally occurring intemucleoside linkages. Such non-naturally internucleoside linkages are often selected over naturally occurring forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases. In a specific embodiment, the modification comprises a methyl group.

[0302] Nucleic acid molecules can have one or more modified intemucleoside linkages. As defined in this specification, oligonucleotides having modified intemucleoside linkages include intemucleoside linkages that retain a phosphorus atom and intemucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.

[0303] Modifications to nucleic acid molecules can include modifications wherein one or both terminal nucleotides is modified.

[0304] One suitable phosphorus-containing modified intemucleoside linkage is the phosphorothioate intemucleoside linkage. A number of other modified oligonucleotide backbones (intemucleoside linkages) are known in the art and may be useful in the context of this embodiment.299713335.1 - 50 -

[0305] Representative U.S. patents that teach the preparation of phosphorus-containing internucleoside linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243, 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;5,527,899; 5,721,218; 5,672,697 5,625,050, 5,489,677, and 5,602,240 each of which is herein incorporated by reference.

[0306] Modified oligonucleoside backbones (intemucleoside linkages) that do not include a phosphorus atom therein have internucleoside linkages that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having amide backbones; and others, including those having mixed N, O, S and CH2 component parts.

[0307] Representative U.S. patents that teach the preparation of the above non- phosphorous-containing oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of which is herein incorporated by reference.

[0308] Nucleic acid molecules can also contain one or more modified or substituted sugar moieties. The base moieties are maintained for hybridization with an appropriate nucleic acid target compound. Sugar modifications can impart nuclease stability, binding affinity or some other beneficial biological property to the oligomeric compounds.

[0309] Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2', 3' or 4' positions, sugars having substituents in place of one or more hydrogen atoms of the sugar, and sugars having a linkage between any two other atoms in the sugar. A large number of sugar modifications are known in the art, sugars modified at the 2' position and those which have a bridge between any 2 atoms of the sugar (such that the sugar is bicyclic) are particularly useful in this embodiment. Examples of sugar modifications useful in this embodiment include, but are not limited to compounds comprising a sugar substituent group such as: OH; F; O-, S-, or N-alkyl; or O-alkyl-O-alkyl,299713335.1 - 51 -wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. Particularly suitable are: 2-methoxyethoxy (also known as 2'- O-methoxyethyl, 2'-M0E, or 2'-OCH2CH2OCH3), 2'-O-methyl (2'-O-CH3), 2'-fluoro (2'-F), or bicyclic sugar modified nucleosides having a bridging group connecting the 4' carbon atom to the 2' carbon atom wherein example bridge groups include — CH2— O— , — (CH2)2— O— or — CH2-N(R3)-O wherein R3 is H or C1-C12 alkyl.

[0310] One modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2'-M0E side chain (Baker etal., J. Biol. Chem., 1997, 272, 11944- 12000). One of the immediate advantages of the 2'-M0E substitution is the improvement in binding affinity, which is greater than many similar 2' modifications such as O-methyl, O- propyl, and O-aminopropyl. Oligonucleotides having the 2'-M0E substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

[0311] 2'-Sugar substituent groups may be in the arabino (up) position or ribo (down) position. One 2'-arabino modification is 2'-F. Similar modifications can also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of which is herein incorporated by reference in its entirety.

[0312] Nucleic acid molecules can also contain one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions which are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to the oligomeric compounds. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also299713335.1 - 52 -referred to herein as heterocyclic base moieties include other synthetic and natural nucleobases, many examples of which such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, 7- deazaguanine and 7-deazaadenine among others.

[0313] Additional modifications to nucleic acid molecules are disclosed in U.S. Patent Publication 2009 / 0221685, which is hereby incorporated by reference. Also disclosed herein are additional suitable conjugates to the nucleic acid molecules.2. Barcodes

[0314] In some embodiments, the oligos used in the methods described herein (either first, second, bridging, detection / retrieval etc. . .) may comprise a barcode. The barcode region can be specific to a particular protein or to a particular family member of a family of proteins / enzymes.

[0315] The barcoded region may be used to specifically identify one protein of interest. The barcode region can be a polynucleotide of at least, at most, or exactly 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200 or more (or any range derivable therein) nucleotides in length. The barcode may comprise or further comprise one or more universal PCR regions, adaptors, linkers, or a combination thereof.

[0316] The barcode region or at least a portion thereof is a polynucleotide sequence that can be used to identify the specific activated protein of interest. In embodiments relating to a population of cells, determining the identity of the barcode is done by determining the nucleotide sequence of the barcode. As discussed herein, methods may involve sequencing one or more barcode regions or nucleic acid regions or having such regions sequenced.

[0317] The unique portions of the barcodes may be continuous along the length of the barcode sequence or the barcode may include stretches of nucleic acid sequence that are not unique to any one barcode. The barcodes and / or index regions are quantified or determined by methods known in the art, including quantitative sequencing (e.g., using an Illumina® sequencer) or quantitative hybridization techniques (e.g., microarray hybridization technology or using a Luminex® bead system). Sequencing methods are further described herein.D. Molecular Labels

[0318] The oligonucleotides, nucleic acid molecules, primers, probes, antibodies, and retrieval tag / binding component molecules in the compositions and methods described herein299713335.1 - 53 -may include one or more labels. Nucleic acid molecules can be labeled by incorporating moieties detectable by one or more means including, but not limited to, spectroscopic, photochemical, biochemical, immunochemical, or chemical assays. The method of linking or conjugating the label to the nucleotide or oligonucleotide depends on the type of label(s) used and the position of the label on the nucleotide or oligonucleotide.

[0319] As used herein, “labels” are chemical or biochemical moieties useful for labeling a nucleic acid. “Labels” include, for example, fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionucleotides, enzymes, substrates, cofactors, inhibitors, nanoparticles, magnetic particles, and other moieties known in the art. Labels are capable of generating a measurable signal and may be covalently or noncovalently joined to an oligonucleotide or nucleotide.

[0320] In some embodiments, the molecules may be labeled with a “fluorescent dye” or a “fluorophore.” As used herein, a “fluorescent dye” or a “fluorophore” is a chemical group that can be excited by light to emit fluorescence. Some fluorophores may be excited by light to emit phosphorescence. Dyes may include acceptor dyes that are capable of quenching a fluorescent signal from a fluorescent donor dye. Dyes that may be used in the disclosed methods include, but are not limited to, the following dyes sold under the following trade names: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-JOE; 7- Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2- methoxyacridine); Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC; AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET; Bimane; Bisbenzamide; Bisbenzimide299713335.1 - 54 -(Hoechst); Blancophor FFG; Blancophor SV; BOBO™-1; B0B0™-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy FL; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; Calcium Orange; Calcofluor White; Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP — Cyan Fluorescent Protein; CFP / YFP FRET; Chlorophyll; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.18; Cy3.5™; Cy3™; Cy5.18; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Diehl orodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di- 16-ASP); Diehl orodihydrofluorescein Diacetate (DCFH); DiD — Lipophilic Tracer; DiD (DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DiIC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY- 635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC ; Ethidium Bromide; Ethidium homodimer- 1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); FluorRuby; FluorX; FM 1-43™; FM 4-46; Fura Red™; Fura RedTm / Fluo-3; Fura-2; Fura- 2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxy coumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO-JO-1; JO- PRO- 1; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue- White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;299713335.1 - 55 -Ly soSensor Green; Ly soSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag- Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; NED™; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline; Procion Yellow; Propidium lodid (PI); PYMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R- phy cocyanine; R-phycoerythrin (PE); RsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™; sgBFP™ (super glow BFP); sgGFP™; sgGFP™ (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; Spectrum Aqua; Spectrum Green; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3- sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; TET™; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite;299713335.1 - 56 -Uranine B; Uvitex SFC; VIC®; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO-3; YOYO-1; YOYO-3; and salts thereof.

[0321] Fluorescent dyes or fluorophores may include derivatives that have been modified to facilitate conjugation to another reactive molecule. As such, fluorescent dyes or fluorophores may include amine-reactive derivatives such as isothiocyanate derivatives and / or succinimidyl ester derivatives of the fluorophore.

[0322] The nucleic acid molecules of the disclosed compositions and methods may be labeled with a quencher. Quenching may include dynamic quenching (e.g., by FRET), static quenching, or both. Illustrative quenchers may include Dabcyl. Illustrative quenchers may also include dark quenchers, which may include black hole quenchers sold under the tradename “BHQ” (e.g., BHQ-0, BHQ-1, BHQ-2, and BHQ-3, Biosearch Technologies, Novato, Calif). Dark quenchers also may include quenchers sold under the tradename “QXL™” (Anaspec, San Jose, Calif.). Dark quenchers also may include DNP-type non-fluorophores that include a 2,4- dinitrophenyl group.

[0323] The labels can be conjugated to molecules directly or indirectly by a variety of techniques. Depending upon the precise type of label used, the label can be located at the 5' or 3' end of the oligonucleotide or N or C-terminus of a peptide / polypeptide or located internally in the oligonucleotide's nucleotide sequence. Using commercially available phosphoramidite reagents, one can produce nucleic acid molecules containing functional groups (e.g., thiols or primary amines) at either terminus, for example by the coupling of a phosphoramidite dye to the 5' hydroxyl of the 5' base by the formation of a phosphate bond, or internally, via an appropriately protected phosphoramidite.

[0324] Molecules may also incorporate functionalizing reagents having one or more sulfhydryl, amino or hydroxyl moi eties into the nucleic acid sequence. For example, a 5' phosphate group can be incorporated as a radioisotope by using polynucleotide kinase and [y32P]ATP to provide a reporter group. Biotin can be added to the 5' end by reacting an aminothymidine residue, introduced during synthesis, with an N-hydroxysuccinimide ester of biotin. Labels at the 3' terminus, for example, can employ polynucleotide terminal transferase to add the desired moiety, such as for example, cordycepin, 35S-dATP, and biotinylated dUTP.

[0325] Oligonucleotide derivatives are also available as labels. For example, etheno-dA and etheno-A are known fluorescent adenine nucleotides which can be incorporated into a reporter.299713335.1 - 57 -Similarly, etheno-dC is another analog that can be used in reporter synthesis. The reporters containing such nucleotide derivatives can be hydrolyzed to release much more strongly fluorescent mononucleotides by the polymerase's 5' to 3' nuclease activity as nucleic acid polymerase extends a primer during PCR.V. Assay MethodsE. Ligation Methods

[0326] Aspects of the disclosure include the ligation of nucleic acids. In some embodiments, the methods include ligation of the first oligonucleotide and the second oligo when the first and second oligonucleotides are in close proximity to each other.

[0327] In some embodiments, the ligation of the oligos utilizes splint ligation. Splint ligation includes the addition of one or more splint or bridging oligos, which may be RNA or DNA, that can base-pair with the oligo of the antibody-oligo construct and the oligo linked to the retrieval tag binding component. It is contemplated that many different ligases can be used in the methods of the disclosure, depending on the end composition of the oligos to be ligated. For example, the antibody -linked oligo and the retrieval tag binding component-linked oligo may have a 3’ hydroxyl and a 5 ’phosphate that are in close proximity together due to the association of the molecular construct and the protein of interest. A ligase can then be added to ligate the free 3 ’OH and 5 ’phosphate to form a single strand that can then be primed for amplification and / or probed with a fluorescent probe.

[0328] It is contemplated that any suitable ligase may be used and easily selected by one skilled in the art. Exemplary ligases include E. coli DNA ligase, T4 DNA ligase, mammalian ligases, and thermostable ligases. Embodiments of the disclosure may also include incubation of one or more assay components with a phosphatase. For example, embodiments include incubation of one or more of the molecular construct, the antibody-oligo construct, the retrieval tag binding component-oligo construct, a primer, or probe with a phosphatase. The incubation may be a pre-incubation, meaning that it takes place prior to contact with the composition.

[0329] In some embodiments, the methods further comprise the addition of a uracil-specific excision reagent (USER) enzyme that generates a single nucleotide gap at the location of a uracil and can be utilized for breading down the splint / bridging oligos.

[0330] After ligation of the oligos, the double-stranded nucleic acid can then undergo a preamplification step, followed by a further amplification technique. In some embodiments, the one or more bridging oligos may form a circle that can then be used in rolling circle299713335.1 - 58 -amplification (see, for example, FIG. IB). In some embodiments, the pre-amplification step and further amplification technique comprises polymerase chain reaction. Accordingly, methods of the disclosure may include one or more of the following steps: contacting the composition with a polymerase (e.g., DNA or RNA polymerase), contacting the composition with one or more primers, contacting the composition with a buffer solution, contacting the composition with bivalent cations such as magnesium or manganese, and / or contacting the composition with deoxynucleoside triphosphates.F. Sequencing

[0331] Aspects of the disclosure may include sequencing nucleic acids to determine the expression level of total protein or to determine / quantify the amount of an amplified region of an oligo of the disclosure, such as a first or second (or further oligo) or barcode region thereof. Described below are exemplary methods for performing such sequencing reactions.Massively parallel signature sequencing (MPSS).

[0332] The first of the next-generation sequencing technologies, massively parallel signature sequencing (or MPSS), was developed in the 1990s at Lynx Therapeutics. MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides. This method made it susceptible to sequence-specific bias or loss of specific sequences. Because the technology was so complex, MPSS was only performed 'in-house' by Lynx Therapeutics and no DNA sequencing machines were sold to independent laboratories. Lynx Therapeutics merged with Solexa (later acquired by Illumina) in 2004, leading to the development of sequencing-by- synthesis, a simpler approach acquired from Manteia Predictive Medicine, which rendered MPSS obsolete. However, the essential properties of the MPSS output were typical of later "next-generation" data types, including hundreds of thousands of short DNA sequences. In the case of MPSS, these were typically used for sequencing cDNA for measurements of gene expression levels. Indeed, the powerful Illumina HiSeq2000, HiSeq2500 and MiSeq systems are based on MPSS.2. Polony sequencing.

[0333] The Polony sequencing method, developed in the laboratory of George M. Church at Harvard, was among the first next-generation sequencing systems and was used to sequence a full genome in 2005. It combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an A. coli genome299713335.1 - 59 -at an accuracy of >99.9999% and a cost approximately 1 / 9 that of Sanger sequencing. The technology was licensed to Agencourt Biosciences, subsequently spun out into Agencourt Personal Genomics, and eventually incorporated into the Applied Biosystems SOLiD platform, which is now owned by Life Technologies.3. 454 pyrosequencing.

[0334] A parallelized version of pyrosequencing was developed by 454 Life Sciences, which has since been acquired by Roche Diagnostics. The method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picoliter-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other.4. Illumina (Solexa) sequencing.

[0335] Solexa, now part of Illumina, developed a sequencing method based on reversible dye-terminators technology, and engineered polymerases, that it developed internally. The terminated chemistry was developed internally at Solexa and the concept of the Solexa system was invented by Balasubramanian and Klennerman from Cambridge University's chemistry department. In 2004, Solexa acquired the company Manteia Predictive Medicine in order to gain a massivelly parallel sequencing technology based on "DNA Clusters", which involves the clonal amplification of DNA on a surface. The cluster technology was co-acquired with Lynx Therapeutics of California. Solexa Ltd. later merged with Lynx to form Solexa Inc.

[0336] In this method, DNA molecules and primers are first attached on a slide and amplified with polymerase so that local clonal DNA colonies, later coined "DNA clusters", are formed. To determine the sequence, four types of reversible terminator bases (RT -bases) are added and non-incorporated nucleotides are washed away. A camera takes images of the fluorescently labeled nucleotides, then the dye, along with the terminal 3' blocker, is chemically removed from the DNA, allowing for the next cycle to begin. Unlike pyrosequencing, the DNA chains are extended one nucleotide at a time and image acquisition can be performed at a delayed moment, allowing for very large arrays of DNA colonies to be captured by sequential images taken from a single camera.299713335.1 - 60 -

[0337] Decoupling the enzymatic reaction and the image capture allows for optimal throughput and theoretically unlimited sequencing capacity. With an optimal configuration, the ultimately reachable instrument throughput is thus dictated solely by the analog-to-digital conversion rate of the camera, multiplied by the number of cameras and divided by the number of pixels per DNA colony required for visualizing them optimally (approximately 10 pixel s / colony). In 2012, with cameras operating at more than 10 MHz A / D conversion rates and available optics, fluidics and enzymatics, throughput can be multiples of 1 million nucleotides / second, corresponding roughly to one human genome equivalent at lx coverage per hour per instrument, and one human genome re-sequenced (at approx. 3 Ox) per day per instrument (equipped with a single camera).5. SOLiD sequencing.

[0338] Applied Biosystems' (now a Thermo Fisher Scientific brand) SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing single copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing. This sequencing by ligation method has been reported to have some issue sequencing palindromic sequences.6. Ion Torrent semiconductor sequencing.

[0339] Ion Torrent Systems Inc. (now owned by Thermo Fisher Scientific) developed a system based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerization of DNA, as opposed to the optical methods used in other sequencing systems. A microwell containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.299713335.1 - 61 -7. DNA nanoball sequencing.

[0340] DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. The company Complete Genomics uses this technology to sequence samples submitted by independent researchers. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run and at low reagent costs compared to other next generation sequencing platforms. However, only short sequences of DNA are determined from each DNA nanoball which makes mapping the short reads to a reference genome difficult. This technology has been used for multiple genome sequencing projects.8. Heliscope single molecule sequencing.

[0341] Heliscope sequencing is a method of single-molecule sequencing developed by Helicos Biosciences. It uses DNA fragments with added poly-A tail adapters which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Heliscope sequencer. The reads are short, up to 55 bases per run, but recent improvements allow for more accurate reads of stretches of one type of nucleotides. This sequencing method and equipment were used to sequence the genome of the Ml 3 bacteriophage.9. Single molecule real time (SMRT) sequencing.

[0342] SMRT sequencing is based on the sequencing by synthesis approach. The DNA is synthesized in zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. According to Pacific Biosciences, the SMRT technology developer, this methodology allows detection of nucleotide modifications (such as cytosine methylation). This happens through the observation of polymerase kinetics. This approach allows reads of 20,000 nucleotides or more, with average read lengths of 5 kilobases.]299713335.1 - 62 -G. Protein Assays

[0343] In some embodiments, the gene or protein expression of a protein of interest is measured. Methods for measuring transcription and / or translation of a particular gene sequence or biomarker are well known in the art. See, for example, Ausubel, Current Protocols in Molecular Biology, 1987-2006, John Wiley & Sons; and Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, 2000.

[0344] Polypeptides from the protein of interest encoded by a gene can be detected and / or quantified by any methods known to those of skill in the art from samples as described herein. In some embodiments, antibodies can also be used to detect polypeptides / proteins of interest. Antibodies to a protein of interest can be produced using well known techniques (see, e.g., Harlow & Lane, 1988 and Harlow & Lane, 1999; Coligan, 1991; Goding, 1986; and Kohler & Milstein, 1975). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing rabbits or mice (see, e.g., Huse et al., 1989; Ward et al., 1989).

[0345] Once specific antibodies are available, the expression of a protein of interest can be detected by a variety of immunoassay methods. For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (1991). Moreover, the immunoassays of certain aspects can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (1980); and Harlow & Lane, supra).

[0346] Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen. The labeling agent may itself be one of the moieties comprising the antibody / antigen complex. Thus, the labeling agent may be a labeled polypeptide or a labeled antibody that binds the protein of interest. Alternatively, the labeling agent may be a third moiety, such as a secondary antibody, that specifically binds to the antibody / antigen complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the labeling agent. These proteins exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, e.g., Kronval et al., 1973; Akerstrom et al., 1985). The labeling agent can be modified with a detectable moiety, such as299713335.1 - 63 -biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art.

[0347] Commonly used assays include noncompetitive assays, e.g., sandwich assays, and competitive assays. In competitive assays, the amount of polypeptide present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) polypeptide of interest displaced (competed away) from an antibody that binds by the unknown polypeptide present in a sample. Commonly used assay formats include immunoblots, which are used to detect and quantify the presence of protein in a sample. Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., 1986).

[0348] Any suitable method can be used to detect one or more of the markers described herein. Successful practice can be achieved with one or a combination of methods that can detect and, preferably, quantify the markers. These methods include, without limitation, hybridization-based methods, including those employed in biochip arrays, mass spectrometry (e.g., laser desorption / ionization mass spectrometry), fluorescence (e.g., sandwich immunoassay), surface plasmon resonance, ellipsometry and atomic force microscopy. Expression levels of markers (e.g., polynucleotides or polypeptides) are compared by procedures well known in the art, such as RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and / or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, or colorimetric assays. Methods may further include, one or more of electrospray ionization mass spectrometry (ESLMS), ESLMS / MS, ESI- MS / (MS)n, matrix- assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDLTOF- MS), desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS)11, atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.299713335.1 - 64 -

[0349] Detection methods may include the use of a biochip array. Biochip arrays include protein and polynucleotide arrays. The protein of interest may be captured on the biochip array and subjected to analysis to detect the level of the protein in a sample.H. Nucleic Acid Assays

[0350] Aspects of the methods include assaying nucleic acids to determine expression levels. Arrays can be used to detect differences between two samples. An array comprises a solid support with nucleic acid probes attached to the support. Arrays typically comprise a plurality of different nucleic acid probes that are coupled to a surface of a substrate in different, known locations. These arrays, also described as "microarrays" or colloquially "chips" have been generally described in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 and Fodor et al., 1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is used in certain aspects, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are hereby incorporated in their entirety for all purposes.

[0351] In addition to the use of arrays and microarrays, it is contemplated that a number of difference assays could be employed to analyze expressed nucleic acids. Such assays include, but are not limited to, nucleic amplification, polymerase chain reaction, quantitative PCR, RT- PCR, in situ hybridization, digital PCR, dd PCR (digital droplet PCR), nCounter (nanoString), BEAMing (Beads, Emulsions, Amplifications, and Magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (Tagged-Amplicon deep sequencing), PAP (Pyrophosphorolysis-activation polymerization), next generation RNA sequencing, northern hybridization, hybridization protection assay (HPA)(GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and / or Bridge Litigation Assay (Genaco).

[0352] Embodiments of the disclosure relate to determining the expression of a protein of interest or of a nucleic acid comprising a barcode or protein of interest-specific region. The299713335.1 - 65 -expression or abundance level can be determined by measuring the levels of RNA transcripts of a protein of interest or the abundance of amplified nucleic acids comprising a barcode. Suitable methods for this purpose include, but are not limited to, RT-PCR, Northern Blot, in situ hybridization, Southern Blot, slot-blotting, nuclease protection assay and oligonucleotide arrays.

[0353] In certain aspects, RNA isolated from cells can be amplified to cDNA or cRNA before detection and / or quantitation. The isolated RNA can be either total RNA or mRNA. The RNA amplification can be specific or non-specific. In some embodiments, the amplification is specific in that it specifically amplifies a gene for a protein of interest. In some embodiments, the amplification and / or reverse transcriptase step excludes or includes random priming. Suitable amplification methods include, but are not limited to, reverse transcriptase PCR, isothermal amplification, ligase chain reaction, and Qbeta replicase. The amplified nucleic acid products can be detected and / or quantitated through hybridization to labeled probes. In some embodiments, detection may involve fluorescence resonance energy transfer (FRET) or some other kind of quantum dots.

[0354] Amplification primers or hybridization probes can be prepared to be complementary to a barcode region or oligo described herein. The term "primer" or “probe” as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process and / or pairing with a single strand of an oligo of the disclosure, or portion thereof. Typically, primers are oligonucleotides from ten to twenty and / or thirty nucleic acids in length, but longer sequences can be employed. Primers may be provided in double-stranded and / or single-stranded form, although the single-stranded form is preferred.

[0355] The use of a probe or primer of between 13 and 100 nucleotides, particularly between 17 and 100 nucleotides in length, or in some aspects up to 1-2 kilobases or more in length, allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over contiguous stretches greater than 20 bases in length may be used to increase stability and / or selectivity of the hybrid molecules obtained. One may design nucleic acid molecules for hybridization having one or more complementary sequences of 20 to 30 nucleotides, or even longer where desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragment by chemical means or by introducing selected sequences into recombinant vectors for recombinant production.299713335.1 - 66 -

[0356] In one embodiment, each probe / primer comprises at least 15 nucleotides. For instance, each probe can comprise at least or at most 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein). They may have these lengths and have a sequence that is identical or complementary to a gene described herein. Particularly, each probe / primer has relatively high sequence complexity and does not have any ambiguous residue (undetermined "n" residues). The probes / primers can hybridize to the target gene, including its RNA transcripts, under stringent or highly stringent conditions. In some embodiments, because each of the biomarkers has more than one human sequence, it is contemplated that probes and primers may be designed for use with each of these sequences. For example, inosine is a nucleotide frequently used in probes or primers to hybridize to more than one sequence. It is contemplated that probes or primers may have inosine or other design implementations that accommodate recognition of more than one human sequence for a particular biomarker.

[0357] For applications requiring high selectivity, one will typically desire to employ relatively high stringency conditions to form the hybrids. For example, relatively low salt and / or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50°C to about 70°C. Such high stringency conditions tolerate little, if any, mismatch between the probe or primers and the template or target strand and would be particularly suitable for isolating specific genes or for detecting specific mRNA transcripts. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0358] In one embodiment, quantitative RT-PCR (such as TaqMan, ABI) is used for detecting and comparing the levels or abundance of nucleic acids in samples. Quantitative RT- PCR involves reverse transcription (RT) of RNA to cDNA followed by relative quantitative PCR (RT-PCR). The concentration of the target DNA in the linear portion of the PCR process is proportional to the starting concentration of the target before the PCR was begun. By determining the concentration of the PCR products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived may be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundances is true in299713335.1 - 67 -the linear range portion of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the sampling and quantifying of the amplified PCR products may be carried out when the PCR reactions are in the linear portion of their curves. In addition, relative concentrations of the amplifiable cDNAs may be normalized to some independent standard, which may be based on either internally existing RNA species or externally introduced RNA species. The abundance of a particular mRNA species may also be determined relative to the average abundance of all mRNA species in the sample.

[0359] In one embodiment, the PCR amplification utilizes one or more internal PCR standards. The internal standard may be an abundant housekeeping gene in the cell or it can specifically be GAPDH, GUSB and P-2 microglobulin. These standards may be used to normalize expression levels so that the expression levels of different gene products can be compared directly. A person of ordinary skill in the art would know how to use an internal standard to normalize expression levels.

[0360] A problem inherent in some samples is that they are of variable quantity and / or quality. This problem can be overcome if the RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is similar or larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target. This assay measures relative abundance, not absolute abundance of the respective mRNA species.

[0361] In another embodiment, the relative quantitative RT-PCR uses an external standard protocol. Under this protocol, the PCR products are sampled in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling can be empirically determined for each target cDNA fragment. In addition, the reverse transcriptase products of each RNA population isolated from the various samples can be normalized for equal concentrations of amplifiable cDNAs.

[0362] A nucleic acid array can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more different polynucleotide probes, which may hybridize to different and / or the same biomarkers. Multiple probes for the same gene can be used on a single nucleic acid array. Probes for other disease genes can also be299713335.1 - 68 -included in the nucleic acid array. The probe density on the array can be in any range. In some embodiments, the density may be 50, 100, 200, 300, 400, 500 or more probes / cm2.

[0363] Specifically contemplated are chip-based nucleic acid technologies such as those described by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization (see also, Pease et al., 1994; and Fodor et al, 1991). It is contemplated that this technology may be used in conjunction with evaluating the expression level of one or more cancer biomarkers with respect to diagnostic, prognostic, and treatment methods.

[0364] Certain embodiments may involve the use of arrays or data generated from an array. Data may be readily available. Moreover, an array may be prepared in order to generate data that may then be used in correlation studies.

[0365] Representative methods and apparatus for preparing a microarray have been described, for example, in U.S. Patent Nos. 5,143,854; 5,202,231; 5,242,974; 5,288,644; 5,324,633; 5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,432,049; 5,436,327;5,445,934; 5,468,613; 5,470,710; 5,472,672; 5,492,806; 5,525,464; 5,503,980; 5,510,270;5,525,464; 5,527,681; 5,529,756; 5,532,128; 5,545,531; 5,547,839; 5,554,501; 5,556,752;5,561,071; 5,571,639; 5,580,726; 5,580,732; 5,593,839; 5,599,695; 5,599,672; 5,610;287;5,624,711; 5,631,134; 5,639,603; 5,654,413; 5,658,734; 5,661,028; 5,665,547; 5,667,972;5,695,940; 5,700,637; 5,744,305; 5,800,992; 5,807,522; 5,830,645; 5,837,196; 5,871,928;5,847,219; 5,876,932; 5,919,626; 6,004,755; 6,087,102; 6,368,799; 6,383,749; 6,617,112;6,638,717; 6,720,138, as well as WO 93 / 17126; WO 95 / 11995; WO 95 / 21265; WO 95 / 21944; WO 95 / 35505; WO 96 / 31622; WO 97 / 10365; WO 97 / 27317; WO 99 / 35505; WO 09923256; WO 09936760; W00138580; WO 0168255; WO 03020898; WO 03040410; WO 03053586; WO 03087297; WO 03091426; WO03100012; WO 04020085; WO 04027093; EP 373 203; EP 785 280; EP 799 897 and UK 8 803 000; the disclosures of which are all herein incorporated by reference.VI. Exemplary UtilitiesI. Diagnostic and Therapeutic methods

[0366] Method aspects of the disclosure may be used in diagnostics to evaluate a protein of interest in a patient, for example a protein of interest in a specified functional form. For299713335.1 - 69 -example, methods of the disclosure provide for a method of evaluating a protein of interest in a patient in a specified functional form, said method comprising: (i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; (ii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises a epitope tag binding component and a first oligonucleotide; (iii) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (iv) incubating the composition under conditions sufficient for being ligated and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and (v) detecting ligated or annealed first and second oligonucleotide; or the method comprising: (i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; (ii) contacting the composition with an epitope tag binding component capable of binding the epitope tag; (iii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide; (iv) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide; (v) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide to the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and (vi) detecting ligated and / or annealed first and second oligonucleotides.

[0367] In certain aspects, methods involve obtaining a sample from a subject. The methods of obtaining provided herein may include methods of biopsy such as fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In certain embodiments the sample is obtained from a biopsy from ovarian or prostate tissue by any of the biopsy methods previously mentioned. In other embodiments the sample may be obtained from any of the tissues provided herein that include but are not limited to gall bladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source including but not limited to299713335.1 - 70 -blood, sweat, hair follicle, buccal tissue, tears, menses, feces, or saliva. In certain aspects the sample is obtained from cystic fluid or fluid derived from a tumor or neoplasm. In yet other embodiments the cyst, tumor or neoplasm is ovarian or prostate. In certain aspects of the current methods, any medical professional such as a doctor, nurse or medical technician may obtain a biological sample for testing. Yet further, the biological sample can be obtained without the assistance of a medical professional.

[0368] A sample may include but is not limited to, tissue, cells, or biological material from cells or derived from cells of a subject. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods including but not limited to: scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen.

[0369] The sample may be obtained by methods known in the art. In certain embodiments the samples are obtained by biopsy. In other embodiments the sample is obtained by swabbing, scraping, phlebotomy, or any other methods known in the art. In some cases, the sample may be obtained, stored, or transported using components of a kit of the present methods. In some cases, multiple samples, such as multiple colorectal samples may be obtained for diagnosis by the methods described herein. In other cases, multiple samples, such as one or more samples from one tissue type (for example colon) and one or more samples from another tissue (for example buccal) may be obtained for diagnosis by the methods. In some cases, multiple samples such as one or more samples from one tissue type (e.g., rectal) and one or more samples from another tissue (e.g., cecum) may be obtained at the same or different times. Samples may be obtained at different times are stored and / or analyzed by different methods. For example, a sample may be obtained and analyzed by routine staining methods or any other cytological analysis methods.

[0370] In some embodiments the biological sample may be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects a molecular profiling business may consult on which assays or tests are most appropriately indicated. In further aspects of the current methods, the patient or subject may obtain a biological sample for testing299713335.1 - 71 -without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0371] In other cases, the sample is obtained by an invasive procedure including but not limited to: biopsy, needle aspiration, or phlebotomy. The method of needle aspiration may further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. In some embodiments, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0372] General methods for obtaining biological samples are also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, which is herein incorporated by reference in its entirety, describes general methods for biopsy and cytological methods. In one embodiment, the sample is a fine needle aspirate of a colorectal or a suspected colorectal tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of an ultrasound, X-ray, or other imaging device.

[0373] In some embodiments of the present methods, the molecular profiling business may obtain the biological sample from a subject directly, from a medical professional, from a third party, or from a kit provided by a molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, a medical professional, or a third party acquires and sends the biological sample to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers, and excipients for storage and transport of the biological sample to the molecular profiling business.

[0374] In some embodiments of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. An individual may alternatively obtain a sample through the use of an over the counter (OTC) kit. An OTC kit may contain a means for obtaining said sample as described herein, a means for storing said sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately. A sample suitable for use by the molecular profiling business may be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and / or adequacy are provided.299713335.1 - 72 -

[0375] In some embodiments, the subject may be referred to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may likewise obtain a biological sample for testing or refer the individual to a testing center or laboratory for submission of the biological sample. In some cases the medical professional may refer the subject to a testing center or laboratory for submission of the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample.

[0376] The cancers referred to in the methods of the disclosure include, but are not limited to, tumors of all types, locations, sizes, and characteristics. Exemplary cancer types include, pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS- related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, childhood cerebellar or cerebral basal cell carcinoma, bile duct cancer, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal tumors brain tumor, visual pathway and hypothalamic glioma, breast cancer, specific breast cancers such as ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma of the breast, medullary carcinoma of the breast, mucinous carcinoma of the breast, papillary carcinoma of the breast, cribriform carcinoma of the breast, invasive lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, male breast cancer, paget’s disease of the nipple, phyllodes tumors of the breast, recurrent and / or metastatic breast, cancer, luminal A or B breast cancer, triple-negative / basal-like breast cancer, and HER2-enriched breast cancer, lymphoid cancer, bronchial adenomas / carcinoids, tracheal cancer, Burkitt lymphoma, carcinoid tumor, childhood carcinoid tumor, gastrointestinal carcinoma of unknown primary, central nervous system lymphoma, primary cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, childhood cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's, childhood extragonadal Germ cell tumor, extrahepatic bile duct cancer, eye cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor: extracranial, extragonadal, or ovarian, gestational trophoblastic tumor, glioma of the brain stem, glioma, childhood cerebral astrocytoma, childhood visual pathway and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck299713335.1 - 73 -cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, childhood intraocular melanoma, islet cell carcinoma (endocrine pancreas), kaposi sarcoma, kidney cancer (renal cell cancer), laryngeal cancer , leukemia, acute lymphoblastic (also called acute lymphocytic leukemia) leukemia, acute myeloid (also called acute myelogenous leukemia) leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia) leukemia, chronic myelogenous (also called chronic myeloid leukemia) leukemia, hairy cell lip and oral cavity cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphomas, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, NonHodgkin (an old classification of all lymphomas except Hodgkin's) lymphoma, primary central nervous system lymphoma, Waldenstrom macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, childhood medulloblastoma, intraocular (eye) melanoma, merkel cell carcinoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative diseases, chronic myelogenous leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant, fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial- stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood pituitary adenoma, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, childhood Salivary gland cancer Sarcoma, Ewing family of tumors, Kaposi sarcoma, soft tissue sarcoma, uterine sezary syndrome sarcoma, skin cancer (nonmelanoma), skin cancer (melanoma), skin carcinoma, Merkel cell small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer with occult primary, metastatic stomach cancer, supratentorial primitive neuroectodermal tumor, childhood T-cell lymphoma, testicular cancer, throat cancer, thymoma, childhood thymoma, thymic carcinoma, thyroid cancer,299713335.1 - 74 -urethral cancer, uterine cancer, endometrial uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, childhood vulvar cancer, and wilms tumor (kidney cancer).J. Drug Discovery and Spatial Detection of Specified Functional Forms of Proteins

[0377] The current methods of the disclosure may also be used to evaluate activity modifiers. This is depicted in Example 1 of the application. The methods of the disclosure allow for the quantification of small molecule target engagement in live cells. The disclosed methods allow for a way to detect and quantify target engagement in live cells. The methods can be used to quantify active enzymes with high spatial resolution.VII. Kits

[0378] Certain aspects of the present disclosure also concern kits containing nucleic acids (e.g., oligos), vectors, molecular constructs, antibodies, ligation, primer extension, and amplification reagents, and / or retrieval tag / retrieval tag binding component pairs of the disclosure. The kits may be used to implement the methods of the disclosure. In some embodiments, kits can be used to evaluate a specified functional form of a protein in a composition, such as a composition of cells or extracts thereof. In some embodiments, the kits can be used to evaluate proteins in live cells, according to methods of the disclosure. In certain embodiments, a kit contains, contains at least or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1,000 or more nucleic acid probes, primers, or synthetic RNA molecules, or any value or range and combination derivable therein. In some embodiments, there are kits for evaluating the activation of or engagement of a receptor by a ligand. In some embodiments, universal probes or primers are included for amplifying, identifying, or sequencing a barcode or receptor. Such reagents may also be used to generate or test host cells that can be used in screens.

[0379] In certain embodiments, the kits may comprise materials for analyzing cell morphology and / or phenotype, such as histology slides and reagents, histological stains, alcohol, buffers, tissue embedding mediums, paraffin, formaldehyde, and tissue dehydrant.

[0380] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means.

[0381] Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would299713335.1 - 75 -be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more.

[0382] Kits for using probes, polypeptide or polynucleotide detecting agents of the disclosure for drug discovery are contemplated.

[0383] In certain aspects, negative and / or positive control agents are included in some kit embodiments. The control molecules can be used to verify specific binding of a molecular construct or antibody to a protein of interest, or an amplification or ligation control, for example.

[0384] Embodiments of the disclosure include kits for analysis of a pathological sample by assessing a nucleic acid or polypeptide profile for a sample comprising, in suitable container means, two or more RNA probes or primers for detecting expressed polynucleotides. Furthermore, the probes or primers may be labeled. Labels are known in the art and also described herein. In some embodiments, the kit can further comprise reagents for labeling probes, nucleic acids, and / or detecting agents. The kit may also include labeling reagents, including at least one of amine-modified nucleotide, poly(A) polymerase, and poly(A) polymerase buffer. Labeling reagents can include an amine-reactive dye. Kits can comprise any one or more of the following materials: enzymes, reaction tubes, buffers, detergent, primers, probes, antibodies. In some embodiments, these kits include the needed apparatus for performing RNA extraction, RT-PCR, and gel electrophoresis. Instructions for performing the assays can also be included in the kits.

[0385] The kits may further comprise instructions for using the kit for assessing expression, means for converting the expression data into expression values and / or means for analyzing the expression values to generate ligand / receptor interaction data.

[0386] Kits may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a composition which includes a probe that is useful for the methods of the disclosure. The kit may comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.299713335.1 - 76 -VIII. ExamplesThe following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1 -Modularity of the system.

[0387] Various POIs with GFP epitope tags were generated for use in the system (FIG. 4). Other POIs and tags are also generated. Example tags are provided in FIG 4. Activity probes are generated based on the POI being tested. Recognition probes are generated based on the other components of the system. The oligonucleotides depicted in FIGs 7-12 are generated. Oligonucleotides are selected (interchangeable) depending on intended use (imaging, multiplexing, sequencing, etc) and / or the sequencing platform.Example 2. Kinase Activity Probes

[0388] The probes X4K, X8K and X12K (having different length linkers) were generated and used in ADPL readout. X8K and X12K yielded productive signal, while X4K did not. Results show that linker length and overall molecular properties of the probes can be important (FIG.13).Example 3. Specific probes and family wide probes

[0389] In an experiment, the POI was SNAP fused with GFP and the activity probe was benzylguanine biotin. GFP shows expression of the SNAP protein. A modular ADPL signal was seen on addition of the activity probe but was lost with the addition of an inhibitor of the POI or one of the recognition elements of the system (FIG 14 left panel).

[0390] Under the same conditions, the POI was switched to PAFAH2 and the activity probe was switched to FP-bio. A modular ADPL signal was seen on addition of the activity probe but was lost with the addition of an inhibitor of the POI or one of the recognition elements of the system right panel.

[0391] Increasing signal was observed on addition of increasing activity probe and decreasing signal on addition of increasing concentration of inhibitor.299713335.1 - 77 -Example 4. Modular ADPL with kinases

[0392] A recombinant protein of CDK2 fused to eftp was made and overexpressed in Hela cells. The cells were treated with the chemical probe and a ADPL signal was detected. A known CDK2 inhibitor was added to the cells and the signal was lost. When any of the recognition elements (DP binding reagent and Epitope binding reagent) are removed, a signal is not generated from the probes. The signal was quantified from the CDK2 overexpressed HeLa cells (FIG. 15 A and B)

[0393] A recombinant protein of LIMK1 fused to eftp was made and overexpressed in Hela cells. The cells were treated with the chemical probe and a ADPL signal was detected. A known LIMK1 inhibitor was added to the cells and the signal was lost. When any of the recognition elements (DP binding reagent and Epitope binding reagent) are removed, a signal is not generated from the probes. The signal was quantified from the LIMK1 overexpressed HeLa cells (FIG. 15 C and D).Example 5. Validation of a POI-gfp fusion cell lines

[0394] POI-gfp cell lines were generated and visualized. Results are shown in FIG. 16. Images of gfp expressing cells are shown in FIG. 16. The experiments confirmed expression of the protein and probe labeling.Example 6. Modular ADPL signal on treating with ibrutinib

[0395] The inventors used a selective inhibitor of Burton’s Tyrosine kinase(BTK) implicated in Chronic Myeloid Lukemia to test if modular ADPL is able to identify selective target engagement of ibrutinib with BTK.

[0396] Modular ADPL signal was generated following the SPARC protocol under noinhibitor conditions. Treatment with the pan-kinase inhibitor XO44 completely ablated the signal, and a similar loss of signal was observed with the selective BTK inhibitor ibrutinib (FIG. 18 A). Quantification confirmed that the decrease in signal was statistically significant in both XO44- and ibrutinib-treated samples (FIG. 18B). Furthermore, a dose-response experiment with ibrutinib yielded an ICso of 8 nM (FIG. 18C), which was within experimental error of the reported literature value. This demonstrates that the ADPL platform can also be applied to estimate compound ICso values.

[0397] Using modular ADPL the inventors were not only able to estimate ICso’s but were also able to understand the kinetics of target engagement suing a time course study (FIG. 19A).299713335.1 - 78 -The inventors also verified inhibition and reliability of the overexpression system using western blot (FIG. 19B). However, the IC50 estimated by the western blot was much higher which indicates that the modular ADPL platform in much more sensitive and reliable in estimating the ICsos of the compounds.* * *

[0398] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0399] Further applicable details may be found in PCT Application No. PCT / US2018 / 062231, filed November 21, 2018, and titled “Chemical Probe-Dependent Evaluation of Protein Activity and Uses Thereof,” which is hereby incorporated by reference in its entirety. Further applicable details may be found in U.S. Provisional Applications entitled “Kinase Inhibitor Compositions and Therapeutic Uses Thereof,” (Attorney Docket No.: ARCD.P0855US.P1) and “Reversible and Irreversible Kinome Probes and Uses Thereof,” (Attorney Docket No.: ARCD.P0854US.P1), both filed on September 18, 2024, and both of which are incorporated by reference in their entireties herein.REFERENCESThe following references and the publications referred to throughout the specification, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.1. Agapakis, C.M., Boyle, P.M. & Silver, P.A. 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Claims

1. CLAIMS1. A system for evaluating a protein of interest comprising:(i) a protein of interest fused to an epitope tag;(ii) an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises an epitope-binding component and a first oligonucleotide;(iii) an activity probe, wherein the activity probe comprises a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; and(iv) a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval -binding tag comprises a second oligonucleotide.

2. A system for evaluating a protein of interest comprising:(i) a protein of interest fused to an epitope tag;(ii) an epitope tag binding component capable of binding the epitope tag;(iii) an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide;(iv) an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest; and(v) a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval -binding tag comprises a second oligonucleotide.

3. The system of any one of claims 1-2, wherein the first and / or second oligonucleotide is single stranded.

4. The system of any one of claims 1-3, wherein the first and / or second oligonucleotides comprise a DNA barcode.299713335.1 - 86 -5. The system of any one of claims 1-4, further comprising one or more bridging oligonucleotides, wherein at least one of the one or more bridging oligonucleotides comprises complementary regions to both the first and second oligonucleotide.

6. The system of any one of claim 1-5, wherein the system further comprises one or more reagents for rolling circle amplification.

7. The system of any one of claims 1-6, further comprising a recognition oligonucleotide conjugated to a label, wherein the recognition oligonucleotide is complementary to the first oligonucleotide, the second oligonucleotide or at least one of the one or more bridging oligonucleotides.

8. The system of claim 7, wherein the label comprises a flourescent protein, a pull down tag or a split recognition system.

9. The system of any one of claims 1-8, further comprising a first labeled primer and / or second labeled primer, wherein the first labeled primer is specifically complementary to the first oligonucleotide and non-complementary to the second oligonucleotide and the second labeled primer is specifically complementary to the second oligonucleotide and non-complementary to the first oligonucleotide.

10. The system of claim 9, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

11. The system of claim 9 or 10, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

12. The system of any one of claims 1-11, wherein first and the second oligonucleotides are capable of being ligated and / or and annealing when in sufficient proximity to each other.

13. The system of any one of claims 1-12, wherein the protein of interest is an enzyme14. The system of any one of claims 1-13, wherein the protein of interest comprises a serine hydrolase, cysteine protease, kinase, metalloprotease, P-retaining glycosidase, tyrosine phosphatase, or cytochrome P450.299713335.1 - 87 -15. The system of any one of claims 1-14, wherein the protein of interest is a DNA repair enzyme.

16. The system of any one of claims 1-15, wherein the protein of interest is a non-enzyme.

17. The system of any one of claims 1-16, the epitope tag comprises a fluorescent protein, a pull down tag a split recognition system, or a protein tag for covalent labeling, optionally, wherein the protein tags for covalent labeling comprise snap tag, or halo tag.

18. The system of claim 17, wherein the fluorescent protein comprises GFP, RFP, or mCherry.

19. The system of claim 17, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

20. The system of claim 17, wherein the split recognition system comprises split gfp or spytag and catcher.

21. The system of any one of claims 1-20, wherein the epitope tag is fused to the protein of interest by combining genetic material.

22. The system of any one of claims 1-21, wherein protein of interest fused to the epitope tag is a recombinant protein.

23. The system of any one of claims 1-22, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

24. The system of any one of claims 1-23, wherein the targeting group comprises a flourophosphonate, diphenylphosphonate, sulfonyl fluoride, acyloxymethyl ketone, phenoxymethylketone, vinyl sulfone, epoxide, halomethylketone, alpha-haloester, alpha-haloamide, a, P-unsaturated ester, a, P-unsaturated ketone, diazomethylketone, acyl phosphate, acylphosphonate, hydroxamate, carbamate, ester, thioester, 2-deoxy-2- fluoro glycoside, a-bromobenzylphosphonate, 2-ethynylnaphthalene, or acrylamide, butyneamide, crotonamide, chloroacetamide, alpha-methylchloroacetamide.

25. The system of any one of claims 1-24, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are one or more of the following retrieval tag and retrieval tag299713335.1 - 88 -binding component pairs: biotin and streptavidin, biotin and avidin, biotin and antibiotin, desthiobiotin and streptavidin, desthiobiotin and avidin, desthiobiotin and antibiotin, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne , a tetrazine and a tran-cyclooctene, or an epitope and an epitope-specific antibody.

26. The system of any one of claims 1-25, wherein the targeting group is selected based on the protein family of interest.

27. The system of any one of claims 1-26, wherein the protein of interest is a serineO hydrolase and the targeting group comprises28. The system of any one of claims 1-27, wherein the protein of interest is a kinase andthe targeting group comprises29. The system of any one of claims 1-28, wherein the protein of interest is a cysteineprotease and the targeting group comprises one of30. The system of any one of claims 1-29, wherein the targeting group is linked to the retrieval tag with a linker.

31. The system of claim 30, wherein the linker is organic.

32. The system of claim 30 or 31, wherein the linker comprises an aliphatic linker.

33. The system of any one of claims 1-32, wherein the retrieval tag comprises an orthogonal recognition element.299713335.1 - 89 -34. The system of any one of claims 1-33, wherein the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, 06-benzylguanine, SNAP protein, an alkyne, an azide, a cyclooctyne a tetrazine, a trans-cyclooctene, a peptide epitope or a peptide epitope-specific antibody.

35. The system of any one of claims 1-34, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

36. The system of claim 35, wherein the specialized functional form is an active form.

37. The system of any one of claims 1-36, wherein the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form.

38. The system of claim 37, wherein the non-specialized functional form is an inactive form.

39. The system of any one of claims 1-38, wherein the system further comprises a nonactivity probe capable of binding the protein of interest in non-specialized functional form.

40. A method for evaluating a target protein of interest comprising the system of any one of claims 1-39.

41. The method of claim 40, wherein the evaluating comprises measuring activity and / or quantity.

42. An epitope tag recognition element according to the system of any one of claims 1-41.

43. An epitope tag recognition element comprising an epitope-binding component and a barcode.

44. The epitope tag recognition element of claim 43, wherein the epitope-binding component is interchangeable with at least another epitope-binding component.

45. An activity probe according to the system of any one of claims 1-41.

46. An activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest.299713335.1 - 90 -47. The activity probe of claim 46, wherein the targeting group is interchangeable with at least another targeting group.

48. The activity probe of claim 46 or 47, wherein the targeting group is selected based on the protein of interest.

49. A method for evaluating a target protein of interest, the method comprising:(i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest;(ii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises a epitope tag binding component and a first oligonucleotide(iii) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide;(iv) incubating the composition under conditions sufficient for being ligated and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and(v) detecting the ligated or annealed first and second oligonucleotide.

50. The method of claim 49, wherein step (i) occurs in a live cell and steps (ii) - (v) occur after fixing and / or lysing of the cell.

51. A method for evaluating a target protein of interest, the method comprising:(i) contacting a composition comprising a protein of interest fused to an epitope tag with an activity probe comprising a targeting group operatively linked to a retrieval tag, wherein the targeting group is capable of binding to the protein of interest;(ii) contacting the composition with an epitope tag binding component capable of binding the epitope tag;299713335.1 - 91 -(iii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide;(iv) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide;(v) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and(vi) detecting the ligated and / or annealed first and second oligonucleotides.

52. The method of claim 51, wherein steps (iii) and (iv) are performed:(a) consecutively with (iii) before (iv);(b) consecutively with (iv) before (iii); or(c) concurrently.

53. The method of claim 51, wherein step (i) occurs in a live cell and steps (ii) - (vi) occur after fixing and / or lysing of the cell.

54. The method of any one of claims 49-52, wherein the protein of interest is in a cell.

55. The method of any one of claims 49-54, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialized functional form.

56. The method of claim 52, wherein the specialized functional form is an active form of the protein of interest.

57. The method of any one of claims 49-56, wherein the detecting the ligated or annealed oligonucleotides comprises imaging, qPCR or a sequencing based readout.

58. The method of any one of claims 49-57, wherein the method further comprises determining abundance of the target protein of interest299713335.1 - 92 -59. The method of claim 58, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

60. The method of claim 58 or 59, wherein determining the abundance of the target protein or interest comprises primer extension and / or PCR amplification of the ligated and / or annealed first and second oligonucleotides.

61. The method of any one of claims 49-60, wherein the steps are performed in chronological order.

62. The method of any one of claims 49-61, further comprising contacting the composition with one or more bridging oligonucleotides, wherein at least one of the one or more bridging oligonucleotides comprises complementary regions to both the first and second oligonucleotide.

63. The method of any one of claim 49-62, wherein the method further comprises performing rolling circle amplification after detecting the ligated and / or annealed first and second oligonucleotides.

64. The method of any one of claims 49-63, further comprising contacting the composition with one or more recognition oligonucleotides conjugated to a label, wherein the recognition oligonucleotide nucleotide is complementary to the first oligonucleotide, the second oligo nucleotide or at least one of the one or more bridging oligonucleotides.

65. The method of claim 64, wherein the label comprises a fluorescent protein, a pull down tag or a split recognition system.

66. The method of any one of claims 49-65, further comprising contacting the composition with a first labeled primer and / or second labeled primer, wherein the first labeled primer is specifically complementary to the first oligonucleotide and non-complementary to the second oligonucleotide and the second labeled primer is specifically complementary to the second oligonucleotide and non-complementary to the first oligonucleotide.

67. The method of claim 66, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.299713335.1 - 93 -68. The method of claim 66 or 67, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

69. The method of any one of claims 49-68, wherein first and the second oligonucleotides are capable of being ligated and / or and annealing when in sufficient proximity to each other in the presence of ligation reagents.

70. The method of any one of claims 49-69, wherein the method further comprises spatially detecting the target protein of interest.

71. The method of any one of claims 49-70, wherein the activity probe covalently modifies, or is capable of covalently modifying the proteins of interest.

72. The method of any one of claims 49-71, wherein the composition comprises fewer than 5000 cells.

73. The method of any one of claim 49-72, wherein the composition comprises less than 1 pg of total protein.

74. The method of any one of claims 49-73, wherein the method further comprises detecting a cellular marker.

75. The method of any one of claims 49-74, wherein the method further comprises determining the total amount of target protein of interest.

76. The method of claim 76, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

77. The method of claim 75 or 76, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the specialized functional form and non-specialized functional forms of the protein.

78. The method of any one of claims 49-77, wherein the method excludes performing liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and / or mass spectrometry.299713335.1 - 94 -79. The method of any one of claims 49-78, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are one or more of the following retrieval tag and retrieval tag binding component pairs: biotin and streptavidin, biotin and avidin, biotin and antibiotin, desthiobiotin and streptavidin, desthiobiotin and avidin, desthiobiotin and antibiotin, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne , a tetrazine and a tran-cyclooctene, or an epitope and an epitope-specific antibody.

80. The method of any one of claims 49-79, wherein the first and / or second oligonucleotide is single-stranded.

81. The method of any one of claims 49-80, wherein the first and / or second oligonucleotide comprises a DNA barcode.

82. The method of any one of claims 49-81, wherein the protein of interest is an enzyme.

83. The method of any one of claims 49-82, wherein the protein of interest is a serine hydrolase, cysteine proteases, kinases, metalloproteases, P-retaining glycosidases, tyrosine phosphatases, or cytochrome P450s,84. The method of any one of claims 49-83, wherein the protein of interest is a DNA repair enzyme.

85. The method of any one of claims 49-84, wherein the protein of interest is a non-enzyme.

86. The method of any one of claims 49-85, the epitope tag comprises a fluorescent protein, a pull down tag, a split recognition system, or a protein tag for covalent labeling, optionally, wherein the protein tag for covalent labeling comprises snap tag or halo tag.

87. The method of claim 86, wherein the fluorescent protein comprises GFP, RFP, or mCherry.

88. The method of claim 86, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

89. The method of claim 86, wherein the split recognition system comprises split gfp or spytag and catcher.299713335.1 - 95 -90. The method of any one of claims 49-89, wherein the epitope tag is fused to the protein of interest by combining genetic material.

91. The method of any one of claims 49-89, wherein the protein of interest fused to the epitope tag is a recombinant protein.

92. The method of any one of claims 49-91, wherein the protein of interest is introduced into a cell by transfection.

93. The method of claim 92, wherein the cell is a live cell94. The method of any one of claims 49-93, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

95. The method of any one of claims 49-94, wherein the targeting group comprises a flourophosphonate, diphenylphosphonate, sulfonyl fluoride, acyloxymethyl ketone, phenoxymethylketone, vinyl sulfone, epoxide, halomethylketone, alpha-haloester, alpha-haloamide, a, P-unsaturated ester, a, P-unsaturated ketone, diazomethylketone, acyl phosphate, acylphosphonate, hydroxamate, carbamate, ester, thioester, 2-deoxy-2- fluoro glycoside, a-bromobenzylphosphonate, 2-ethynylnaphthalene, acrylamide, butyneamide, crotonamide, chloroacetamide, or alpha-methylchloroacetamide.

96. The method of any one of claims 49-95, wherein the targeting group is selected based on the protein family of interest.

97. The method of any one of claims 49-96, wherein the protein of interest is a serineO hydrolase and the targeting group comprises98. The method of any one of claims 49-96, wherein the protein of interest is a kinase andthe targeting group comprises299713335.1 - 96 -99. The method of any one of claims 49-96, wherein the protein of interest is a cysteineprotease and the targeting group comprises one of100. The method of any one of claims 49-99, wherein the targeting group is linked to the retrieval tag with a linker.

101. The method of claim 100, wherein the linker is organic.

102. The method of claim 100 or 101, wherein the linker comprises an aliphatic linker.

103. The method of any one of claims 49-102, wherein the retrieval tag comprises an orthogonal recognition element.

104. The method of any one of claims 49-103, wherein the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, O6-benzylguanine, SNAP protein, an alkyne, an azide, a cyclooctyne a tetrazine, a trans-cyclooctene, a peptide epitope or a peptide epitope-specific antibody.

105. The method of any one of claims 49-104, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

106. The method of claim 105, wherein the specialized functional form is an active form.

107. The method of any one of claims 49-106, wherein the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form.

108. The method of claim 107, wherein the non-specialized functional form is an inactive form.

109. The method of any one of claims 49-108, wherein the system further comprises a nonactivity probe capable of binding the protein of interest in non-specialized functional form.299713335.1 - 97 -110. A method of competitive profiling comprising the method of any one of claims 49- 109, wherein step (i) further comprises contacting the composition with a non-activity probe capable of binding the protein of interest in non-specialized functional form.

111. A method of analyzing two more proteins of interest using the method of any one of claims 49-109, wherein the label and the recognition oligos are different for each protein.

112. A method of determining a half maximal inhibitory concentration (ICso) value of a compound of interest against a protein of interest, the method comprising:(i) contacting a composition comprising the protein of interest fused to an epitope tag with an activity probe comprising the compound of interest operatively linked to a retrieval tag, wherein the compound of interest is capable of binding to the protein of interest;(ii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag, wherein the epitope tag recognition element comprises a epitope tag binding component and a first oligonucleotide(iii) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide;(iv) incubating the composition under conditions sufficient for being ligated and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and(v) detecting the ligated or annealed first and second oligonucleotide.

113. A method of determining a half maximal inhibitory concentration (ICso) value of a compound of interest against a protein of interest, the method comprising:(i) contacting a composition comprising the protein of interest fused to an epitope tag with an activity probe comprising the compound of interest operatively linked to a retrieval tag, wherein the compound is capable of binding to the protein of interest;299713335.1 - 98 -(ii) contacting the composition with an epitope tag binding component capable of binding the epitope tag;(iii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a first oligonucleotide;(iv) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element comprises a second oligonucleotide;(v) incubating the composition under conditions sufficient for ligating and / or annealing of the first oligonucleotide and the second oligonucleotide when the first and second oligonucleotides are in close proximity to each other; and(vi) detecting the ligated and / or annealed first and second oligonucleotides.299713335.1 - 99 -

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