Reversible and irreversible kinome probes and uses thereof

Abstract

Aspects of the disclosure relate to chemical probes for the detection of proteins of interest. The chemical probes can be employed in activity-dependent proximity ligation methods for quantifying proteins of interest in single cells. In some embodiments, the chemical probes can be employed in identifying enzymes in single cells.

Description

REVERSIBLE AND IRREVERSIBLE KINOME PROBES AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 696,341, 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 / 696,271 filed September 18, 2024 entitled “Methods and Compositions for Activity- Dependent Protein Labeling” and U.S. Provisional Application Serial No. 63 / 696,346 filed September 18, 2024 entitled “Kinase Inhibitor Compositions and Therapeutic Uses Thereof’ both of which are incorporated by reference in their entireties herein.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_P0854WO_sequence_listing.xml and is 10,602 bytes in size.TECHNICAL FIELD

[0004] The current disclosure relates to the field of molecular biology, therapeutic methods, and diagnostics.BACKGROUND

[0005] The maturation of broad-scale “-omic” profiling methods and platforms has enabled the quantitative comparison of biological signal regulation at each step within the central dogma. In addition to providing unprecedented views of transcriptome-wide and proteome-wide regulation, these studies have unambiguously shown that mRNA transcript levels do not reliably predict protein abundance. Further complicating the situation, in principle the mere presence of a protein does not provide any information about the functional state of that protein, which can be impacted by posttranslational modifications, protein-protein or proteinmetabolite binding, spatial compartmentalization, and other factors. Activity-based protein profiling methods utilize chemical probes upstream of common detection methods, such as299555014.1 - 1 -liquid chromatography tandem mass spectrometry (LC-MS / MS) or gel-based protein separation, to specifically detect and quantify only active subpopulations of the proteome of interest.

[0006] Family-wide chemical proteomic probes are particularly useful because they can report on the activity of large swaths of the active proteome simultaneously, with published examples available for serine hydrolases, kinases, metalloproteases, cysteine proteases, and other functionalities that regulate protein activity. Although large sections of the proteome can be interrogated using these probes, commonly used gel- and MS-based detection platforms place significant limitations on the biological contexts that can be efficiently studied. Gel-based profiling is readily accessible but is inherently low-throughput, suffers from poor resolution, and has a narrow dynamic range for low-abundance target proteins and input proteome. MSbased platforms, on the other hand, are multiplexed and target-agnostic, enabling profiling across entire families. However, these attributes are accompanied by several trade-offs, including laborious and expensive sample preparation (days per sample), the need for large quantities of input proteome (typically several milligrams) for a single analysis, and the capability of analyzing only one sample at a time per instrument, which limits parallel and reproducible sample analyses. Therefore, while ideal for discovery mode activity-based profiling, these strategies are poorly suited for rapid, ultrasensitive, and multifamily activity profiling that could be useful for complex and limited abundance samples like patient tissues, cells, and fluids. Indeed, chemical proteomic approaches have not been widely adopted for clinical samples, despite their significant potential to diagnose disease and ultimately track and guide patient treatment decisions.SUMMARY

[0007] To address the inherent shortcomings of existing chemical proteomic technologies and to more readily enable translational applications, the present inventors developed a gel- and MS-free chemical proteomic platform that in principle can overcome limitations of existing platforms while providing 3 features: (1) broad and immediate compatibility with existing family-wide chemical proteomic probes to measure active proteins in native samples, rather than overall protein abundance; (2) signal deconvolution, superior dynamic range, and high sensitivity through signal amplification, to allow measurement of low abundance proteins and small quantities of input proteome; and (3) rapid, multiplexed, parallel, and simultaneous analysis of active targets within and between multiple protein families in diverse sample types.299555014.1 - 2 -

[0008] Some aspects of the present disclosure are therefore directed to chemical probes for the detection of proteins of interest. In some aspects, the chemical probes can be employed in activity-dependent proximity ligation (ADPL) methods for quantifying proteins of interest in single cells. In some aspects, the chemical probes comprise a reactive group that can covalently bind to an epitope tag of a side chain of an amino acid of a protein of interest and a retrieval tag that can be used to covalently bind the chemical probe to a retrieval tag recognition element. In some aspects, the chemical probes is a compound of formula I:where A is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom; Ri, R2, R3, and R4 are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide; Rs, Re, R7, and Rs are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide, wherein one of Rs, Re, R7, and Rs is optionally replaced by X; wherein X comprises, consists of, or consists essentially of a phenyl ring comprising a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the indazole phenyl ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof; Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-299555014.1 - 3 -2-one, N-hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the piperazine ring through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof. In some aspects, a linker as disclosed herein, e.g., Li and L2, can have a length that is any one of, less than, greater than, or between 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, or 100 atoms in length. In some aspects, any of the aforementioned functional groups is specifically excluded.

[0009] In some aspects, the compound is further defined as a compound of formula II:In some aspects, the compound is further defined as a compound of formula III:(III).299555014.1 - 4 -

[0010] In some aspects, the compound is further defined as a compound of formula IV:(iv).

[0011] In some aspects, the compound is further defined as a compound of formula V:

[0012] In some aspects, the compound is further defined as a compound of formula VI:

[0013] In some aspects, the compound of formula I is further defined as one of:299555014.1 - 5 -

[0014] In some aspects, the chemical probes is a compound of formula VII:299555014.1 -6-wherein A is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom; Ri and R2 are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide; R3, R4, Rs, and Re are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide, wherein one of R3, R4, Rs, and Re is optionally replaced by X; wherein X comprises, consists of, or consists essentially of a phenyl ring comprising a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the indolin-2-one phenyl ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof; Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-2-one, N- hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the piperazine ring through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally299555014.1 - 7 -comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof. In some aspects, a linker as disclosed herein, e.g., Li and L2, can have a length that is any one of, less than, greater than, or between 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, or 100 atoms in length. In some aspects, any of the aforementioned functional groups is specifically excluded.

[0015] In some aspects, the compound of formula VII is further defined as a compound of formula VIII:(VIII).

[0016] In some aspects, the compound of formula VII is one of299555014.1 - 8 -

[0017] In some aspects, the chemical probes is a compound of formula IX:wherein A s oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom; wherein X comprises, consists of, or consists essentially of a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the piperazine ring through a linker LI; wherein LI is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof; Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-2-one, N-hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the amine through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride299555014.1 - 9 -linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof. In some aspects, a linker as disclosed herein, e.g., Li and L2, can have a length that is any one of, less than, greater than, or between 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, or 100 atoms in length. In some aspects, any of the aforementioned functional groups is specifically excluded.

[0018] In some aspects, compound of formula (IX) is one of299555014.1 - 10 -

[0019] In some aspects, the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that reversibly covalently bind to the amino acid side chain moiety or epitope tag. In some aspects, the moiety that reversibly covalently binds to an amino acid side chain moiety or epitope tag is an aldehyde. In some aspects, the aldehyde is protected as a 1,3- dioxolane. In some aspects, the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that irreversibly covalently bind to the amino acid side chain moiety or epitope tag. In some aspects, the moiety that irreversibly covalently binds to an amino acid side chain moiety is hydroxyl, epoxide, fluorosulfonyl, ethenesulfonyl, substituted or unsubstituted benzoate ester, sulfurofluoridate, ethenesulfonate, fluorophosphonate, or ethenephosphonate. In some aspects, the amino acid side chain moiety or epitope tag comprises a moiety that comprises, consists of, or consists essentially of a hydroxyl group, an amine group, or a thiol group.

[0020] Some aspects of the present disclosure are directed to a method for evaluating a protein of interest. In some aspects, the method comprises the steps of contacting a composition comprising a protein of interest fused to an epitope tag with a compound of formula (I) or formula (VIII) or formula (IX), contacting the composition with a retrieval tag recognition element capable of binding to moiety Z of the compound of formula (I) or formula (VIII) or formula (IX), wherein the retrieval tag recognition element comprises a second oligonucleotide; incubating the composition under conditions sufficient for the epitope tag to covalently bind to the moiety that covalently binds to the epitope tag of the compound of formula (I) or formula (VIII) or formula (IX), and for the retrieval tag recognition element to covalently bind to moiety Z of the compound of formula (I) or formula (VIII) or formula (IX), and detecting a complex comprising the protein of interest, the compound of claim 1, and the retrieval tag recognition element, wherein the protein of interest is covalently bound to the299555014.1 - 11 -compound of formula (I) or formula (VIII) or formula (IX) and the retrieval tag recognition element is covalently bound to the compound of formula (I) or formula (VIII) or formula (IX).

[0021] In some aspects of the method, the first and / or second oligonucleotide single stranded. The first and / or second oligonucleotide can include a DNA barcode. In some the first and the second oligonucleotides are capable of ligation 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 spontaneous ligation and / or annealing of the first and / or the second oligonucleotide. In some aspects, the protein of interest is an enzyme. The protein of interest can comprise, 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.

[0022] In some aspects, the epitope tag includes a florescent protein, a pull down tag or a split recognition system. 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 spy tag and / or the like.

[0023] In some aspects, the epitope tag is fused to the protein of interest by genetic manipulation. In some aspects, the protein of interest fused to the epitope tag is a recombinant protein. In some aspects, the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, aptamer and / or the like.

[0024] In some aspects, the retrieval tag and / or retrieval tag recognition element are selected from 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 trans-cyclooctene, an epitope and an epitope-specific antibody, and the like.

[0025] In some aspects, the protein of interest is a serine hydrolase and the compound of formula (I) or formula (VIII) comprises an fluorophosphonate. In some aspects, the protein of interest is a kinase and the compound of formula (I) or formula (VIII) or formula (IX) comprises a fluorosulfonyl. In some aspects, the protein of interest is a cysteine protease and299555014.1 - 12 -the compound of formula (I) or formula (VIII) or formula (IX) comprises one of ethenesulfonyl, ethenesulfonate, ethenephosphonate, epoxide, and substituted or unsubstituted benzoate ester. In some aspects, the compound of formula (I) or formula (VIII) or formula (IX) 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. In some aspects, the retrieval tag comprises an orthogonal recognition element. In some aspects, the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, 06- benzylguanine, SNAP protein, an alkyne, an azide, a cyclooctyne a tetrazine, a transcyclooctene, a peptide epitope, a peptide epitope-specific antibody, and / or the like.

[0026] In some aspects, 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 aspects, the specified functional form is an active form of the protein. In some aspects, 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, the specialized functional form is an active form.

[0027] In some aspects, 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, the non-specialized functional form is an inactive form. In some aspects, the method further comprises employing 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.

[0028] Some aspects of the disclosure relate to methods for evaluating activity and / or abundance of the protein of interest. 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. The probes disclosed herein can299555014.1 - 13 -be synthesized by appending a retrieval tag and / or an epitope tag to a molecule that has affinity for a protein of interest. Exemplary molecules with affinity for proteins of interest may be found in U.S. Provisional Application entitled “Kinase Inhibitor Compositions and Therapeutic Uses Thereof,” (Attorney Docket No.: ARCD.P0855US.P1), the entirety of which is incorporated by reference.

[0029] Some aspects of the disclose relate to an activity probe comprising compound of formula (I) or formula (VIII) or formula (IX) capable of binding to the protein of interest operatively linked to a retrieval tag. 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 small molecule is interchangeable with at least another small molecule. In some aspects, the small molecule is selected based on the protein of interest.

[0030] In some aspects of the method, the protein of interest is in a cell. In some aspects of the disclosure, the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialize functional form. In some aspects of the disclosure, the specialized functional form is an active form of the protein of interest. In some aspects of the disclosure, the detecting the ligated or annealed oligonucleotides comprises imaging, qPCR, a sequencing based readout, and / or the like. 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 of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides. 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 proximity-dependent manner enabled by the action of a DNA polymerase (e.g. 3' exonuclease-capable polymerase). In some aspects of the disclosure, the steps are performed in chronological order.

[0031] 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 complementary regions to both the first and second oligonucleotide. In some aspects of the disclosure, the method further comprises performing299555014.1 - 14 -rolling circle amplification after the last step. 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 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 florescent protein, a pull down tag or a split recognition system.

[0032] 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 differentially 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.

[0033] In some aspects of the disclosure, wherein first and the second oligonucleotides are capable of ligation and / or and annealing when in sufficient proximity to each other. In some aspects of the disclosure, the method or the system further includes spatially detecting the target protein of interest. In some aspects of the disclosure, the composition comprises fewer than 5000 cells. In some embodiments, the composition comprises fewer 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.

[0034] In some aspects of the disclosure, the method further comprises the detection of a cellular marker. Such methods may include one or more of the following steps: addition of a marker-specific antibody, detection of binding of the marker-specific antibody, detection of the binding of the marker-specific antibody by immunofluorescence, isolating cells comprising the299555014.1 - 15 -detected binding by cell sorting (e.g., fluorescence activated cell sorting, FACS), flow cytometry of the cells, and / or culturing of the isolated cells.

[0035] 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. 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 non-specialized functional forms of the protein.

[0036] In some aspects of the disclosure, the method excludes one or more of liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry. In some aspects of the disclosure, retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are selected from 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, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a transcyclooctene, and an epitope and an epitope-specific antibody.

[0037] Some aspects are directed to a method of competitive profiling comprising any of the methods herein, wherein step (i) further comprises contacting the composition with a nonactivity probe capable of binding the protein of interest in non-specialized functional form. Some aspects are directed to a method 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.

[0038] In some aspects, the method further comprises obtaining a biological sample from a patient. In some aspects, the biological sample comprises blood, serum, or tissue. In some embodiments, the biological sample comprises cancerous cells. In some aspects, the cancerous cells comprise ovarian or prostate cancerous cells. In some embodiments, the cancerous cells are from a cancer described herein. In some aspects, the method further comprises determining the level or abundance of the protein of interest in the specified functional form in the biological sample.299555014.1 - 16 -

[0039] In some aspects, 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 aspects, 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 aspects, 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 aspects, the non-aggressive cancer comprises stage I or stage II cancer. In some aspects, the non-aggressive ovarian or prostate cancer comprises stage I or stage II ovarian or prostate cancer. In some aspects, diagnosing the patient based on the determined level of protein of interest in the specified functional form. In some aspects, 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 aspects, 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 biological sample from the patient. In some aspects, 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 aspects, 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.

[0040] In some aspects, the retrieval tag comprises biotin, streptavidin, avidin, anti-biotin, desthiobiotin, O6-benzylguanine, SNAP protein, alkyne, azide, cyclooctatriene, tetrazine, trans-cyclooctadiene, a peptide epitope or a peptide epitope-specific antibody. In some embodiments, the retrieval tag binding component comprises biotin, streptavidin, avidin, antibiotin, desthiobiotin, O6-benzylguanine, SNAP protein, alkyne, azide, cyclooctatriene, tetrazine, trans-cyclooctadiene, a peptide epitope or a peptide epitope-specific antibody.

[0041] Certain aspects of the present invention are characterized through the following enumerated aspects.

[0042] Aspect l is a compound of formula I:299555014.1 - 17 -

[0043] (I) wherein: A is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom; Ri, R2, R3, and R4 are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide; Rs, Re, R7, and Rs are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide, wherein one of Rs, Re, R7, and Rs is optionally replaced by X; wherein X comprises, consists of, or consists essentially of a phenyl ring comprising a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the indazole phenyl ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof; Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-2-one, N-hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the piperazine ring through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety299555014.1 - 18 -that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof.

[0044] Aspect 2 is the compound of aspect 1, wherein an aldehyde group is protected as a 1,3- di oxolane.

[0045] Aspect 3 is the compound of aspect 1, wherein the compound is further defined as a compound of formula

[0046] Aspect 4 is the compound of aspect 1, wherein the compound is further defined as acompound of formula III: (III).

[0047] Aspect 5 is the compound of aspect 1, wherein the compound is further defined as acompound of formula IV:299555014.1 - 19 -

[0048] Aspect 6 the compound of aspect 1, wherein the compound is further defined as a compound of formula V:

[0049] Aspect 7 is the compound of aspect 1, wherein the compound is further defined as a compound of formula VI:

[0050] Aspect 8 is the compound of aspect 1, wherein the compound is further defined as:299555014.1 - 20 -

[0051] Aspect 9 is the compound of aspect 1, wherein the compound is further defined as:

[0052] Aspect 10 is the compound of aspect 1, wherein the compound is further defined as:

[0053] Aspect 11 is the compound of aspect 1, wherein the compound is further defined as:299555014.1 - 21 -

[0054] Aspect 12 is the compound of aspect 1, wherein the compound is further defined as:

[0055] Aspect 13 is the compound of aspect 1, wherein the compound is further defined as:[wherein A is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom; Ri and R2 are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic299555014.1 - 22 -acid, nitrate, nitrite, nitrile, isocyanate, or azide; R3, R4, Rs, and Re are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide, wherein one of R3, R4, Rs, and Re is optionally replaced by X; wherein X comprises, consists of, or consists essentially of a phenyl ring comprising a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the indolin-2-one phenyl ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof; Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline- 2-one, N-hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the piperazine ring through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof.

[0057] Aspect 15 is the compound of aspect 14, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that reversibly covalently binds to the amino acid side chain moiety or epitope tag.

[0058] Aspect 16 is the compound of aspect 15, wherein the moiety that reversibly covalently binds to an amino acid side chain moiety or epitope tag is an aldehyde.

[0059] Aspect 17 is the compound of aspect 16, wherein the aldehyde is protected as a 1,3- di oxolane.299555014.1 - 23 -

[0060] Aspect 18 is the compound of aspect 14, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that irreversibly covalently binds to the amino acid side chain moiety or epitope tag.

[0061] Aspect 19 is the compound of aspect 18, wherein the moiety that irreversibly covalently binds to an amino acid side chain moiety or epitope tag is hydroxyl, fluorosulfonyl, epoxide, substituted or unsubstituted benzoate ester, ethenesulfonyl, sulfurofluoridate, ethenesulfonate, fluorophosphonate, or ethenephosphonate.

[0062] Aspect 20 is the compound of aspect 14, wherein the amino acid side chain moiety or epitope tag comprises a moiety that comprises, consists of, or consists essentially of a hydroxyl group, an amine group, or a thiol group.

[0063] Aspect 21 is the compound of aspect 14, wherein the compound is further defined as acompound of formula VIII: VIII.299555014.1 - 24 -

[0064] Aspect 22 is the compound of aspect 14, wherein the compound is one of:

[0065] Aspect 23 is a method for evaluating a protein of interest, the method comprising: (i) contacting a composition comprising a protein of interest fused to an epitope tag with a compound of aspect 1; (ii) contacting the composition with a retrieval tag recognition element capable of binding to moiety Z of the compound of aspect 1, wherein the retrieval tag recognition element comprises a second oligonucleotide; (iii) incubating the composition under conditions sufficient for the epitope tag to covalently bind to the moiety that covalently binds to the epitope tag of the compound of aspect 1, and for the retrieval tag recognition element to covalently bind to moiety Z of the compound of aspect 1; (iv) detecting a complex comprising the protein of interest, the compound of aspect 1, and the retrieval tag recognition element, wherein the protein of interest is covalently bound to the compound of aspect 1 and the retrieval tag recognition element is covalently bound to the compound of aspect 1.

[0066] Aspect 24 is the method of aspect 23, wherein the protein of interest and / or the retrieval tag each independently comprise a ligated or annealed oligonucleotide.

[0067] Aspect 25 is the method of aspect 24, wherein the protein of interest is in a cell.299555014.1 - 25 -

[0068] Aspect 26 is the method of any one of aspects 23-25, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialized functional form.

[0069] Aspect 27 is the method of aspect 26, wherein the specialized functional form is an active form of the protein of interest.

[0070] Aspect 28 is the method of any one of aspects 23-27, wherein the detecting the ligated or annealed oligonucleotide comprises imaging, qPCR, or a sequencing-based readout.

[0071] Aspect 29 is the method of any one of aspects 23-28, further comprising determining an abundance of the target protein of interest.

[0072] Aspect 30 is the method of aspect 29, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0073] Aspect 31 is the method of aspect 29 or 30, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed oligonucleotide.

[0074] Aspect 32 is the method of any one of aspects 23-31, wherein the steps are performed in chronological order.

[0075] Aspect 33 is the method of any one of aspects 23-32, 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.

[0076] Aspect 34 is the method of any one of aspects 23-33, wherein the method further comprises performing rolling circle amplification after the last step.

[0077] Aspect 35 is the method of any one of aspects 23-34, 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.

[0078] Aspect 36 is the method of aspect 35, wherein the label comprises a fluorescent protein, a pull down tag, or a split recognition system.299555014.1 - 26 -

[0079] Aspect 37 is the method of any one of aspects 23-36, 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.

[0080] Aspect 38 is the method of aspect 37, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

[0081] Aspect 39 is the method of aspect 37 or 38, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

[0082] Aspect 40 is the method of any one of aspects 23-39, wherein the first and the second oligonucleotides are capable of ligation and / or annealing when in sufficient proximity to each other.

[0083] Aspect 41 is the method of any one of aspects 23-40, wherein the method further comprises spatially detecting the target protein of interest.

[0084] Aspect 42 is the method of any one of aspects 23-41, wherein the composition comprises less than 5,000 cells.

[0085] Aspect 43 is the method of any one of aspects 23-42, wherein the composition comprises less than 1 pg of total protein.

[0086] Aspect 44 is the method of any one of aspects 23-43, wherein the method further comprises detection of a cellular marker.

[0087] Aspect 45 is the method of any one of aspects 23-44, wherein the method further comprises determining a total amount of target protein of interest.

[0088] Aspect 46 is the method of aspect 45, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

[0089] Aspect 47 is the method of aspect 45 or 46, 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.299555014.1 - 27 -

[0090] Aspect 48 is the method of any one of aspects 23-47, wherein the method excludes one or more of liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry.

[0091] Aspect 49 is the method of any one of aspects 23-48, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are selected from 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 antibiotin, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a trans-cyclooctene, and an epitope and an epitope-specific antibody.

[0092] Aspect 50 is the method of any one of aspects 23-49, wherein the first and / or second oligonucleotide are single stranded.

[0093] Aspect 51 is the method of any one of aspects 23-50, wherein the first and / or second oligonucleotide comprises a DNA barcode.

[0094] Aspect 52 is the method of any one of aspects 23-51, wherein the protein of interest is an enzyme.

[0095] Aspect 53 is the method of any one of aspects 23-52, wherein the protein of interest is a serine hydrolase, cysteine protease, kinase, metalloprotease, P-retaining glycosidase, tyrosine phosphatase, or cytochrome P450.

[0096] Aspect 54 is the method of any one of aspects 23-53, wherein the protein of interest is a DNA repair enzyme.

[0097] Aspect 55 is the method of any one of aspects 23-54, wherein the protein of interest is a non-enzyme.

[0098] Aspect 56 is the method of any one of aspects 23-55, wherein the epitope tag comprises a fluorescent protein, a pull down tag, or a split recognition system.

[0099] Aspect 57 is the method of aspect 56, wherein the fluorescent protein comprises GFP, RFP, or mCherry.299555014.1 - 28 -

[0100] Aspect 58 is the method of aspect 56, wherein the pull down tag comprises GST, flag, MBP, His, or HA.

[0101] Aspect 59 is the method of aspect 56, wherein the split recognition system comprises a spy tag.

[0102] Aspect 60 is the method of any one of aspects 54-59, wherein the epitope tag is fused to the protein of interest by genetic manipulation.

[0103] Aspect 61 is the method of any one of aspects 54-60, wherein the protein of interest fused to the epitope tag is a recombinant protein.

[0104] Aspect 62 is the method of any one of aspects 54-61, wherein the recombinant protein is introduced into a cell by transfection.

[0105] Aspect 63 is the method of aspect 62, wherein the cell is a live cell.

[0106] Aspect 64 is the method of any one of aspects 54-63, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

[0107] Aspect 65 is the method of any one of aspects 23-64, wherein the protein of interest is a serine hydrolase and the compound of claim 1 comprises a fluorophosphonate.

[0108] Aspect 66 is the method of any one of aspects 23-65, wherein the protein of interest is a kinase and the compound of claim 1 comprises a fluorosulfonyl.

[0109] Aspect 67 is the method of any one of aspects 23-66, wherein the protein of interest is a cysteine protease and the compound of claim 1 comprises one of ethenesulfonyl, ethenesulfonate, ethenephosphonate, epoxide, and / or a substituted or unsubstituted benzoate ester.

[0110] Aspect 68 is the method of any one of aspects 23-67, wherein the retrieval tag comprises an orthogonal recognition element.

[0111] Aspect 69 is the method of any one of aspects 23-68, 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.299555014.1 - 29 -

[0112] Aspect 70 is the method of any one of aspects 23-68, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

[0113] Aspect 71 is the method of aspect 70, wherein the specialized functional form is an active form.

[0114] Aspect 72 is the method of any one of aspects 23-69, wherein the activity probe does not bind to the protein of interest when the protein of interest is in a non-specialized functional form.

[0115] Aspect 73 is the method of aspect 72, wherein the non-specialized functional form is an inactive form.

[0116] Aspect 74 is the method of any one of aspects 23-73, wherein the system further comprises a non-activity probe capable of binding the protein of interest in a non-specialized functional form.

[0117] Aspect 75 is a method for evaluating a protein of interest, the method comprising (i) contacting a composition comprising a protein of interest fused to an epitope tag with a compound of claim 14; (ii) contacting the composition with a retrieval tag recognition element capable of binding to moiety Z of the compound of claim 14, wherein the retrieval tag recognition element comprises a second oligonucleotide; (iii) incubating the composition under conditions sufficient for the epitope tag to covalently bind to the moiety that covalently binds to the epitope tag of the compound of claim 14, and for the retrieval tag recognition element to covalently bind to moiety Z of the compound of claim 14; (iv) detecting a complex comprising the protein of interest, the compound of claim 1, and the retrieval tag recognition element, wherein the protein of interest is covalently bound to the compound of claim 14 and the retrieval tag recognition element is covalently bound to the compound of claim 14.

[0118] Aspect 76 is the method of aspect 75, wherein the protein of interest and / or the retrieval tag each independently comprise a ligated or annealed oligonucleotide.

[0119] Aspect 77 is the method of aspect 76, wherein the protein of interest is in a cell.

[0120] Aspect 78 is the method of any one of aspects 75-77, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialized functional form.299555014.1 - 30 -

[0121] Aspect 79 is the method of aspect 78, wherein the specialized functional form is an active form of the protein of interest.

[0122] Aspect 80 is the method of any one of aspects 75-79, wherein detecting the ligated or annealed oligonucleotide comprises imaging, qPCR, or a sequencing-based readout.

[0123] Aspect 81 is the method of any one of aspects 75-80, wherein the method further comprises determining the abundance of the target protein of interest.

[0124] Aspect 82 is the method of aspect 81, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0125] Aspect 83 is the method of aspect 81 or 82, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed oligonucleotide.

[0126] Aspect 84 is the method of any one of aspects 75-83, wherein the steps are performed in chronological order.

[0127] Aspect 85 is the method of any one of aspects 75-84, 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.

[0128] Aspect 86 is the method of any one of aspects 75-85, wherein the method further comprises performing rolling circle amplification after the last step.

[0129] Aspect 87 is the method of any one of aspects 75-86, 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.

[0130] Aspect 88 is the method of aspect 87, wherein the label comprises a fluorescent protein, a pull down tag, or a split recognition system.

[0131] Aspect 89 is the method of any one of aspects 75-88, further comprising contacting the composition with a first labeled primer and / or second labeled primer, wherein the first labeled299555014.1 - 31 -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.

[0132] Aspect 90 is the method of aspect 89, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

[0133] Aspect 91 is the method of aspect 89 or 90, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

[0134] Aspect 92 is the method of any one of aspects 75-91, wherein the first and the second oligonucleotides are capable of ligation and / or annealing when in sufficient proximity to each other.

[0135] Aspect 93 is the method of any one of aspects 75-92, wherein the method further comprises spatially detecting the target protein of interest.

[0136] Aspect 94 is the method of any one of aspects 75-93, wherein the composition comprises less than 5000 cells.

[0137] Aspect 95 is the method of any one of aspects 75-94, wherein the composition comprises less than 1 pg of total protein.

[0138] Aspect 96 is the method of any one of aspects 75-95, wherein the method further comprises detection of a cellular marker.

[0139] Aspect 97 is the method of any one of aspects 75-96, wherein the method further comprises determining a total amount of target protein of interest.

[0140] Aspect 98 is the method of aspect 97, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

[0141] Aspect 99 is the method of aspect 97 or 98, 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.299555014.1 - 32 -

[0142] Aspect 100 is the method of any one of aspects 75-99, wherein the method excludes one or more of liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry.

[0143] Aspect 101 is the method of any one of aspects 75-100, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are selected from 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 antibiotin, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a trans-cyclooctene, and an epitope and an epitope-specific antibody.

[0144] Aspect 102 is the method of any one of aspects 75-101, wherein the first and / or second oligonucleotide are single stranded.

[0145] Aspect 103 is the method of any one of aspects 75-102, wherein the first and / or second oligonucleotide comprises a DNA barcode.

[0146] Aspect 104 is the method of any one of aspects 75-103, wherein the protein of interest is an enzyme.

[0147] Aspect 105 is the method of any one of aspects 75-104, wherein the protein of interest is a serine hydrolase, cysteine protease, kinase, metalloprotease, P-retaining glycosidase, tyrosine phosphatase, or cytochrome P450.

[0148] Aspect 106 is the method of any one of aspects 75-105, wherein the protein of interest is a DNA repair enzyme.

[0149] Aspect 107 is the method of any one of aspects 75-106, wherein the protein of interest is a non-enzyme.

[0150] Aspect 108 is the method of any one of aspects 75-107, wherein the epitope tag comprises a fluorescent protein, a pull-down tag, or a split recognition system.

[0151] Aspect 109 is the method of aspect 108, wherein the fluorescent protein comprises GFP, RFP, or mCherry.299555014.1 - 33 -

[0152] Aspect 110 is the method of aspect 108, wherein the pull-down tag comprises GST, flag, MBP, His, or HA.

[0153] Aspect 111 is the method of aspect 108, wherein the split recognition system comprises a spy tag.

[0154] Aspect 112 is the method of any one of aspects 75-111, wherein the epitope tag is fused to the protein of interest by genetic manipulation.

[0155] Aspect 113 is the method of any one of aspects 75-112, wherein the protein of interest fused to the epitope tag is a recombinant protein.

[0156] Aspect 114 is the method of any one of aspects 75-113, wherein the recombinant protein is introduced into a cell by transfection.

[0157] Aspect 115 is the method of aspect 114, wherein the cell is a live cell.

[0158] Aspect 116 is the method of any one of aspects 75-115, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

[0159] Aspect 117 is the method of any one of aspects 75-116, wherein the protein of interest is a serine hydrolase and the compound of claim 14 comprises a fluorophosphonate.

[0160] Aspect 118 is the method of any one of aspects 75-117, wherein the protein of interest is a kinase and the compound of claim 14 comprises a fluorosulfonyl.

[0161] Aspect 119 is the method of any one of aspects 75-118, wherein the protein of interest is a cysteine protease and the compound of claim 14 comprises one of ethenesulfonyl, ethenesulfonate, ethenephosphonate, epoxide, and substituted or unsubstituted benzoate ester.

[0162] Aspect 120 is the method of any one of aspects 75-119, wherein the retrieval tag comprises an orthogonal recognition element.

[0163] Aspect 121 is the method of any one of aspects 75-120, 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.299555014.1 - 34 -

[0164] Aspect 122 is the method of any one of aspects 75-121, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

[0165] Aspect 123 is the method of aspect 122, wherein the specialized functional form is an active form.

[0166] Aspect 124 is the method of any one of aspects 75-123, wherein the activity probe does not bind to the protein of interest when the protein of interest is in a non-specialized functional form.

[0167] Aspect 125 is the method of aspect 124, wherein the non-specialized functional form is an inactive form.

[0168] Aspect 126 is the method of any one of aspects 75-125, wherein the system further comprises a non-activity probe capable of binding the protein of interest in a non-specialized functional form.

[0169] Aspect 127 is a compound of formula IX:wherein: A is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom; wherein X comprises, consists of, or consists essentially of a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the piperazine ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof; Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-2-one, N-hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the amine through a linker L2; and wherein L2 is a linker selected from the group299555014.1 - 35 -comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof.

[0170] Aspect 128 is the compound of aspect 127, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that reversibly covalently binds to the amino acid side chain moiety or epitope tag.

[0171] Aspect 129 is the compound of aspect 128, wherein the moiety that reversibly covalently binds to an amino acid side chain moiety or epitope tag is an aldehyde.

[0172] Aspect 130 is the compound of aspect 129, wherein the aldehyde is protected as a 1,3- di oxolane.

[0173] Aspect 131 is the compound of aspect 127, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that irreversibly covalently binds to the amino acid side chain moiety or epitope tag.

[0174] Aspect 132 is the compound of aspect 130, wherein the moiety that irreversibly covalently binds to an amino acid side chain moiety or epitope tag is hydroxyl, epoxide, fluorosulfonyl, ethenesulfonyl, substituted or unsubstituted benzoate ester, sulfurofluoridate, ethenesulfonate, fluorophosphonate, or ethenephosphonate.

[0175] Aspect 133 is the compound of aspect 127, wherein the amino acid side chain moiety or epitope tag comprises a moiety that comprises, consists of, or consists essentially of a hydroxyl group, an amine group, or a thiol group.299555014.1 - 36 -

[0176] Aspect 134 is the compound of aspect 127, wherein the compound is one of:

[0177] Aspect 135 is a method for evaluating a protein of interest, the method comprising: (i) contacting a composition comprising a protein of interest fused to an epitope tag with a compound of aspect 127; (ii) contacting the composition with a retrieval tag recognition element capable of binding to moiety Z of the compound of aspect 127, wherein the retrieval tag recognition element comprises a second oligonucleotide; (iii) incubating the composition under conditions sufficient for the epitope tag to covalently bind to the moiety that covalently299555014.1 - 37 -binds to the epitope tag of the compound of aspect 127, and for the retrieval tag recognition element to covalently bind to moiety Z of the compound of aspect 127; (iv) detecting a complex comprising the protein of interest, the compound of aspect 127, and the retrieval tag recognition element, wherein the protein of interest is covalently bound to the compound of aspect 127 and the retrieval tag recognition element is covalently bound to the compound of aspect 127.

[0178] Aspect 136 is the method of aspect 135, wherein the protein of interest and / or the retrieval tag each independently comprise a ligated or annealed oligonucleotide.

[0179] Aspect 137 is the method of aspect 135 or 136, wherein the protein of interest is in a cell.

[0180] Aspect 138 is the method of any one of aspects 135-137, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialized functional form.

[0181] Aspect 139 is the method of aspect 138, wherein the specialized functional form is an active form of the protein of interest.

[0182] Aspect 140 is the method of any one of aspects 135-139, wherein detecting the ligated or annealed oligonucleotide comprises imaging, qPCR, or a sequencing-based readout.

[0183] Aspect 141 is the method of any one of aspects 135-140, wherein the method further comprises determining an abundance of the target protein of interest.

[0184] Aspect 142 is the method of aspect 141, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

[0185] Aspect 143 is the method of aspect 141 or 142, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed oligonucleotide.

[0186] Aspect 144 is the method of any one of aspects 135-143, wherein the steps are performed in chronological order.

[0187] Aspect 145 is the method of any one of aspects 135-144, further comprising contacting the composition with one or more bridging oligonucleotides, wherein at least one of the one or299555014.1 - 38 -more bridging oligonucleotides comprises complementary regions to both the first and second oligonucleotides.

[0188] Aspect 146 is the method of any one of aspects 135-145, wherein the method further comprises performing rolling circle amplification after the last step.

[0189] Aspect 147 is the method of any one of aspects 135-146, 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.

[0190] Aspect 148 is the method of aspect 146, wherein the label comprises a fluorescent protein, a pull-down tag, or a split recognition system.

[0191] Aspect 149 is the method of any one of aspects 135-148, 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.

[0192] Aspect 150 is the method of aspect 149, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

[0193] Aspect 151 is the method of aspect 149 or 150, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

[0194] Aspect 152 is the method of any one of aspects 135-151, wherein the first and the second oligonucleotides are capable of ligation and / or annealing when in sufficient proximity to each other.

[0195] Aspect 153 is the method of any one of aspects 135-152, wherein the method further comprises spatially detecting the target protein of interest.

[0196] Aspect 154 is the method of any one of aspects 135-153, wherein the composition comprises less than 5,000 cells.299555014.1 - 39 -

[0197] Aspect 155 is the method of any one of aspects 135-154, wherein the composition comprises less than 1 pg of total protein.

[0198] Aspect 156 is the method of any one of aspects 135-155, wherein the method further comprises detection of a cellular marker.

[0199] Aspect 157 is the method of any one of aspects 135-156, wherein the method further comprises determining a total amount of target protein of interest.

[0200] Aspect 158 is the method of aspect 157, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

[0201] Aspect 159 is the method of aspect 157 or 158, 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.

[0202] Aspect 160 is the method of any one of aspects 135-159, wherein the method excludes one or more of liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry.

[0203] Aspect 161 is the method of any one of aspects 135-160, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are selected from 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 antibiotin, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a trans-cyclooctene, and an epitope and an epitope-specific antibody.

[0204] Aspect 162 is the method of any one of aspects 135-161, wherein the first and / or second oligonucleotide are single stranded.

[0205] Aspect 163 is the method of any one of aspects 135-162, wherein the first and / or second oligonucleotide comprises a DNA barcode.299555014.1 - 40 -

[0206] Aspect 164 is the method of any one of aspects 135-163, wherein the protein of interest is an enzyme.

[0207] Aspect 165 is the method of any one of aspects 135-164, wherein the protein of interest is a serine hydrolase, cysteine protease, kinase, metalloprotease, P-retaining glycosidase, tyrosine phosphatase, or cytochrome P450.

[0208] Aspect 166 is the method of any one of aspects 135-165, wherein the protein of interest is a DNA repair enzyme.

[0209] Aspect 167 is the method of any one of aspects 135-166, wherein the protein of interest is a non-enzyme.

[0210] Aspect 168 is the method of any one of aspects 135-167, wherein the epitope tag comprises a fluorescent protein, a pull-down tag, or a split recognition system.

[0211] Aspect 169 is the method of aspect 168, wherein the fluorescent protein comprises GFP, RFP, or mCherry.

[0212] Aspect 170 is the method of aspect 168, wherein the pull-down tag comprises GST, a flag, MBP, His, or HA.

[0213] Aspect 171 is the method of aspect 168, wherein the split recognition system comprises a spy tag.

[0214] Aspect 172 is the method of any one of aspects 166-171, wherein the epitope tag is fused to the protein of interest by genetic manipulation.

[0215] Aspect 173 is the method of any one of aspects 166-172, wherein protein of interest fused to the epitope tag is a recombinant protein.

[0216] Aspect 174 is the method of any one of aspects 166-173, wherein the recombinant protein is introduced into a cell by transfection.

[0217] Aspect 175 is the method of aspect 174, wherein the cell is a live cell.

[0218] Aspect 176 is the method of any one of aspects 135-174, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.299555014.1 - 41 -

[0219] Aspect 177 is the method of any one of aspects 135-174, wherein the protein of interest is a serine hydrolase and the compound of aspect 127 comprises a fluorophosphonate.

[0220] Aspect 178 is the method of any one of aspects 135-174, wherein the protein of interest is a kinase and the compound of aspect 127 comprises a fluorosulfonyl.

[0221] Aspect 179 is the method of any one of aspects 166-174, wherein the protein of interest is a cysteine protease and the compound of aspect 127 comprises one of ethenesulfonyl, ethenesulfonate, ethenephosphonate, epoxide, and substituted or unsubstituted benzoate ester.

[0222] Aspect 180 is the method of any one of aspects 135-179, wherein the retrieval tag comprises an orthogonal recognition element.

[0223] Aspect 181 is the method of any one of aspects 135-180, 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.

[0224] Aspect 182 is the method of any one of aspects 135-181, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

[0225] Aspect 183 is the method of aspect 181, wherein the specialized functional form is an active form.

[0226] Aspect 184 is the method of any one of aspects 135-181, wherein the activity probe does not bind to the protein of interest when the protein of interest is in a non-specialized functional form.

[0227] Aspect 185 is the method of aspect 184, wherein the non-specialized functional form is an inactive form.

[0228] Aspect 186 is the method of any one of aspects 135-185, wherein the system further comprises a non-activity probe capable of binding the protein of interest in non-specialized functional form.

[0229] It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention,299555014.1 - 42 -and any method of the invention may be used to produce or to utilize any composition of the invention. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. 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, Detailed Description, Claims, and Brief Description of the Drawings.

[0230] Other objects, 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 and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0231] An object of the presently disclosed subject matter having been stated hereinabove, and which is achieved in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying drawings and examples as best described hereinbelow.BRIEF DESCRIPTION OF THE DRAWINGS

[0232] 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 aspects presented herein.

[0233] FIG. 1: Diagram of probe compounds as disclosed herein. The probes include a reactive group A, a linker B, and a recognition tag C.

[0234] FIG. 2: Exemplary procedure for a probe compound (KW50P) as disclosed herein.

[0235] FIG. 3: 1H NMR of probe compound KW50P.

[0236] FIG. 4: LC / MS traces of synthesis of probe compound KW50P, M+l = 687 g / mol.299555014.1 - 43 -

[0237] FIGS. 5A-5C: (A) Increasing concentration of the kinome probes (KW60P & KW50P) with a purified kinase (ABL) shows a corresponding increase in the kinase bound to the probe as shown by an increase in the amount of protein band when blotted with streptavidin (top panel of the Western blots). Adding a competitive binder (XO44) decreases the binding of the probe, which shows the probe is specifically binding to the active site of ABL. This binding also occurs in protein lysate from HeLa cells (B) and live HeLa cells (B).

[0238] FIGS. 6A-6C: (A) Experimental scheme for SILAC LC-MS protocol to determine probe binding proteins. (B) Plot showing SILAC ratio for identified proteins using the protocol in (A) probing a HeLa lysate with KW60P. (C) Plot showing SILAC ratio for identified proteins using the protocol in (A) probing live HeLa cells with KW60P.

[0239] FIGS. 7A-7B: (A) Volcano plot showing enrichment of KW2449 binding proteins identified from the experiment of Figure 6A. (B) heatmap proteins identified from the experiment of Figure 6A.

[0240] FIGS. 8A-8B: (A) Plot showing SILAC ratio for identified proteins using the protocol in Figure 6A probing live HeLa cells with XO44-4. (B) Dots showing kinases identified as binding to XO44-4 in the kinome tree.

[0241] FIGS. 9A-9B: Irreversible covalent kinomeprobe XO44-4 is compatible with activitydependent proximity ligation (ADPL) platform. (A) ADPL with XO44-4 as the small molecule warhead in three different cell lines. (B) mean intensity of the imaging from (A).

[0242] FIG. 10: Chemical structures of probe compounds XO44-4, XO44-8, and XO44-12.

[0243] FIGS. 11A-11B: Increased linker lengths between retrieval tag and pharmacophore results in increased signal to noise intensities for ADPL readouts. (A) Intensity of signal using an imaging ADPL platoform. (B) Intensity of signal using a soluble ADPL platoform.

[0244] FIGS. 12A-12B: XO44-12 allows increased sensitivity of primary imaging using ADPL platform. (A) Imaging ADPL against HER-2 using the XO44-12 probe in various cell lines. (B) mean intensity of (A).

[0245] FIG. 13: Chemical structures of probe compounds S50P and S52P.

[0246] FIG. 14: Exemplary procedure for a probe compound (S50P) as disclosed herein.299555014.1 - 44 -

[0247] FIG. 15: Exemplary procedure for a probe compound (S52P) as disclosed herein.

[0248] FIG. 16: 1H NMR of probe compound S52P.

[0249] FIG. 17: S50P chemical probe can covalently label proteins in lysate after 2 hour incubation at 37C.

[0250] FIG. 18: S52P chemical probe can covalently label proteins in HEK lysate after 2 hour incubation at 37C.

[0251] FIGS. 19A-19D: Figure 19A Chemical structures of probe compound PCA84.

[0252] FIGS. 20A-20B: Kinome probes show more selective kinase scope in AML cell lines.

[0253] FIG. 21: Kinome probes show differential effect on various cancer cell lines.

[0254] FIGS. 22A-22B: Kinome probes show differential effect on growth inhibition for various cancer cell lines.

[0255] FIGS. 23: Kinome probes show highly potent and selective growth inhibition of AML cell lines.

[0256] FIG. 24: Chemical structures of exemplary compounds.

[0257] FIG. 25: Chemical structures of exemplary compounds.DETAILED DESCRIPTION

[0258] The present inventors recently reported an imaging-based activity-dependent proximity ligation platform, termed ADPL imaging, which enables spatially resolved detection of active enzymes in single cells using family-wide probes. While the concept behind this approach addresses several shortcomings, the imaging-based format places limitations on samples types that can be interrogated (i.e., incompatible with biofluids), as well as low processing throughput involved with sample preparation and microscopy. Disclosed herein is an updated platform referred to as soluble activity-dependent proximity ligation (sADPL). sADPL profiling can quantify active enzymes at picogram levels of whole proteome from cells, blood, and primary patient tissue samples. This multiplexed activity-based approach can be applied to directly quantify small molecule-protein target engagement in vivo, as well as molecular phenotyping299555014.1 - 45 -of disease states through direct quantitative profiling of active enzyme biomarkers in tissue samples.

[0259] 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.

[0260] “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. In some embodiments it is contemplated that a numerical value discussed herein may be used with the term “about” or “approximately.”

[0261] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. “Consisting essentially of’ in the context of pharmaceutical compositions of the disclosure is intended to include all the recited active agents and excludes any additional non-recited active agents, but does not exclude other components of the composition that are not active ingredients. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or299555014.1 - 46 -achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

[0262] The terms “protein”, “polypeptide” and “peptide” are used interchangeably herein when referring to a gene product or functional protein.

[0263] The terms “oligonucleotide” and “oligo” are used interchangeably and refer to short DNA or RNA molecules.

[0264] 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.

[0265] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0266] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0267] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives as well as “and / or.” As used herein “another” may mean at least a second or more.

[0268] As used in this specification and claim(s), 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”) 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. It is contemplated that any embodiment set forth with the term “comprising” may also be substituted with the word “consisting of’ for “comprising.”

[0269] 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.299555014.1 - 47 -

[0270] 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.

[0271] Other objects, 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 preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0272] The presently disclosed subject matter will now be described more fully hereinafter with reference to the accompanying Examples, in which representative embodiments are shown. The presently disclosed subject matter can, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the embodiments to those skilled in the art.

[0273] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are now described. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0274] Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist, unless indicated otherwise.299555014.1 - 48 -Chemical Definitions

[0275] As used herein, the term “nitro” means -NO2; the term “amino” means — NH2; the term “halo” or “halogen” designates -F, -Cl, -Br or -I; the term “mercapto” means -SH; the terms “cyano” and “nitrile” mean -CN; the term “azido” means -N3; the term “silyl” means -SiHs, and the term “hydroxy” means -OH.

[0276] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a linear (i.e., unbranched) or branched carbon chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons, which may be fully saturated, monounsaturated, or polyunsaturated. An unsaturated alkyl group includes those having one or more carbon-carbon double bonds (alkenyl) and those having one or more carbon-carbon triple bonds (alkynyl). The groups, -CH3 (Me, methyl), - CH2CH3 (Et, ethyl), -CH2CH2CH3 (n-Pr, n-propyl), propenyl, -CH(CH3)2 (iso-Pr, iso-propyl), - CH2CH2CH2CH3 (n-Bu, n-butyl), butenyl, -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 (isobutyl), -C(CH3)3 (tert-butyl), -CH2C(CH3)3 (neo-pentyl), propargyl, are all non-limiting examples of alkyl groups. Specifically included within the definition of “alkyl” are those alkyl groups that are optionally substituted.

[0277] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a linear or branched chain having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, S, P, and Si. The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the heteroalkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive. The following groups are all non-limiting examples of heteroalkyl groups: trifluoromethyl, -CH2F, -CH2CI, -CH2Br, -CH2OH, -CH2OCH3, -CH2OCH2CF3, - CH2OC(O)CH3, -CH2NH2,-CH2NHCH3, -CH2N(CH3)2, -CH2CH2CI, -CH2SCH3, -CH2CH2OH, -CH2CH2OC(O)CH3, -CH2CH2NHCO2C(CH3)3, and -CH2Si(CH3)3. Specifically included within the definition of “heteroalkyl” are those heteroalkyl groups that are optionally substituted.

[0278] The terms “cycloalkyl” and “heterocyclyl,” by themselves or in combination with other terms, means cyclic versions of “alkyl” and “heteroalkyl”, respectively. Additionally, for heterocyclyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. A cycloalkyl group and a heteroalkyl group can include one or more carbon-carbon double bonds, and / or one or more carbon-carbon triple bonds. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, and299555014.1 - 49 -cycloheptyl groups. Examples of heterocyclic groups include indole, azetidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2- oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, hexahydrodiazepinyl, 4-piperidonyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3 -di oxolane, tetrahydro- 1,1 -di oxothienyl, and the like. Specifically included within the definition of “cycloalkyl” are those cycloalkyl groups that are optionally substituted. Specifically included within the definition of “heterocyclyl” are those heterocycle groups that are optionally substituted.

[0279] The term “aryl” includes aromatic groups, and is not limited to phenyl, a-naphthyl, P- naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenaphthenyl. In certain aspects an aryl group is a phenyl group. Specifically included within the definition of “aryl” are those aromatic groups that are optionally substituted. For example, in some aspects of the present invention, the “aryl” groups are optionally substituted with from 1 to 5 substituents selected from the group consisting of hydrogen, hydroxy, aryl, acyl, Ci-Ce alkyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, amino substituted by one or two Ci-Ce alkyl groups, cyano, halogen, nitro, and trihalomethyl. In some aspects of the present invention, for example, in some aspects wherein the aryl group is phenyl, the aryl groups are optionally substituted with from 1 to 5 substituents selected from the group consisting of hydrogen, hydroxy, aryl, acyl, Ci-Ce alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, amino, amino substituted by one or two Ci-Ce alkyl groups, cyano, halogen, nitro, and trihalomethyl.

[0280] The term “heteroaryl” refers to an aryl group that contains one to four heteroatoms selected from N, O, and S. Specifically included within the definition of “heteroaryl” are those heteroaryl groups that are optionally substituted. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 4-azaindole, 5-azaindole, 6-azaindole, 7-azaindole, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3 -pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-299555014.1 - 50 -isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5- quinoxalinyl, 3 -quinolyl, and 6-quinolyl.

[0281] The term “alkoxy” means a group having the structure -OR', where R' is an optionally substituted alkyl or cycloalkyl group. The term “heteroalkoxy” similarly means a group having the structure -OR, where R is a heteroalkyl or heterocyclyl. Specifically included within the definition of “alkoxy” are those alkoxy groups that are optionally substituted.

[0282] Various groups, including alkyl, heteroalky, cycloalkyl, heterocyclyl, aryl, heteroaryl, and alkoxy, are described herein as substituted or unsubstituted (z.e., optionally substituted). Optionally substituted groups may include one or more substituents independently selected from: -CF3, -OCF3, halogen, nitro, cyano, hydroxy, amino, amido, mercapto, formyl, carboxy, oxo, acyl, carbamoyl, silyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, alkoxy, alkenyloxy, alkynloxy, aryloxy, acyloxy, alkylthio, alkylamino, alkenylamino, alkynlamino, arylamino, (alkyl)2amino, alkylthio, alkenylthio, alkynylthio, arylthio, alkylsulfinyl, sulfonyl, alkylsulfonyl, alkenyl sulfonyl, alkynylsulfonyl,, arylsulfonyl, acylthio, alkylsilyl, phosphate, phosphonate, phosphinate, cycloalkyl, heterocyclyl, aryl, and heteroaryl. In certain aspects the substituents may be further substituted with one or more substituents.

[0283] The term “amino” means a group having the structure -NR'R", where R' and R" are independently hydrogen or an optionally substituted acyl, alkyl, heteroalkyl, cycloalkyl, or heterocyclyl group. The term “amino” includes primary, secondary, and tertiary amines.

[0284] The term “acyl” includes straight-chain acyl, branched-chain acyl, cycloacyl, cyclic acyl, heteroatom -unsubstituted acyl, heteroatom-substituted acyl, alkylcarbonyl, alkoxycarbonyl and aminocarbonyl groups. The groups, -CHO, -C(O)CH3, -C(O)CH2CH3, - C(O)CH2CH2CH3, -C(O)CH(CH3)2, -C(O)CH(CH2)2, -C(O)C6H5, -C(O)C6H4CH3, - C(O)CeH4CH2CH3, and -COCeH3(CH3)2, are non-limiting examples of heteroatom- unsubstituted acyl groups. The term “heteroatom-substituted acyl” refers to a radical, having a single carbon atom as the point of attachment, the carbon atom being part of a carbonyl group, further having a linear or branched, cyclic or acyclic structure, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and299555014.1 - 51 -S. The groups, -C(O)CH2CF3, -CO2H, -CO2, -CO2CH3, -CO2CH2CH3, -CO2CH2CH2CH3, - CO2CH(CH3)2, -CO2CH(CH2)2, -C(0)NH2(carbamoyl), -C(O)NHCH3, -C(O)NHCH2CH3, - CONHCH(CH3)2,-CONHCH(CH2)2, -CON(CH3)2, and -CONHCH2CF3, are non-limiting examples of heteroatom-substituted acyl groups.

[0285] The term “alkoxy” includes straight-chain alkoxy, branched-chain alkoxy, cycloalkoxy, cyclic alkoxy, heteroatom -unsubstituted alkoxy, heteroatom-substituted alkoxy, heteroatom- unsubstituted alkoxy, and heteroatom-substituted alkoxy. The term “heteroatom -unsubstituted alkoxy” refers to a group, having the structure -OR, in which R is a heteroatom -unsubstituted alkyl. Heteroatom -unsubstituted alkoxy groups include: -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, and -OCH(CH2)2. The term “heteroatom-substituted alkoxy” refers to a group, having the structure -OR, in which R is a heteroatom-substituted alkyl. For example, -OCH2CF3 is a heteroatom-substituted alkoxy group.

[0286] The term “alkenyloxy” includes straight-chain alkenyloxy, branched-chain alkenyloxy, cycloalkenyloxy, cyclic alkenyloxy, heteroatom -unsubstituted alkenyloxy, and heteroatom- substituted alkenyloxy. The term “heteroatom -unsubstituted alkenyloxy” refers to a group, having the structure — OR, in which R is a heteroatom -unsubstituted alkenyl. The term “heteroatom-substituted alkenyloxy” refers to a group, having the structure -OR, in which R is a heteroatom-substituted alkenyl.

[0287] The term “alkynyloxy” includes straight-chain alkynyloxy, branched-chain alkynyloxy, cycloalkynyloxy, cyclic alkynyloxy, heteroatom -unsubstituted alkynyloxy, and heteroatom- substituted alkynyloxy. The term “heteroatom -unsubstituted alkynyloxy” refers to a group, having the structure -OR, in which R is a heteroatom -unsubstituted alkynyl. The term “heteroatom-substituted alkynyloxy” refers to a group, having the structure -OR, in which R is a heteroatom-substituted alkynyl.

[0288] The term “aryloxy” includes heteroatom -unsubstituted aryloxy, heteroatom-substituted aryloxy, heteroaryloxy, and heterocyclic aryloxy groups. The term “heteroatom -unsubstituted aryloxy” refers to a group, having the structure -OAr, in which Ar is a heteroatom -unsubstituted aryl. A non-limiting example of a heteroatom -unsubstituted aryloxy group is -OCeHs. The term “heteroatom-substituted aryloxy” refers to a group, having the structure -OAr, in which Ar is a heteroatom-substituted aryl.299555014.1 - 52 -

[0289] The term “acyloxy” includes straight-chain acyloxy, branched-chain acyloxy, cycloacyloxy, cyclic acyloxy, heteroatom -unsubstituted acyloxy, heteroatom-substituted acyloxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The term “heteroatom-unsubstituted acyloxy” refers to a group, having the structure -OAc, in which Ac is a heteroatom -unsubstituted acyl. For example, -OC(O)CH3 is a non-limiting example of a heteroatom -unsubstituted acyloxy group. The term “heteroatom- substituted acyloxy” refers to a group, having the structure -OAc, in which Ac is a heteroatom- substituted acyl. For example, -OC(O)OCH3 and -OC(O)NHCH3 are non-limiting examples of heteroatom -unsubstituted acyloxy groups.

[0290] The term “alkylamino” includes straight-chain alkylamino, branched-chain alkylamino, cycloalkylamino, cyclic alkylamino, heteroatom -unsubstituted alkylamino, and heteroatom- substituted alkylamino. The term “heteroatom -unsubstituted alkylamino” refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, 4 or more hydrogen atoms, a total of 1 nitrogen atom, and no additional heteroatoms. The term “heteroatom -unsubstituted alkylamino” includes groups, having the structure -NHR, in which R is a heteroatom -unsubstituted alkyl. A heteroatom -unsubstituted alkylamino group would include -NHCH3, -NHCH2CH3, -NHCH2CH2CH3, -NHCH(CH3)2, -NHCH(CH2)2, -NHCH2CH2CH2CH3, -NHCH(CH3)CH2CH3, -NHCH2CH(CH3)2, - NHC(CH3)3,-N(CH3)2, -N(CH3)CH2CH3, -N(CH2CH3)2, N-pyrrolidinyl, and N-piperidinyl. The term “heteroatom-substituted alkylamino” refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two saturated carbon atoms attached to the nitrogen atom, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The term “heteroatom-substituted alkylamino” includes groups, having the structure -NHR, in which R is a heteroatom-substituted alkyl.

[0291] The term “alkenylamino” includes straight-chain alkenylamino, branched-chain alkenylamino, cycloalkenylamino, cyclic alkenylamino, heteroatom -unsubstituted alkenylamino, heteroatom-substituted alkenylamino, dialkenylamino, and alkyl(alkenyl)amino299555014.1 - 53 -groups. The term “heteroatom-unsubstituted alkenylamino” refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one nonaromatic carbon-carbon double bond, 4 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. The term “heteroatom -unsubstituted alkenylamino” includes groups, having the structure -NHR, in which R is a heteroatom- unsubstituted alkenyl. The term “heteroatom-substituted alkenylamino” refers to a radical, having a single nitrogen atom as the point of attachment and at least one nonaromatic carboncarbon double bond, but no carbon-carbon triple bonds, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The term “heteroatom-substituted alkenylamino” includes groups, having the structure -NHR, in which R is a heteroatom-substituted alkenyl.

[0292] The term “alkynylamino” includes straight-chain alkynylamino, branched-chain alkynylamino, cycloalkynylamino, cyclic alkynylamino, heteroatom -unsubstituted alkynylamino, heteroatom-substituted alkynylamino, dialkynylamino, alkyl(alkynyl)amino, and alkenyl(alkynyl)amino groups. The term “heteroatom -unsubstituted alkynylamino” refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, containing at least one carbon-carbon triple bond, at least one hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. The term “heteroatom- unsubstituted alkynylamino” includes groups, having the structure -NHR, in which R is a heteroatom -unsubstituted alkynyl. The term “heteroatom-substituted alkynylamino” refers to a radical, having a single nitrogen atom as the point of attachment, further having one or two carbon atoms attached to the nitrogen atom, further having at least one nonaromatic carboncarbon triple bond, further having a linear or branched, cyclic or acyclic structure, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the nitrogen atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The term “heteroatom- substituted alkynylamino” includes groups, having the structure NHR, in which R is a heteroatom-substituted alkynyl.299555014.1 - 54 -

[0293] The term “arylamino” includes heteroatom -unsubstituted arylamino, heteroatom- substituted arylamino, heteroarylamino, heterocyclic arylamino, and alkyl(aryl)amino groups. The term “heteroatom -unsubstituted arylamino” refers to a radical, having a single nitrogen atom as the point of attachment, further having at least one aromatic ring structure attached to the nitrogen atom, wherein the aromatic ring structure contains only carbon atoms, 6 or more hydrogen atoms, a total of one nitrogen atom, and no additional heteroatoms. The term “heteroatom -unsubstituted arylamino” includes groups, having the structure -NHR, in which R is a heteroatom-unsubstituted aryl. The term “heteroatom-substituted arylamino” refers to a radical, having a single nitrogen atom as the point of attachment, at least one hydrogen atom, at least one additional heteroatoms, that is, in addition to the nitrogen atom at the point of attachment, wherein at least one of the carbon atoms is incorporated into one or more aromatic ring structures, further wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The term “heteroatom-substituted arylamino” includes groups, having the structure -NHR, in which R is a heteroatom-substituted aryl.

[0294] The term “amido” includes straight-chain amido, branched-chain amido, cycloamido, cyclic amido, heteroatom -unsubstituted amido, heteroatom-substituted amido, alkylcarbonylamino, arylcarbonylamino, alkoxycarbonylamino, aryloxycarbonylamino, acylamino, alkylaminocarbonylamino, arylaminocarbonylamino, and ureido groups. The term “heteroatom -unsubstituted amido” refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, 1 or more hydrogen atoms, a total of one oxygen atom, a total of one nitrogen atom, and no additional heteroatoms. The term “heteroatom -unsubstituted amido” includes groups, having the structure -NHR, in which R is a heteroatom -unsubstituted acyl. The group, -NHC(O)CH3, is a non-limiting example of a heteroatom -unsubstituted amido group. The term “heteroatom-substituted amido” refers to a radical, having a single nitrogen atom as the point of attachment, further having a carbonyl group attached via its carbon atom to the nitrogen atom, further having a linear or branched, cyclic or acyclic structure, 0, 1, or more than one hydrogen atom, at least one additional heteroatom in addition to the oxygen of the carbonyl group, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S. The term “heteroatom-substituted amido” includes groups, having the structure -NHR, in which R is a299555014.1 - 55 -heteroatom -unsubstituted acyl. The group, -NHCO2CH3, is a non-limiting example of a heteroatom-substituted amido group.

[0295] The term “alkylthio” includes straight-chain alkylthio, branched-chain alkylthio, cycloalkylthio, cyclic alkylthio, heteroatom -unsubstituted alkylthio, and heteroatom- substituted alkylthio. The term “heteroatom -unsubstituted alkylthio” refers to a group, having the structure-SR, in which R is a heteroatom -unsubstituted alkyl. The group, -SCH3, is an example of a heteroatom -unsubstituted alkylthio group. The term “heteroatom-substituted alkylthio” refers to a group, having the structure -SR, in which R is a heteroatom- substituted alkyl.

[0296] The term “alkenylthio” includes straight-chain alkenylthio, branched-chain alkenylthio, cycloalkenylthio, cyclic alkenylthio, heteroatom -unsubstituted alkenylthio, and heteroatom- substituted alkenylthio. The term “heteroatom -unsubstituted alkenylthio” refers to a group, having the structure -SR, in which R is a heteroatom -unsubstituted alkenyl. The term “heteroatom-substituted alkenylthio” refers to a group, having the structure -SR, in which R is a heteroatom-substituted alkenyl.

[0297] The term “alkynylthio” includes straight-chain alkynylthio, branched-chain alkynylthio, cycloalkynylthio, cyclic alkynylthio, heteroatom -unsubstituted alkynylthio, and heteroatom-substituted alkynylthio. The term “heteroatom -unsubstituted alkynylthio” refers to a group, having the structure -SR, in which R is a heteroatom -unsubstituted alkynyl. The term “heteroatom-substituted alkynylthio” refers to a group, having the structure -SR, in which R is a heteroatom-substituted alkynyl.

[0298] The term “arylthio” includes heteroatom -unsubstituted arylthio, heteroatom-substituted arylthio, heteroaryl thio, and heterocyclic arylthio groups. The term “heteroatom -unsubstituted arylthio” refers to a group, having the structure -SAr, in which Ar is a heteroatom -unsubstituted aryl. The group, -SCeHs, is an example of a heteroatom-unsubstituted arylthio group. The term “heteroatom-substituted arylthio” refers to a group, having the structure -SAr, in which Ar is a heteroatom-substituted aryl.

[0299] The term “acylthio” includes straight-chain acylthio, branched-chain acylthio, cycloacylthio, cyclic acylthio, heteroatom -unsubstituted acylthio, heteroatom-substituted acylthio, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, and carboxylate groups. The term “heteroatom-unsubstituted acylthio” refers to a group, having the299555014.1 - 56 -structure -SAc, in which Ac is a heteroatom-unsubstituted acyl. The group, -SCOCH3, is an example of a heteroatom -unsubstituted acylthio group. The term “heteroatom-substituted acylthio” refers to a group, having the structure -SAc, in which Ac is a heteroatom-substituted acyl.

[0300] The term “alkylsilyl” includes straight-chain alkylsilyl, branched-chain alkylsilyl, cycloalkylsilyl, cyclic alkylsilyl, heteroatom -unsubstituted alkylsilyl, and heteroatom- substituted alkylsilyl. The term “heteroatom -unsubstituted alkylsilyl” refers to a radical, having a single silicon atom as the point of attachment, further having one, two, or three saturated carbon atoms attached to the silicon atom, further having a linear or branched, cyclic or acyclic structure, 5 or more hydrogen atoms, a total of 1 silicon atom, and no additional heteroatoms. An alkylsilyl group includes dialkylamino groups. The groups, -Si(CH3)3 and - Si(CH3)2C(CH3)3, are non-limiting examples of heteroatom -unsubstituted alkylsilyl groups. The term “heteroatom-substituted alkylsilyl” refers to a radical, having a single silicon atom as the point of attachment, further having at least one, two, or three saturated carbon atoms attached to the silicon atom, no carbon-carbon double or triple bonds, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, all of which are nonaromatic, 0, 1, or more than one hydrogen atom, and at least one additional heteroatom, that is, in addition to the silicon atom at the point of attachment, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.

[0301] The term “phosphonate” includes straight-chain phosphonate, branched-chain phosphonate, cyclophosphonate, cyclic phosphonate, heteroatom -unsubstituted phosphonate, and heteroatom-substituted phosphonate. The term “heteroatom-unsubstituted phosphonate” refers to a radical, having a single phosphorous atom as the point of attachment, further having a linear or branched, cyclic or acyclic structure, 2 or more hydrogen atoms, a total of three oxygen atom, and no additional heteroatoms. The three oxygen atoms are directly attached to the phosphorous atom, with one of these oxygen atoms doubly bonded to the phosphorous atom. The groups, -P(O)(OH)2, -P(O)(OH)OCH3,-P(O)(OH)OCH2CH3, -P(O)(OCH3)2, and - P(O)(OH)(OC6HS) are non-limiting examples of heteroatom -unsubstituted phosphonate groups. The term “heteroatom-substituted phosphonate” refers to a radical, having a single phosphorous atom as the point of attachment, further having a linear or branched, cyclic or acyclic structure, 2 or more hydrogen atoms, three or more oxygen atoms, three of which are299555014.1 - 57 -directly attached to the phosphorous atom, with one of these three oxygen atoms doubly bonded to the phosphorous atom, and further having at least one additional heteroatom in addition to the three oxygen atoms, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.

[0302] The term “phosphinate” includes straight-chain phosphinate, branched-chain phosphinate, cyclophosphinate, cyclic phosphinate, heteroatom -unsubstituted phosphinate, and heteroatom-substituted phosphinate. The term “heteroatom -unsubstituted phosphinate” refers to a radical, having a single phosphorous atom as the point of attachment, further having a linear or branched, cyclic or acyclic structure, 2 or more hydrogen atoms, a total of two oxygen atom, and no additional heteroatoms. The two oxygen atoms are directly attached to the phosphorous atom, with one of these oxygen atoms doubly bonded to the phosphorous atom. The groups,-P(O)(OH)H, -P(O)(OH)CH3, -P(O)(OH)CH2CH3, -P(O)(OCH3)CH3, and -P(O)(OC6H5)H are non-limiting examples of heteroatom -unsubstituted phosphinate groups. The term “heteroatom-substituted phosphinate” refers to a radical, having a single phosphorous atom as the point of attachment, further having a linear or branched, cyclic or acyclic structure, further having a total of n carbon atoms, 2 or more hydrogen atoms, two or more oxygen atoms, two of which are directly attached to the phosphorous atom, with one of these two oxygen atoms doubly bonded to the phosphorous atom, and further having at least one additional heteroatom in addition to the two oxygen atoms, wherein each additional heteroatom is independently selected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.

[0303] Any apparently unfulfilled valency is to be understood to be properly filled by hydrogen atom(s). For example, a compound with a substituent of -O or -N is to be understood to be - OH or -NH2, respectively.

[0304] The term “geminal” refers to a carbon atom to which two atoms or functional groups are attached. A geminal diol, for example, is a compound in which two hydroxyl groups are attached to the same carbon atom.

[0305] Any genus, subgenus, or specific compound discussed herein is specifically contemplated as being excluded from any aspect described herein.299555014.1 - 58 -

[0306] Compounds described herein may be prepared synthetically using conventional organic chemistry methods known to those of skill in the art and / or are commercially available (e.g., ChemBridge Co., San Diego, Calif.; MCule, Palo Alto, CA; etc.).

[0307] The claimed invention is also intended to encompass salts of any of the compounds of the present invention. The term “salt(s)” as used herein, is understood as being acidic and / or basic salts formed with inorganic and / or organic acids and bases. Zwitterions (internal or inner salts) are understood as being included within the term “salt(s)” as used herein, as are quaternary ammonium salts such as alkylammonium salts. Nontoxic, pharmaceutically acceptable salts are preferred, although other salts may be useful, as for example in isolation or purification steps during synthesis. Salts include, but are not limited to, sodium, lithium, potassium, amines, tartrates, citrates, hydrohalides, phosphates and the like. A salt may be a pharmaceutically acceptable salt, for example. Thus, pharmaceutically acceptable salts of compounds of the present invention are contemplated.

[0308] The term “pharmaceutically acceptable salts,” as used herein, refers to salts of compounds of this invention that are substantially non-toxic to living organisms. Typical pharmaceutically acceptable salts include those salts prepared by reaction of a compound of this invention with an inorganic or organic acid, or an organic base, depending on the substituents present on the compounds of the invention.

[0309] Non-limiting examples of inorganic acids which may be used to prepare pharmaceutically acceptable salts include: hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid and the like. Examples of organic acids which may be used to prepare pharmaceutically acceptable salts include: aliphatic mono- and dicarboxylic acids, such as oxalic acid, carbonic acid, citric acid, succinic acid, phenyl- heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids and the like. Pharmaceutically acceptable salts prepared from inorganic or organic acids thus include hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydrofluoride, acetate, propionate, formate, oxalate, citrate, lactate, p- toluenesulfonate, methanesulfonate, maleate, and the like.299555014.1 - 59 -

[0310] Suitable pharmaceutically acceptable salts may also be formed by reacting the agents of the invention with an organic base such as methylamine, ethylamine, ethanolamine, lysine, ornithine and the like.

[0311] Pharmaceutically acceptable salts include the salts formed between carboxylate or sulfonate groups found on some of the compounds of this invention and inorganic cations, such as sodium, potassium, ammonium, or calcium, or such organic cations as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium.

[0312] Derivatives of compounds of the present invention are also contemplated. In certain aspects, “derivative” refers to a chemically modified compound that still retains the desired effects of the compound prior to the chemical modification. Such derivatives may have the addition, removal, or substitution of one or more chemical moieties on the parent molecule. Non-limiting examples of the types modifications that can be made to the compounds and structures disclosed herein include the addition or removal of lower alkanes such as methyl, ethyl, propyl, or substituted lower alkanes such as hydroxymethyl or aminomethyl groups; carboxyl groups and carbonyl groups; hydroxyls; nitro, amino, amide, and azo groups; sulfate, sulfonate, sulfono, sulfhydryl, sulfonyl, sulfoxido, phosphate, phosphono, phosphoryl groups, and halide substituents. Additional modifications can include an addition or a deletion of one or more atoms of the atomic framework, for example, substitution of an ethyl by a propyl; substitution of a phenyl by a larger or smaller aromatic group. Alternatively, in a cyclic or bicyclic structure, heteroatoms such as N, S, or O can be substituted into the structure instead of a carbon atom.

[0313] Compounds of the present invention may contain one or more asymmetrically- substituted carbon or nitrogen atoms, and may be isolated in optically active or racemic form. Thus, all chiral, diastereomeric, racemic form, epimeric form, and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. Compounds may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some aspects, a single diastereomer is obtained. The chiral centers of the compounds of the present invention can have the S- or the R-configuration, as defined by the IUPAC 1974 Recommendations. Compounds may be of the D- or L-form, for example. It is well known in the art how to prepare and isolate such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic form, normal, reverse-phase, and chiral299555014.1 - 60 -chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from chiral starting materials or by deliberate synthesis of target chiral centers. Compounds of the present invention may occur as a hydrate, a compound containing an equivalent of water in the form of an H2O molecule, or polyhydrate, a compound containing more than one equivalent of water in the form of H2O molecules.

[0314] In addition, atoms making up the compounds of the present invention are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include13C and14C.

[0315] As noted above, compounds of the present invention may exist in prodrug form. As used herein, “prodrug” is intended to include any covalently bonded carriers which release the active parent drug or compounds that are metabolized in vivo to an active drug or other compounds employed in the methods of the invention in vivo when such prodrug is administered to a subject. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.), the compounds employed in some methods of the invention may, if desired, be delivered in prodrug form. Thus, the invention contemplates prodrugs of compounds of the present invention as well as methods of delivering prodrugs. Prodrugs of the compounds employed in the invention may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound.

[0316] Accordingly, prodrugs include, for example, compounds described herein in which a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively. Other examples include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.

[0317] It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and299555014.1 - 61 -use are presented in Handbook of Pharmaceutical Salts: Properties, Selection and Use (2002), which is incorporated herein by reference.Assay ComponentsMolecular Construct

[0318] Embodiments of the disclosure relate to methods and compositions comprising a molecular construct comprising: a small molecule capable of binding to the protein of interest operatively linked to a retrieval tag. The small molecule 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.

[0319] The concentration of the molecular construct in the compositions and methods of the disclosure may be at least, at most, or exactly 1x10-10, 1x10-9, 1x10-8, 1x10-7, 1x10-6, 1x10- 5, 1x10-4, 1x10-3, 1x10-2, 1x10-1, 1, 1x102, 1x103, 1x104, 1x105, 1x106, 1x107, 1x108, 1x109, 1x1010, 1x10-2, 1x10-1, or 1, 1x1010 (or any derivable range therein) pM, nM, pM, mM, M, cM, or dM. In some embodiments, the molecular construct is cell permeable.Targeting Groups (“small molecules capable of binding the protein of interest”)

[0320] 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 function al 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 Km of at least, at most, or exactly 1x10-10, 1x10-9, 1x10-8, 1x10-7, 1x10-6, 1x10-5, 1x10-4, 1x10-3, 1x10-2, 1x10-1, 1, 1x102, 1x103, 1x104, 1x105, 1x106, 1x107, 1x108, 1x109, 1x1010, 1x10-2, 1x10- 1, or 1, 1x1010 (or any derivable range therein) pM, nM, pM, mM, M, cM, or dM.299555014.1 - 62 -

[0321] 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, acylphosphonate, N-hydroxysuccinimide, ester and others can be appended onto kinase- binding small molecule scaffolds and used for ADPL kinase profiling as well.

[0322] Further examples of family-wide activity probes useful in the methods and compositions of the disclosure include:299555014.1 - 63 -299555014.1 -64-299555014.1 -65-299555014.1 -66-Retrieval Tags and Retrieval Tag Binding Component Pairs

[0323] Exemplary retrieval tags and retrieval tag binding component (partner) pairs are shown in the table below. It is contemplated that the retrieval tag and retrieval tag binding component / partner may be interchangeable. For example, a compound identified herein as a299555014.1 - 67 -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.

[0324] 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).Linker

[0325] In order to conjugate two molecules, such as a retrieval tag to a targeting group (“small molecule”) or an oligo to an antibody or to a retrieval tag binding component (“binder”), the following exemplary techniques and linking reagents can be applied.

[0326] 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 groups forming covalent bonds, but also includes chemical groups leading to an ionic interaction or hydrogen bonds with one or more components of the assay.299555014.1 - 68 -

[0327] The functional groups or the linking molecules bearing them may be selected from 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.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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 as phospholipids or derivatised polysaccharides, which can be crosslinked with each other, are useful.299555014.1 - 69 -

[0332] 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).OligosNucleic Acids

[0333] 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 be employed, e.g., where processing by enzymes is called for, usually oligonucleotides consisting of natural nucleotides are required.299555014.1 - 70 -

[0334] 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.

[0335] Specific examples of nucleic acid molecules include nucleic acid molecules containing modified, i.e., non-naturally occurring internucleoside 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.

[0336] Nucleic acid molecules can have one or more modified internucleoside linkages. As defined in this specification, oligonucleotides having modified intemucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside 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.

[0337] Modifications to nucleic acid molecules can include modifications wherein one or both terminal nucleotides is modified.

[0338] 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.299555014.1 - 71 -

[0339] 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.

[0340] Modified oligonucleoside backbones (intemucleoside linkages) that do not include a phosphorus atom therein have intemucleoside linkages that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having amide backbones; and others, including those having mixed N, O, S and CH2 component parts.

[0341] 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.

[0342] 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.

[0343] 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 compounds299555014.1 - 72 -comprising a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; or O-alkyl-O- alkyl, 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.

[0344] One modification that imparts increased nuclease resistance and a very high binding affinity to nucleotides is the 2'-M0E side chain (Baker et al., 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).

[0345] 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.

[0346] 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,299555014.1 - 73 -“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 also 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.

[0347] 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.Barcodes

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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.299555014.1 - 74 -Molecular Labels

[0352] The oligonucleotides, nucleic acid molecules, primers, probes, antibodies, and retrieval tag / binding component molecules in the compositions and methods described herein 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.

[0353] 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.

[0354] 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;299555014.1 - 75 -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; Bisbenzimide (Hoechst); Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-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-299555014.1 - 76 -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; Ly soSensor Green; LysoSensor 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™;299555014.1 - 77 -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; 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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 an299555014.1 - 78 -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.

[0359] 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. 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.Assay MethodsLigation Methods

[0360] Aspects of the disclosure include the ligation of nucleic acids. In some embodiments, the methods include ligating the first oligonucleotide to the second oligo when the first and second oligonucleotides are in close proximity to each other.

[0361] 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.

[0362] 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.299555014.1 - 79 -

[0363] 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.

[0364] 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 circle amplification. 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.Sequencing

[0365] 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.

[0366] Massively parallel signature sequencing (MPSS).

[0367] 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 beadbased 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,299555014.1 - 80 -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.Polony sequencing.

[0368] 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 E. coli genome 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.454 pyrosequencing.

[0369] 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.Illumina (Solexa) sequencing.

[0370] Solexa, now part of Illumina, developed a sequencing method based on reversible dyeterminators 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.299555014.1 - 81 -

[0371] 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.

[0372] 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).SOLiD sequencing.

[0373] 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.299555014.1 - 82 -lon Torrent semiconductor sequencing.

[0374] 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.DNA nanoball sequencing.

[0375] 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.Heliscope single molecule sequencing.

[0376] 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.299555014.1 - 83 -Single molecule real time (SMRT) sequencing.

[0377] 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.]Protein Assays

[0378] 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.

[0379] 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).

[0380] 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, the299555014.1 - 84 -immunoassays of certain aspects can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (1980); and Harlow & Lane, supra).

[0381] 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 as 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.

[0382] 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).

[0383] 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, hybridizationbased 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,299555014.1 - 85 -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), ESI-MS / 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 (SELDI-TOF-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), APCLMS / MS, APCI-(MS)11, atmospheric pressure photoionization mass spectrometry (APPLMS), APPLMS / 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.

[0384] 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.Nucleic Acid Assays

[0385] 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.299555014.1 - 86 -

[0386] 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).

[0387] 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. The 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.

[0388] 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.

[0389] 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 of299555014.1 - 87 -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.

[0390] 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.

[0391] 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.

[0392] 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. It299555014.1 - 88 -is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0393] In one embodiment, quantitative RT-PCR (such as TaqMan, AB I) 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 in 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.

[0394] 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.

[0395] 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 that299555014.1 - 89 -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.

[0396] 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.

[0397] 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 be 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.

[0398] 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.

[0399] 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.

[0400] 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;299555014.1 - 90 -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.Exemplary UtilitiesDiagnostic and Therapeutic methods

[0401] 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. For 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 biological sample from a patient with (a) 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; and (b) an activity probe comprising a small molecule capable of binding to the protein of interest operatively linked to a retrieval tag; (ii) contacting the composition with a retrieval tag recognition element capable of binding to the retrieval tag, wherein the retrieval tag recognition element retrieval tag binding component comprises a second oligonucleotide; (iii) incubating the composition under conditions sufficient for the ligation 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 / or (iv) detecting the ligated or annealed first and second oligonucleotide; or the method comprising: (i) contacting a a biological sample from a patient with (a) an epitope tag binding component capable of binding the epitope tag; and (b) an activity probe comprising a small molecule capable of binding to the protein of interest operatively linked to a retrieval tag; (ii) contacting the composition with an epitope tag recognition element capable of binding the epitope tag binding component, wherein the epitope tag recognition element comprises a299555014.1 - 91 -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 the ligation 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 / or (v) detecting the ligated and / or annealed first and second oligonucleotides.

[0402] 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 to 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.

[0403] 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.

[0404] 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 some299555014.1 - 92 -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.

[0405] 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 testing 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.

[0406] 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.

[0407] 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.

[0408] 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,299555014.1 - 93 -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.

[0409] 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.

[0410] 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.

[0411] 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,299555014.1 - 94 -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 neck 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, nasopharyngeal299555014.1 - 95 -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, urethral cancer, uterine cancer, endometrial uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, childhood vulvar cancer, and wilms tumor (kidney cancer).Drug Discovery and Spatial Detection of Specified Functional Forms of Proteins

[0412] The current methods of the disclosure may also be used to evaluate activity modifiers. 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.Kits

[0413] 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, 43,299555014.1 - 96 -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.

[0414] 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.

[0415] 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. 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 would be in a solution with other components. Concentrations of components may be provided as lx, 2x, 5x, lOx, or 20x or more. Kits for using probes, polypeptide or polynucleotide detecting agents of the disclosure for drug discovery are contemplated. 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.

[0416] 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.299555014.1 - 97 -

[0417] 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.

[0418] 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.

[0419] After review of the disclosure herein of the presently disclosed subject matter, one of ordinary skill in the art can tailor the dosages to an individual subject, taking into account the particular formulation, method of administration to be used with the composition, and nature of the target to be treated. Such adjustments or variations, as well as evaluation of when and how to make such adjustments or variations, are well known to those of ordinary skill in the art.EXAMPLES

[0420] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

[0421] Referring to the schematic in Figure 1, two probe core structures are depicted, an exemplary procedure for the synthesis of compound KW50P is outlined. The initial ethyl 4- (bromomethyl)benzoate can include different substituents on the phenyl ring. In the exemplary procedure depicted in Figure 2, hydrogen atoms are included as aryl ring substituents. Reaction of ethyl 4-(bromomethyl)benzoate with triphenyl phosphine yields the triphenyl phosphine ylide salt. Wittig reaction with the indazole carbaldehyde yields the trans olefin intermediate ester. BOC protection of the -NH- amine followed by demethylation of the ester yields the corresponding acid. Conversion of the carboxylic acid to the NHS ester is then performed, and299555014.1 - 98 -the resulting activated ester is reacted with piperazine dethiobiotin. In the final step, Suzuki coupling with aryl fluorosulfonyl boronic acid yields the final irreversible probe product KW50P. Figure 2 includes a 7H NMR spectra of probe KW50P. LC / MS was used to corroborate the mass of probe KW50P. As seen in Figure 3, the M+l peak of KW50P is 687 g / mol.

[0422] Figure 4 includes chemical structures of compounds XO44-4, XO44-8, and XO44-12. These compounds differ by the length of the linker that links the biotin moiety to the triazole linkr. Figure 5 includes chemical structures of the two compounds, S50P and S52P. An exemplary procedure for the synthesis of irreversible probe compound S50P is depicted in Figure 6. Bromination of indolin-2-one is accomplished using N-bromosuccinimide. Pd(dppf)C12 is used to convert the brominated indolin-2-one intermediate into the corresponding boronate ester. Suzuki coupling between the boronate ester and para-bromo benzenesulfonylfluoride yields an intermediate indolin-2-one benzenesulfonylfluoride. Coupling of mono-BOC-protected piperazine with 5-formyl-2,4-dimethyl-lH-pyrrole-3- carboxylic acid to yield the pyrrole piperazine amide intermediate. This intermediate is reacted with TMS propargyl methylenebromide to yield the TMS-protected propargyl intermediate. Coupling of the TMS-protected propargyl intermediate and the indolin-2-one benzenesulfonylfluoride yields a TMS protected intermediate which is then deprotected to provide irreversible probe compound S50P.

[0423] An exemplary procedure for the synthesis of irreversible probe compound S52P is depicted in Figure 7. Coupling of mono-BOC-protected piperazine with propargyl methylenebromide yields BOC-protected ethylpropargyl piperazine. After removal of the BOC group, the deprotected ethylpropargyl piperazine is coupled with 5-formyl-2,4-dimethyl-lH- pyrrole-3 -carboxylic acid. The resulting product is reacted with 5-hydroxyindolin-2-one, and the resulting product is reacted with 4-(bromomethyl)benzenesulfonyl fluoride to yield irreversible probe compound S52P. Figure 8 includes a H NMR spectra of probe S52P.299555014.1 - 99 -REFERENCESAll references listed herein including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and / or compositions employed herein.1. Netea, M. G. et al. Defining trained immunity and its role in health and disease. Nat. Rev. Immunol. 20, 375-388 (2020).2. Arts, R. J. W. et al. Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity. Cell Metab. 24, 807-819 (2016).3. Netea, M. G. & van Crevel, R. BCG-induced protection: Effects on innate immune memory. Semin. Immunol. 26, 512-517 (2014).4. Aaby, P. et al. Randomized Trial of BCG Vaccination at Birth to Low-Birth-Weight Children: Beneficial Nonspecific Effects in the Neonatal Period J. Infect. Dis. 204, 245-252 (2011).5. Bekkering, S. et al. In Vitro Experimental Model of Trained Innate Immunity in Human Primary Monocytes. Clin. Vaccine Immunol. CVI 23, 926-933 (2016).6. Kleinnijenhuis, J. et al. Bacille Calmette-Guerin induces NOD2-dependent nonspecific protection from reinfection via epigenetic reprogramming of monocytes. Proc. Natl. Acad. Sci. 109, 17537-17542 (2012).7. Cheng, S.-C. et al. mTOR- and HIF-1 -mediated aerobic glycolysis as metabolic basis for trained immunity. Science 345, 1250684-1250684 (2014).8. van der Heijden, C. D. C. C. et al. Aldosterone induces trained immunity: the role of fatty acid synthesis. Cardiovasc. Res. 116, 317-328 (2020).9. van der Heijden, C. D. C. C. et al. Catecholamines Induce Trained Immunity in Monocytes In Vitro and In Vivo. Circ. Res. 127, 269-283 (2020).10. Edgar, L. et al. 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Estimating cell diffusivity and cell proliferation rate by interpreting IncuCyte ZOOMTM assay data using the Fisher-Kolmogorov model. BMC Syst. Biol. 9, 38 (2015).299555014.1 - 101 -21. Ajit, J. et al. Temporal Control of Trained Immunity via Encapsulated Release of P-Glucan Improves Therapeutic Applications. Adv. Healthc. Mater. 11, 2200819 (2022).22. Ciarlo, E. et al. Trained Immunity Confers Broad- Spectrum Protection Against Bacterial Infections. J. Infect. Dis. 222, 1869-1881 (2020).23. Oxidized Low-Density Lipoprotein Induces Long-Term Proinflammatory Cytokine Production and Foam Cell Formation via Epigenetic Reprogramming of Monocytes | Arteriosclerosis, Thrombosis, and Vascular Biology. https: / / www.ahajournals.org / doi / 10.1161 / ATVBAHA.114.303887.24. Marodi, L. Innate cellular immune responses in newborns. Clin. Immunol. 118, 137-144 (2006).25. Montecino-Rodriguez, E., Berent-Maoz, B. & Dorshkind, K. Causes, consequences, and reversal of immune system aging. J. Clin. Invest. 123, 958-965 (2013).26. Chan, G. C.-F., Chan, W. K. & Sze, D. M.-Y. The effects of P-glucan on human immune and cancer cells. J. Hematol. Oncol. J Hematol Oncol 2, 25 (2009).27. Hesseling, A. C. et al. The risk of disseminated Bacille Calmette-Guerin (BCG) disease in HIV-infected children. Vaccine 25, 14-18 (2007).28. Norouzi, S., Aghamohammadi, A., Mamishi, S., Rosenzweig, S. D. & Rezaei, N. Bacillus Calmette-Guerin (BCG) complications associated with primary immunodeficiency diseases. J. Infect. 64, 543-554 (2012).29. Netea, M. G. & Meer, J. W. M. van der. Trained Immunity: An Ancient Way of Remembering. Cell Host Microbe 21, 297-300 (2017).30. Mulder, W. J. M., Ochando, J., Joosten, L. A. B., Fayad, Z. A. & Netea, M. G. Therapeutic targeting of trained immunity. Nat. Rev. Drug Discov. 18, 553-566 (2019).31. Benjamini, Y; Krieger, A.M.; Yekutieli, D. Adaptive linear step-up procedures that control the false discovery rate. Biometrika 93(3), 491-507 (2006).32. Bruey, J.-M. et al. Bcl-2 and Bcl-XL Regulate Proinflammatory Caspase-1 Activation by Interaction with NALP1. Cell 129, 45-56 (2007).299555014.1 - 102 -33. Badrichani, A. Z. et al. Bcl-2 and Bcl-XL serve an anti-inflammatory function in endothelial cells through inhibition of NF-KB. J Clin Invest 103, 543-553 (1999).34. Gimenez-Cassina, A. & Danial, N. N. Regulation of mitochondrial nutrient and energy metabolism by BCL-2 family proteins. Trends in Endocrinology & Metabolism 26, 165-175 (2015).35. Yi, C. H. et al. Metabolic Regulation of Protein N-Alpha-Acetylation by BclxL Promotes Cell Survival. Cell 146, 607-620 (2011).36. Vander Heiden, M. G. et al. Bcl-xl Promotes the Open Configuration of the Voltagedependent Anion Channel and Metabolite Passage through the Outer Mitochondrial Membrane. Journal of Biological Chemistry 276, 19414-19419 (2001).37. Chen, Y. et al. Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential. Journal of Cell Biology 195, 263-276 (2011).38. Tao, Z.-F. et al. Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity. ACS Med Chem Lett 5, 1088-1093 (2014).39. Mitroulis, I. et al. Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity. Cell 172, 147-161. el2 (2018).40. B. A. Bailey, L. J. Ochyl, S. P. Schwendeman, J. J. Moon, Adv. Healthcare Mater. 2017, 6, 1601418.It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.299555014.1 - 103 -

Claims

CLAIMSWhat is claimed is:

1. A compound of formula I:wherein:A is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom;Ri, R2, R3, and R4 are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide;Rs, Re, R7, and Rs are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide, wherein one of Rs, Re, R7, and Rs is optionally replaced by X; wherein X comprises, consists of, or consists essentially of a phenyl ring comprising a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the indazole phenyl ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an299555014.1 - 104 -alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof;Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-2- one, N-hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the piperazine ring through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof.

2. The compound of claim 1, wherein an aldehyde group is protected as a 1,3 -di oxolane.

3. The compound of claim 1, wherein the compound is further defined as a compound of formula IE4. The compound of claim 1, wherein the compound is further defined as a compound of formula III:299555014.1 - 105 -5. The compound of claim 1, wherein the compound is further defined as a compound of formula IV:(IV).

6. The compound of claim 1, wherein the compound is further defined as a compound of formula V:

7. The compound of claim 1, wherein the compound is further defined as a compound of formula VI:299555014.1 - 106 -(VI).

8. The compound of claim 1, wherein the compound is further defined as:

9. The compound of claim 1, wherein the compound is further defined as:

10. The compound of claim 1, wherein the compound is further defined as:299555014.1 - 107 -11. The compound of claim 1, wherein the compound is further defined as:

12. The compound of claim 1, wherein the compound is further defined as:299555014.1 - 108 -13. The compound of claim 1, wherein the compound is further defined as:whereinA is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom;Ri and R2 are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide;R3, R4, Rs, and Re are each independently hydrogen, halogen, hydroxyl, alkoxy, thiol, thioalkyl, ester, ether, epoxide, amine, amide, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, amine, alkylamine, sulfoxide, sulfone, sulfinic acid, sulfonic acid, sulfonate ester, sulfonyl, phosphine, phosphate, phosphate ester, aldehyde, carboxylic acid, nitrate, nitrite, nitrile, isocyanate, or azide, wherein one of R3, R4, Rs, and Re is optionally replaced by X;299555014.1 - 109 -wherein X comprises, consists of, or consists essentially of a phenyl ring comprising a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the indolin-2-one phenyl ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof;Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-2- one, N-hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the piperazine ring through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof.

15. The compound of claim 14, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that reversibly covalently bind to the amino acid side chain moiety or epitope tag.

16. The compound of claim 15, wherein the moiety that reversibly covalently binds to an amino acid side chain moiety or epitope tag is an aldehyde.

17. The compound of claim 16, wherein the aldehyde is protected as a 1,3 -di oxolane.299555014.1 - 110 -18. The compound of claim 14, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that irreversibly covalently bind to the amino acid side chain moiety or epitope tag.

19. The compound of claim 18, wherein the moiety that irreversibly covalently binds to an amino acid side chain moiety or epitope tag is hydroxyl, fluorosulfonyl, epoxide, substituted or unsubstituted benzoate ester, ethenesulfonyl, sulfurofluoridate, ethenesulfonate, fluorophosphonate, or ethenephosphonate.

20. The compound of claim 14, wherein the amino acid side chain moiety or epitope tag comprises a moiety that comprises, consists of, or consists essentially of a hydroxyl group, an amine group, or a thiol group.

21. The compound of claim 14, wherein the compound is further defined as a compound of formula VIII:VIII.

22. The compound of formula 14, wherein the compound is one of:299555014.1 - 111 -23. A method for evaluating a protein of interest, the method comprising(i) contacting a composition comprising a protein of interest fused to an epitope tag with a compound of claim 1;(ii) contacting the composition with a retrieval tag recognition element capable of binding to moiety Z of the compound of claim 1, wherein the retrieval tag recognition element comprises a second oligonucleotide;(iii) incubating the composition under conditions sufficient for: the epitope tag to covalently bind to the moiety that covalently binds to the epitope tag of the compound of claim 1, and for the retrieval tag recognition element to covalently bind to moiety Z of the compound of claim 1;(iv) detecting a complex comprising the protein of interest, the compound of claim 1, and the retrieval tag recognition element, wherein the protein of interest is covalently bound to the compound of claim 1 and the retrieval tag recognition element is covalently bound to the compound of claim 1.

24. The method of claim 23, wherein the protein of interest and / or the retrieval tag each independently comprise a ligated or annealed oligonucleotide.

25. The method of claim 24 or 25, wherein the protein of interest is in a cell.

26. The method of any one of claims 23-25, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialize functional form.

27. The method of claim 26, wherein the specialized functional form is an active form of the protein of interest.299555014.1 - 112 -28. The method of any of claims 23-27, wherein the detecting the ligated or annealed oligonucleotide comprises imaging, qPCR or a sequencing based readout.

29. The method of any of claims 23-28, wherein the method further comprising determining an abundance of the target protein of interest.

30. The method of claim 29, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

31. The method of claim 29 or 30, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed oligonucleotide.

32. The method of any of claims 23-31, wherein the steps are performed in chronological order.

33. The method of any one of claims 23-32, 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.

34. The method of any one of claim 23-33, wherein the method further comprises performing rolling circle amplification after the last step.

35. The method of any one of claims 23-34, 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 oligonucleotide or at least one of the one or more bridging oligonucleotides.

36. The method of claim 35, wherein the label comprises a florescent protein, a pull down tag or a split recognition system.

37. The method of any one of claims 23-36, 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.299555014.1 - US -38. The method of claim 37, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

39. The method of claim 37 or 38, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

40. The method of any one of claims 23-39, wherein first and the second oligonucleotides are capable of ligation and / or and annealing when in sufficient proximity to each other.

41. The method of any one of claims 23-40, wherein the method further comprises spatially detecting the target protein of interest.

42. The method of any one of claims 23-41, wherein the composition comprises less than 5,000 cells.

43. The method of any one of claim 23-42, wherein the composition comprises less than 1 pg of total protein.

44. The method of any one of claims 23-43, wherein the method further comprises detection of a cellular marker.

45. The method of any one of claims 23-44, wherein the method further comprises determining a total amount of target protein of interest.

46. The method of claim 45, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

47. The method of claim 45 or 46, wherein 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.

48. The method of any one of claims 23-47, wherein the method excludes one or more of liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry.

49. The method of any one of claims 23-48, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are selected from one or more of the following retrieval tag and retrieval tag binding299555014.1 - 114 -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 transcyclooctene, and an epitope and an epitope-specific antibody.

50. The method of any one of claims 23-49, wherein the first and / or second oligonucleotide are single stranded.

51. The method of any one of claims 23-50, wherein the first and / or second oligonucleotide comprises a DNA barcode.

52. The method of any one of claims 23-51, wherein the protein of interest is an enzyme.

53. The method of any one of claims 23-52, wherein the protein of interest is a serine hydrolase, cysteine proteases, kinases, metalloproteases, P-retaining glycosidases, tyrosine phosphatases, or cytochrome P450s,54. The method of any one of claims 23-53, wherein the protein of interest is a DNA repair enzyme.

55. The method of any one of claims 23-54, wherein the protein of interest is a non-enzyme.

56. The method of any one of claims 23-55, the epitope tag comprises a florescent protein, a pull down tag or a split recognition system.

57. The method of claim 56, wherein the florescent protein comprises GFP, RFP, or mCherry.

58. The method of claim 56, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

59. The method of claim 56, wherein the split recognition system comprises a spy tag.

60. The method of any one of claims 54-59, wherein the epitope tag is fused to the protein of interest by genetic manipulation.

61. The method of any one of claims 54-60, wherein protein of interest fused to the epitope tag is a recombinant protein.299555014.1 - 115 -62. The method of any one of claims 54-61, wherein the recombinant protein is introduced into a cell by transfection.

63. The method of claim 62, wherein the cell is a live cell64. The method of any one of claims 54-63, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

65. The method of any one of claims 23-64, wherein the protein of interest is a serine hydrolase and the compound of claim 1 comprises an fluorophosphonate.

66. The method of any one of claims 23-65, wherein the protein of interest is a kinase and the compound of claim 1 comprises a fluorosulfonyl.

67. The method of any one of claims 23-66, wherein the protein of interest is a cysteine protease and the compound of claim 1 comprises one of ethenesulfonyl, ethenesulfonate, ethenephosphonate, epoxide, and substituted or unsubstituted benzoate ester.

68. The method of any one of claims 23-67, wherein the retrieval tag comprises an orthogonal recognition element.

69. The method of any one of claims 23-68, 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 epitopespecific antibody.

70. The method of any one of claims 23-68, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

71. The method of claim 70, wherein the specialized functional form is an active form.

72. The method of any one of claims 23-69, wherein the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form.

73. The method of claim 72, wherein the non-specialized functional form is an inactive form.

74. The method of any one of claims 23-73, wherein the system further comprises a nonactivity probe capable of binding the protein of interest in non-specialized functional form.299555014.1 - 116 -75. A method for evaluating a protein of interest, the method comprising(i) contacting a composition comprising a protein of interest fused to an epitope tag with a compound of claim 14;(ii) contacting the composition with a retrieval tag recognition element capable of binding to moiety Z of the compound of claim 14, wherein the retrieval tag recognition element comprises a second oligonucleotide;(iii) incubating the composition under conditions sufficient for: the epitope tag to covalently bind to the moiety that covalently binds to the epitope tag of the compound of claim 14, and for the retrieval tag recognition element to covalently bind to moiety Z of the compound of claim 14;(iv) detecting a complex comprising the protein of interest, the compound of claim 1, and the retrieval tag recognition element, wherein the protein of interest is covalently bound to the compound of claim 14 and the retrieval tag recognition element is covalently bound to the compound of claim 14.

76. The method of claim 75, wherein the protein of interest and / or the retrieval tag each independently comprise a ligated or annealed oligonucleotide.

77. The method of claim 76 or 77, wherein the protein of interest is in a cell.

78. The method of any one of claims 75-77, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialize functional form.

79. The method of claim 78, wherein the specialized functional form is an active form of the protein of interest.

80. The method of any of claims 75-79, wherein the detecting the ligated or annealed oligonucleotide comprises imaging, qPCR or a sequencing based readout.

81. The method of any of claims 75-80, wherein the method further comprising determining an abundance of the target protein of interest.

82. The method of claim 81, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.299555014.1 - 117 -83. The method of claim 81 or 82, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed oligonucleotide.

84. The method of any of claims 75-83, wherein the steps are performed in chronological order.

85. The method of any one of claims 75-84, 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.

86. The method of any one of claim 75-85, wherein the method further comprises performing rolling circle amplification after the last step.

87. The method of any one of claims 75-86, 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 oligonucleotide or at least one of the one or more bridging oligonucleotides.

88. The method of claim 87, wherein the label comprises a florescent protein, a pull down tag or a split recognition system.

89. The method of any one of claims 75-88, 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.

90. The method of claim 89, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

91. The method of claim 89 or 90, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

92. The method of any one of claims 75-91, wherein first and the second oligonucleotides are capable of ligation and / or and annealing when in sufficient proximity to each other.299555014.1 - US -93. The method of any one of claims 75-92, wherein the method further comprises spatially detecting the target protein of interest.

94. The method of any one of claims 75-93, wherein the composition comprises less than 5000 cells.

95. The method of any one of claim 75-94, wherein the composition comprises less than 1 pg of total protein.

96. The method of any one of claims 75-95, wherein the method further comprises detection of a cellular marker.

97. The method of any one of claims 75-96, wherein the method further comprises determining a total amount of target protein of interest.

98. The method of claim 97, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

99. The method of claim 97 or 98, wherein 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.

100. The method of any one of claims 75-99, wherein the method excludes one or more of liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry.

101. The method of any one of claims 75-100, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are selected from 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, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a transcyclooctene, and an epitope and an epitope-specific antibody.

102. The method of any one of claims 75-101, wherein the first and / or second oligonucleotide are single stranded.299555014.1 - 119 -103. The method of any one of claims 75-102, wherein the first and / or second oligonucleotide comprises a DNA barcode.

104. The method of any one of claims 75-103, wherein the protein of interest is an enzyme.

105. The method of any one of claims 75-102, wherein the protein of interest is a serine hydrolase, cysteine proteases, kinases, metalloproteases, P-retaining glycosidases, tyrosine phosphatases, or cytochrome P450s,106. The method of any one of claims 75-105, wherein the protein of interest is a DNA repair enzyme.

107. The method of any one of claims 75-106, wherein the protein of interest is a non-enzyme.

108. The method of any one of claims 75-107, the epitope tag comprises a florescent protein, a pull down tag or a split recognition system.

109. The method of claim 108, wherein the florescent protein comprises GFP, RFP, or mCherry.

110. The method of claim 108, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

111. The method of claim 108, wherein the split recognition system comprises a spy tag.

112. The method of any one of claims 75-59, wherein the epitope tag is fused to the protein of interest by genetic manipulation.

113. The method of any one of claims 75-112 wherein protein of interest fused to the epitope tag is a recombinant protein.

114. The method of any one of claims 75-113, wherein the recombinant protein is introduced into a cell by transfection.

115. The method of claim 114, wherein the cell is a live cell116. The method of any one of claims 75-115, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.299555014.1 - 120 -117. The method of any one of claims 75-116, wherein the protein of interest is a serine hydrolase and the compound of claim 14 comprises an fluorophosphonate.

118. The method of any one of claims 75-117, wherein the protein of interest is a kinase and the compound of claim 14 comprises a fluorosulfonyl.

119. The method of any one of claims 75-118, wherein the protein of interest is a cysteine protease and the compound of claim 14 comprises one of ethenesulfonyl, ethenesulfonate, ethenephosphonate, epoxide, and substituted or unsubstituted benzoate ester.

120. The method of any one of claims 75-119, wherein the retrieval tag comprises an orthogonal recognition element.

121. The method of any one of claims 75-120, 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 epitopespecific antibody.

122. The method of any one of claims 75-121, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

123. The method of claim 122, wherein the specialized functional form is an active form.

124. The method of any one of claims 75-123, wherein the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form.

125. The method of claim 124, wherein the non-specialized functional form is an inactive form.

126. The method of any one of claims 75-125, wherein the system further comprises a nonactivity probe capable of binding the protein of interest in non-specialized functional form.

127. A compound of formula IX:299555014.1 - 121 -wherein:A is oxygen or two hydrogen atoms, wherein each hydrogen atom is covalently bound to the benzylic carbon atom; wherein X comprises, consists of, or consists essentially of a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; wherein X is optionally linked to the piperazine ring through a linker Li; wherein Li is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof;Z comprises, consists of, or consists essentially of biotin, desthiobiotin, imidazoline-2-one, N- hydroxysuccinimide (NHS), maleimide, alkyne, azide, imidiester, haloacetyl, pyridyl sulfide, hydrazide, alkoxyamine, diazirine, tetrazine, O6-benzylguanine, a reporter, or a retrieval tag; wherein Z is optionally linked to the amine through a linker L2; and wherein L2 is a linker selected from the group comprising, consisting of, or consisting essentially of a triazole linker, a glycol linker, a polyethylene glycol (PEG) linker, an alkyl linker, an aryl linker, a heteroalkyl linker, a heteroatom linker, an alkylene linker, an amide linker, an ester linker, a thioester linker, an ether linker, a thioether linker, a phosphoester linker, a phosphoramide linker, an anhydride linker, a disulfide linker, or a combination thereof, wherein L2 optionally comprises a moiety that covalently binds to an amino acid side chain moiety or an epitope tag; or an enantiomer, diastereomer, racemic mixture, or salt thereof.

128. The compound of claim 127, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that reversibly covalently bind to the amino acid side chain moiety or epitope tag.299555014.1 - 122 -129. The compound of claim 128, wherein the moiety that reversibly covalently binds to an amino acid side chain moiety or epitope tag is an aldehyde.

130. The compound of claim 129, wherein the aldehyde is protected as a 1,3 -di oxolane.

131. The compound of claim 127, wherein the moiety that covalently binds to an amino acid side chain moiety or epitope tag is a moiety that irreversibly covalently bind to the amino acid side chain moiety or epitope tag.

132. The compound of claim 130, wherein the moiety that irreversibly covalently binds to an amino acid side chain moiety or epitope tag is hydroxyl, epoxide, fluorosulfonyl, ethenesulfonyl, substituted or unsubstituted benzoate ester, sulfurofluoridate, ethenesulfonate, fluorophosphonate, or ethenephosphonate.

133. The compound of claim 127, wherein the amino acid side chain moiety or epitope tag comprises a moiety that comprises, consists of, or consists essentially of a hydroxyl group, an amine group, or a thiol group.

134. The compound of claim 127, wherein the compound is one of:299555014.1 - 123 -135. A method for evaluating a protein of interest, the method comprising(i) contacting a composition comprising a protein of interest fused to an epitope tag with a compound of claim 127;(ii) contacting the composition with a retrieval tag recognition element capable of binding to moiety Z of the compound of claim 127, wherein the retrieval tag recognition element comprises a second oligonucleotide;(iii) incubating the composition under conditions sufficient for: the epitope tag to covalently bind to the moiety that covalently binds to the epitope tag of the compound of claim 127, and for the retrieval tag recognition element to covalently bind to moiety Z of the compound of claim 127;(iv) detecting a complex comprising the protein of interest, the compound of claim 127, and the retrieval tag recognition element, wherein the protein of interest is299555014.1 - 124 -covalently bound to the compound of claim 127 and the retrieval tag recognition element is covalently bound to the compound of claim 127.

136. The method of claim 135, wherein the protein of interest and / or the retrieval tag each independently comprise a ligated or annealed oligonucleotide.

137. The method of claim 135 or 136, wherein the protein of interest is in a cell.

138. The method of any one of claims 135-137, wherein the evaluating comprises evaluating activity, wherein the target protein of interest is in a specialize functional form.

139. The method of claim 138, wherein the specialized functional form is an active form of the protein of interest.

140. The method of any of claims 135-139, wherein the detecting the ligated or annealed oligonucleotide comprises imaging, qPCR or a sequencing based readout.

141. The method of any of claims 135-140, wherein the method further comprising determining an abundance of the target protein of interest.

142. The method of claim 141, wherein determining the abundance of the target protein of interest comprises PCR amplification of the ligated and / or annealed first and second oligonucleotides.

143. The method of claim 141 or 142, wherein determining the abundance of the target protein of interest comprises primer extension and / or PCR amplification of the ligated and / or annealed oligonucleotide.

144. The method of any of claims 135-143, wherein the steps are performed in chronological order.

145. The method of any one of claims 135-144, 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.

146. The method of any one of claim 135-145, wherein the method further comprises performing rolling circle amplification after the last step.299555014.1 - 125 -147. The method of any one of claims 135-146, 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 oligonucleotide or at least one of the one or more bridging oligonucleotides.

148. The method of claim 146, wherein the label comprises a florescent protein, a pull down tag or a split recognition system.

149. The method of any one of claims 135-148, 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.

150. The method of claim 149, wherein the first labeled primer and / or second labeled primer is complementary to a barcode.

151. The method of claim 149 or 150, wherein the first labeled primer and second labeled primer are labeled with differentially detectable molecular labels.

152. The method of any one of claims 135-151, wherein first and the second oligonucleotides are capable of ligation and / or and annealing when in sufficient proximity to each other.

153. The method of any one of claims 135-152, wherein the method further comprises spatially detecting the target protein of interest.

154. The method of any one of claims 135-153, wherein the composition comprises less than 5,000 cells.

155. The method of any one of claim 135-154, wherein the composition comprises less than 1 pg of total protein.

156. The method of any one of claims 135-155, wherein the method further comprises detection of a cellular marker.

157. The method of any one of claims 135-156, wherein the method further comprises determining a total amount of target protein of interest.299555014.1 - 126 -158. The method of claim 157, wherein determining the total amount of target protein of interest comprises directly or indirectly detecting the mRNA transcript of the target protein of interest.

159. The method of claim 157 or 158, wherein 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.

160. The method of any one of claims 135-159, wherein the method excludes one or more of liquid chromatography-mass spectrometry, mass cytometry, imaging mass spectrometry, and mass spectrometry.

161. The method of any one of claims 135-160, wherein the retrieval tag recognition element comprises a retrieval tag binding component, and the retrieval tag and / or retrieval tag binding component are selected from 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, O6-benzylguanine and SNAP protein, an alkyne and an azide, an azide and a cyclooctyne, a tetrazine and a transcyclooctene, and an epitope and an epitope-specific antibody.

162. The method of any one of claims 135-161, wherein the first and / or second oligonucleotide are single stranded.

163. The method of any one of claims 135-162, wherein the first and / or second oligonucleotide comprises a DNA barcode.

164. The method of any one of claims 135-163, wherein the protein of interest is an enzyme.

165. The method of any one of claims 135-164, wherein the protein of interest is a serine hydrolase, cysteine proteases, kinases, metalloproteases, P-retaining glycosidases, tyrosine phosphatases, or cytochrome P450s,166. The method of any one of claims 135-165, wherein the protein of interest is a DNA repair enzyme.

167. The method of any one of claims 135-166, wherein the protein of interest is a non-enzyme.299555014.1 - 127 -168. The method of any one of claims 135-167, the epitope tag comprises a florescent protein, a pull down tag or a split recognition system.

169. The method of claim 168, wherein the florescent protein comprises GFP, RFP, or mCherry.

170. The method of claim 168, wherein the pull down tag comprises GST, a flag, MBP, His, or HA.

171. The method of claim 168, wherein the split recognition system comprises a spy tag.

172. The method of any one of claims 166-171, wherein the epitope tag is fused to the protein of interest by genetic manipulation.

173. The method of any one of claims 166-172, wherein protein of interest fused to the epitope tag is a recombinant protein.

174. The method of any one of claims 166-173, wherein the recombinant protein is introduced into a cell by transfection.

175. The method of claim 174, wherein the cell is a live cell176. The method of any one of claims 135-174, wherein the epitope tag binding component comprises an antibody, nanobody, scFv, recombinant protein, or aptamer.

177. The method of any one of claims 135-174, wherein the protein of interest is a serine hydrolase and the compound of claim 127 comprises an fluorophosphonate.

178. The method of any one of claims 135-174, wherein the protein of interest is a kinase and the compound of claim 127 comprises a fluorosulfonyl.

179. The method of any one of claims 166-174, wherein the protein of interest is a cysteine protease and the compound of claim 127 comprises one of ethenesulfonyl, ethenesulfonate, ethenephosphonate, epoxide, and substituted or unsubstituted benzoate ester.

180. The method of any one of claims 135-179, wherein the retrieval tag comprises an orthogonal recognition element.299555014.1 - 128 -181. The method of any one of claims 135-180, 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 epitopespecific antibody.

182. The method of any one of claims 135-180, wherein the activity probe binds to the protein of interest when the protein of interest is in a specialized functional form.

183. The method of claiml81, wherein the specialized functional form is an active form.

184. The method of any one of claims 135-181, wherein the activity probe does not bind to the protein of interest when the protein of interest is in non-specialized functional form.

185. The method of claim 184, wherein the non-specialized functional form is an inactive form.

186. The method of any one of claims 135-185, wherein the system further comprises a nonactivity probe capable of binding the protein of interest in non-specialized functional form.299555014.1 - 129 -

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