WO2003079014A1 - Tethered activity-based probes and uses thereof - Google Patents

Abstract

Methods and compositions are described for analyzing complex protein mixtures, such as proteomes, using activity-based probes. In particular, probes that specifically react with and bind to the active form of one or more target proteins are employed. Labeled proteins and/or peptides can be sequestered using a tABP covalently bound (“tethered”) to a solid phase, and, upon cleavage of the tether, can be related to the identity, presence, amount, or activity of active members of the desired target protein class. The methods and compositions described herein can be used, for example, to provide diagnostic information concerning pathogenic states, in identifying proteins that may act as therapeutic targets, and in drug discovery.

Description

TETHERED ACTIVITY-BASED PROBES AND USES THEREOF

FIELD OF THE INVENTION

[0001] The field of this invention is analysis of complex protein mixtures, such as proteomes, and more specifically, to tethered activity-based probes and methods for using such probes.

BACKGROUND

[0002] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

[0003] The biological revolution has progressed from genomics to proteomics as the newest frontier for gathering information concerning cellular physiology. Transcription of DNA is an early step in an extended process resulting ultimately in the expression of genomic information as a functional protein. Additional steps can include processing of the initial transcript to mRNA, translation of the mRNA into protein, and posttranslational processing of the protein (e.g., cleavage of the protein into smaller fragments, modification of the protein by glycosylation, methylation, acylation, phosphorylation, etc.). In addition, the protein may be activated or deactivated by interaction with other proteins and/or with small molecules (e.g., cofactors). Regulation of active protein expression can occur at one or more of these steps, and the amount of active protein in the cell at any time will vary widely with the state of the protein in the cell. Thus, the presence of a given gene in a cell's genome, or the total amount of a particular protein in a cell is not necessarily a good prognosticator of the state of the cell, as reflected by the amount of active protein present in the cell.

[0004] h evaluating candidate drugs, the readout should provide an indication how the drug will perform in vivo. For example, an accurate evaluation of a candidate drug can be obtained by using the drug in vivo and determining the effect of the drug on the indication and/or absorption, distribution, metabolism, and excretion ("ADME") studies performed. However, where there are a large number of candidates as are available today from combinatorial libraries, and natural and other sources, substitute procedures must be available to allow for reducing the number of candidate drugs to be studied. To allow for large numbers of candidate drugs to be evaluated, while having reasonable costs and times involved, cellular surrogates are finding use. One can expose cells in culture to candidate drugs. The question then is what should be analyzed to obtain the greatest amount of accurate information relevant to the effect of the drug in the most expedient way.

[0005] There is substantial interest in providing platforms that will provide answers to questions asked about the effect of candidate drugs on cells, tissues, and/or organisms, hi order for platforms to be useful they should be efficient, reliable, and economic, and maximize the information provided and the predictive capability of the results. The ability to analyze samples in parallel and the reproducibility, speed, automation, sensitivity, and specificity of the analysis procedures can all contribute to maximizing the efficiency and reliability of such a platform.

[0006] Numerous methods have been described for analyzing protein compositions. Some typical examples include WO 00/11208, which discusses mass spectrometric methods for analysis of proteins; Cravatt and Sorenson, Current Opinion in Chemical Biology (2000) 4(6): 663-668, which discusses chemical strategies for analyzing protein function; U.S. Patent No. 4,433,051, which discusses the use of α-difluoromethylornithine for use in protein analysis; U.S. Patent No. 6,127,134, which discusses difference gel electrophoresis using matched multiple dyes; Gygi et al., Proc. Natl. Acad. Sci. USA (2000) 97:9390-5, which discusses the use of 2D gel electrophoresis in conjunction with mass spectrometry to analyze yeast proteins; and Aebersold et al., PCT/US99/19415, which discusses digestion of labeled protein samples.

[0007] Complex protein mixtures, such as proteomes, can be difficult to analyze. Not only are there many components in the mixtures, but as samples of these mixtures may be processed, many artifacts can be introduced into the sample, e.g., by hydrolysis of amide bonds, deamination, oxidation and the like. In addition, proteins present in the mixture that originally derived from the same polypeptide sequence may have been subject to differential processing reactions, such as glycosylations, prenylations, etc. Moreover, in analysis procedures in which the proteins in a complex mixture are subject to proteolysis, the total number of components is greatly increased in comparison to the original sample. As a result, numerous fractions in a chromatography procedure or bands in an electrophoretic gel can be related to a single protein in the original sample, greatly complicating the interpretation of the data. It is of interest to find ways to simplify the compositions that are being analyzed to permit a more accurate and robust inteφretation of the observed results.

SUMMARY OF THE INVENTION

[0008] Compositions and methods are provided for the identification of active proteins in a complex protein mixture (e.g., a proteomic mixture), h various aspects, the present invention relates to methods for combining one or more complex protein mixture(s) with tethered activity-based probes ("tABPs") to produce covalent conjugates of the active target proteins with the probes. The activity-based probes comprise a "warhead" (defined hereinafter) directed to various proteins, covalently linked to a solid support. The covalent linkage of the tABP(s) to the solid phase may take place either prior to or after contacting the tABP(s) with the complex protein mixture; in either case, the result is that the tABP -bound active target proteins become tethered to the solid phase via a covalent linkage. Preferably, one or more covalent bonds within the tABPs or between the tABP(s) and the solid phase are cleavable, so that active target proteins that react with the tABP may ultimately be released from the solid support. Additionally, the tABPs preferably comprise a ligand, which may be, directly or indirectly, detectable, e.g. by fluorescence, and which may be used for separation and/or detection.

[0009] Following reaction of the complex protein mixture with one or more tABPs, the resulting protein conjugates may be proteolytically digested to provide probe-labeled peptides. This digestion may occur while the protein conjugates are tethered to the solid phase, or following release by cleavage of the tABP. In preferred embodiments, tABPs are selected such that each active target protein forms a conjugate with a single tABP, most preferably at a single discrete location in the target protein; thus, each conjugate gives rise to a single tABP-labeled peptide. Enrichment separation, or identification of one or more tABP-labeled peptides may be achieved using liquid chromatography and/or electrophoresis. Additionally, mass spectrometry may be employed to identify one or more tABP-labeled peptides by molecular weight and/or amino acid sequence, i particularly preferred embodiments, the sequence information derived from of the tABP-labeled peptide(s) is used to identify the protein from which the peptide originally derived. Variations of these aspects can involve the comparison of two or more proteomes, e.g., with tABPs having different ligands, or, when analysis comprises mass spectrometry, having different isotopic compositions.

[0010] Thus, in a first aspect, the invention relates to methods for determining the presence amount, or activity of one or more active target proteins in a complex protein mixture, most preferably a proteome. In various embodiments, these methods comprise one or more of the following steps: contacting a complex protein mixture with one or more tABPs that specifically binds to one or more active target proteins present in the complex protein mixture and that are or become covalently bound to a solid phase, thereby sequestering the tABP-bound active target proteins to a solid phase; removing one or more non-sequestered components of the complex protein mixture, e.g., by washing the solid phase; and cleaving the tABP(s) to provide tABP- labeled active target protein(s) that may be further analyzed by the screening and/or identification methods described hereinafter.

[0011] h a related aspect, the methods described herein comprise contacting a complex protein mixture with one or more tABPs that specifically binds to one or more active target proteins present in the complex protein mixture and that are or become covalently bound to a solid phase, thereby sequestering the tABP-bound active target proteins to a solid phase; removing one or more non-sequestered components of the complex complex protein mixture, e.g., by washing the solid phase; and cleaving the tABP(s), wherein either before or after cleavage of the tABP, the tABP-labeled active target proteins are proteolyzed to produce a product mixture comprising tABP-labeled peptide(s). [0012] In either aspect, a signal generated from those active target proteins, or peptides thereof, bound to the tABP can be correlated to the presence, amount, or activity of one or more active target proteins present in the original complex protein mixture.

[0013] hi preferred embodiments, tABP(s) and reaction conditions are selected such that the relative ability of an active target protein to become labeled depends on the relative level of activity of that active target protein; the signal obtained from such labeling can be correlated to the activity of the active target proteins in the proteomic mixture. Alternatively, tABPs can be used under conditions in which all active forms of an active target protein are labeled, regardless of the level of activity of the particular active target protein. For example, the time of reaction may be extended so that the labeling reaction goes substantially to completion; the signal obtained from such labeling will be unrelated to the activity of the active target proteins in the proteomic mixture.

[0014] Similarly, in another aspect, the instant invention relates to methods for comparing the presence or amount of one or more active target proteins in two or more complex protein mixtures. In various embodiments, these methods comprise one or more of the following steps: contacting one or more complex protein mixture(s) with one or more tABPs, where the lABP(s) specifically bind to one or more active target proteins present in each complex protein mixture and are or become covalently bound to a solid phase, thereby sequestering the tABP-bound active target proteins to a solid phase; combining the complex protein mixtures following this contacting step to form a combined complex protein mixture; prior to and/or following this combination, removing one or more non-sequestered components of the complex protein mixture(s); and cleaving the tABP(s) to provide tABP-labeled active target proteins that may be further analyzed by the screening and/or identification methods described hereinafter.

[0015] In a related aspect, the methods described herein comprise contacting one or more complex protein mixture(s) with one or more tABPs, where the lABP(s) specifically bind to one or more active target proteins present in each complex protein mixture and are or become covalently bound to a solid phase, thereby sequestering the tABP-bound active target proteins to a solid phase; combining the complex protein mixtures following this contacting step to form a combined complex protein mixture; prior to and/or following this combination, removing one or more non-sequestered components of the complex protein mixrure(s); and cleaving the tABP(s), wherein either before or after cleavage of the tABP(s), the tABP-labeled active target proteins are proteolyzed, to produce one or more product mixtures comprising tABP-labeled peptides.

[0016] In preferred embodiments of the above aspects, the tABP(s) comprise a ligand that can be used to identify those tABP-labeled peptides originating from a particular original complex protein mixture (e.g. two tABPs comprising the same warhead but different fluorescent moieties can be used to label two different complex protein mixtures or two tABPs comprising the same warhead but differing isotopic compositions that can be distinguished by mass spectrometry).

[0017] In yet other preferred embodiments, the methods comprise one or more of the following: using a tABP that is cleavable by photolysis; using a tABP that is cleavable by acid or base hydrolysis; using a tABP that is cleavable by reduction of a disulfϊde bond; using a tABP that is cleavable by hydroxylamine; using a tABP that is cleavable by sodium periodate; and/or using a tABP that is cleavable enzymatically.

[0018] In additional preferred embodiments, the methods further comprise one or more of the following: further sequestering one or more tABP-peptide or tABP- protein conjugates using a receptor that specifically binds to a portion of the tABP; the lABP(s) used comprise a fluorescent moiety, and the receptor is an antibody or antibody fragment that binds to the fluorescent moiety; the tABP(s) used comprise a fluorescent moiety, and the signal generated is a fluorescent signal; the signal generated is a mass spectrum; the tABP(s) used comprise an isotopic label; the separating step(s) employed comprise one or more separation methods selected from the group consisting of affinity separation, gel electrophoresis, capillary electrophoresis, liquid chromatography, HPLC, electrospray ionization and MALDI; one or more active target proteins bound to said probe are bound to a solid support prior to proteolysis; one or more standard proteins are added to the complex protein mixture prior to proteolysis; the standard protein(s) are labeled with an activity-based probe prior to addition to the complex protein mixture; the standard protein(s) are labeled with a tABP comprising a fluorescent moiety that is distinguishable from the tABP(s) contacted with complex protein mixture.

[0019] In yet another aspect, the present invention relates to compositions comprising a plurality of tABPs for analysis of complex protein mixtures, and methods for their use. Particularly preferred embodiments, the plurality of tABPs are present in a plurality of discrete locations. For example, a solid phase can comprise such discrete locations having a particular tABP covalently linked thereto. Preferred are microarrays, in which the plurality of discrete locations are a regular, patterned array. One or more complex protein mixtures may be contacted with one or more locations on this solid phase, whereby active target proteins reactive with the particular tABP(s) contacted bind to the proteins, thereby sequestering the tABP- bound active target proteins. Alternatively, a plurality of solid phases may be used, each of which comprises a particular tABP covalently linked thereto. Such embodiments might consist of an array of beads for example, in which one or more beads serve the function of a discrete location on a patterned array. One or more non- sequestered components of the complex protein mixture may be removed, e.g., by washing the solid phase(s), and the lABP(s) cleaved to provide tABP-labeled active target protein(s) that may be further analyzed by the screening and/or identification methods described hereinafter.

[0020] In a related aspect, the methods described herein comprise using a patterned array of locations on a solid phase each comprise a particular ABP covalently linked thereto. One or more complex protein mixtures may be contacted with one or more locations on this solid phase, whereby active target proteins reactive with the particular ABP(s) contacted bind to the proteins, thereby sequestering the ABP-bound active target proteins. One or more non-sequestered components of the complex protein mixture may then be removed, and the ABP(s) cleaved, wherein either before or after cleavage of the ABP(s), the ABP-labeled active target proteins are proteolyzed, to produce one or more product mixtures comprising ABP-labeled peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 describes two exemplary methods by which active target proteins can be tethered to a solid phase. In figures 1(a) and (b), the tABPs of the present invention are covalently bound to a solid phase prior to and following interaction with an active target protein, respectively. Figures 1(c) through 1(e) show specific embodiments of these exemplary methods.

[0022] Figure 2 describes two exemplary methods by which two or more samples may be compared. In Figures 2(a) and (b), tABPs are used to sequester active target proteins in two different samples. Labels may be used to distinguish between the sequestered proteins originating from the two samples, and may be present on either the sequestered protein (Figure 2(a)) or the tABP (Figure 2(b). Figures 2(c) and (d) show specific embodiments of these exemplary methods.

[0023] Figure 3 describes the use of "click chemistry" methods in the formation of tABPs.

[0024] Figure 4 describes exemplary azide/alkyne click chemistry reaction schemes for the formation of tABPs.

[0025] Figure 5 describes exemplary azide/alkyne click chemistry constructs.

[0026] Figure 6 describes an alternative exemplary click chemistry reaction schemes for the formation of tABPs using a Staudinger ligation.

[0027] Figure 7 describes another exemplary click chemistry reaction scheme.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0028] The subject methods and compositions provide enhanced simplicity and accuracy in identifying changes in active protein composition of a complex protein mixture. Using tABPs that bind to active target proteins and thereby sequester the bound active target proteins to a solid phase, the analysis of complex protein mixtures may be greatly simplified, tABPsas high stringency methods may be used to remove those components of the complex protein mixture not bound to tABP(s) prior to analysis. In addition, the compositions and methods described herein are particularly advantageous when combined with microarray methodology to provide a tABP array.

[0029] The protein profiling platforms described herein can have a number of steps leading to the identification of active target proteins in a complex protein mixture. A complex protein mixture, and preferably two or more complex protein mixtures, e.g., a sample and a control, can be used as obtained from a natural source or as processed, e.g., to remove interfering components and/or enrich the target protein components. Each complex protein mixture to be analyzed is combined under reaction conditions with at least one tABP to produce conjugates with active target proteins. The tABPs used in two or more complex protein mixtures can differ as to the choice of ligand moiety and/or isotopic composition in order for the labeled complex protein mixtures to be directly compared (e.g., in the same capillary of a capillary electrophoresis apparatus or lane in an electrophoresis gel, or in a mass spectrometer).

[0030] The analysis platforms described herein differ as to the methods of enrichment and analysis using liquid chromatography and/or electrophoresis, and/or mass spectrometry for identification and quantitation. The choice of the platform is affected by the size of the sample, the rate of throughput of the samples, the mode of identification, and the need for and level of quantitation.

[0031] The compositions and methods described herein find use for the most part with biological samples, which may have been subject to processing before reaction with the tABPs. "Biological sample" intends a sample obtained from a cell, tissue, or organism. Examples of biological samples include proteins obtained from cells (e.g., mammalian cells, bacterial cells, cultured cells), particularly as a lysate, a biological fluid, such as blood, plasma, serum, urine, bile, saliva, tears, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion), a transudate or exudate (e.g. fluid obtained from an abscess or other site of infection or inflammation), a fluid obtained from a joint (e.g. a normal joint or a joint affected by disease such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), or the like.

[0032] Biological samples may be obtained from any organ or tissue

(including a biopsy or autopsy specimen) or may comprise cells (including primary cells, passaged or cultured primary cells, cell lines, cells conditioned by a specific medium) or medium conditioned by cells. In preferred embodiments, a biological sample is free of intact cells. If desired, the biological sample maybe subjected to prior processing, such as lysis, extraction, subcellular fractionation, and the like. See, Deutscher (ed.), 1990, Methods in Enzymology, vol. 182, pp. 147-238.

[0033] Of particular interest are samples that are "complex protein mixtures."

As used herein, this phrase refers to protein mixtures having at least about 20, more usually at least about 50, even 100 or more different proteins, where the particular distribution of proteins is of interest. An example of such a complex protein mixture is a proteome, as defined hereinafter. Complex protein mixtures may be obtained from cells that are normal or abnormal in some particular, where the abnormality is informative as to treatment, status, disease, or the like, can be analyzed using the methods of the subject invention.

[0034] The term "proteome" as used herein refers to a complex protein mixture obtained from a biological sample. Preferred proteomes comprise at least about 5% of the total repertoire of proteins present in a biological sample (e.g., the cells, tissue, organ, or organism from which a lysate is obtained; the serum or plasma, etc.), preferably at least about 10%, more preferably at least about 25%, even more preferably about 75%, and generally 90% or more, up to and including the entire repertoire of proteins obtainable from the biological sample. Thus the proteome may be obtained from an intact cell, a lysate, a microsomal fraction, an organelle, a partially extracted lysate, biological fluid, and the like. The proteome will be a mixture of proteins, generally having at least about 20 different proteins, usually at least about 50 different proteins and in most cases 100 different proteins or more. [0035] Generally, the sample will have at least about 1 x 10^"11 g of protein, and may have 1 g of protein or more, preferably at a concentration in the range of about 0.1 - 50 mg/ml. For screening applications, the sample will typically be between about 1 x 10^"11 g and about 1 x 10^"3 g of protein, preferably between about 1 x 10^"6 g and 1 x 10^"4 g of protein. For identification of labeled active target proteins, the sample will typically be between about 1 x 10^"9 g and about 1 g of protein, preferably between about 1 x 10^"4 g and 1 x 10^"1 g of protein. The term "about" in this context refers to +/- 10% of the amount listed.

[0036] The sample may be adjusted to the appropriate buffer concentration and pH, if desired. One or more tABPs may then be added, each at a concentration in the range of about InM to 20mM, preferably 10 nM to ImM, most preferably 10 nm to 100 μM. After incubating the reaction mixture, generally for a time for the reaction to go substantially to completion, generally for about 0.11 — 60 minutes, at a temperature in the range of about 5 - 40°C, preferably about 10°C to about 30°C , most preferably about 20°C , the reaction may be quenched.

[0037] In one aspect of the invention, the method provides for quantitative measurement of active target proteins in biological fluids, cells or tissues. Moreover, the same general strategy can be broadened to achieve the proteome-wide, qualitative and quantitative analysis of the state of activity of proteins, by employing tABPs with differing specificity for reaction with proteins. The methods and compositions of this invention can be used to identify proteins of low abundance that are active in complex protein mixtures and can be used to selectively analyze specific groups or classes of proteins, such as membrane or cell surface proteins, or proteins contained within organelles, sub-cellular fractions, or biochemical fractions such as immunoprecipitates. Further, these methods can be applied to analyze differences in expressed proteins in different cell states. For example, the methods and reagents herein can be employed in diagnostic assays for the detection of the presence or the absence of one or more active proteins indicative of a disease state, such as cancer.

[0038] The subject method can be used for a variety of purposes. The method can be used in the diagnosis of disease, the response of cells to an external agent, e.g. a drug, staging diseases, such as neoplasia, identifying cell differentiation and maturation, identifying new proteins, screening for active drugs, determining side effects of drugs, identifying allelic response, identifying useful probes from combinatorial libraries, etc.

[0039] The system uses tABPs specific for the active form of a protein or a group of proteins, usually directed to an active site on such proteins, and combines one or a mixture of probes, depending on the specificity of the probes and the variety in the group or groups of proteins to be assayed. In the present invention, it is not necessary that there be no reaction of a tABP with inactive target protein(s). Rather, a tABP is defined as being "specific for," as "specifically reacting with," or as

"specifically binding to," active target protein(s) if the tABP provides at least about twice the amount of signal from tABP labeling of active protein when compared to an equivalent amount of inactive target protein. Preferably the signal obtained from active target protein(s) will be at least about five fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater than that obtained from an equivalent amount of inactive target protein.

[0040] The term "target protein" as used herein refers to one or more proteins, an active site of which becomes labeled by one or more tABPs when the target protein is in its "active" form. The reaction mixture can provide conditions under which the tABP(s) react substantially preferentially with active target proteins. Particularly preferred target proteins are enzymes; other preferred target proteins include receptors, transcription factors, G-proteins, and the like.

[0041] The term "active target protein" refers to a target protein that is in its native conformation and is able to interact with an entity with which it normally interacts, e.g. enzyme with substrate and co factor, receptor with ligand, etc., e.g. phosphorylated active form as compared to unphosphorylated inactive form and vice versa. In effect, the protein is in the form in which it can carry out its biological function. [0042] The term "inactivated" as used herein refers to a sample that has been treated so that at least a portion of target proteins that were active in the original sample are rendered inactive. An "inactive protein" can result from various mechanisms such as denaturation, inhibitor binding, either covalently or non- covalently, mutation, secondary processing, e.g. phosphorylation or dephosphorylation, etc. Functional states of proteins or enzymes as described herein maybe distinct from the level of abundance of the same proteins or enzymes. Inactivated samples may be used to validate the activity-specific binding of tABPs as described herein.

[0043] The term "untreated" as used herein refers to a sample that has not been exposed to one or more conditions as compared to a second sample not exposed to such conditions. An untreated sample may be a sample that has not been inactivated; alternatively, an untreated sample may be one not exposed to one or more molecules (e.g., drug lead compounds) in a screening assay. Thus the compostions and methods described herein may comprise comparing a complex protein mixture obtained from cell(s), tissue(s), or organism(s) treated with one or more compounds (e.g., lead compounds in drug discovery) to a complex protein mixture obtained from cell(s), tissue(s), or organism(s) not so treated. tABP-labeled peptides from the two samples may be compared for relative signal intensity. Such methods may indicate alterations in active protein content due to the treatment regimen. Additionally, such methods can also differentiate between treatments that act by direct inhibition of specific proteins ("primary effects") versus treatments that affect active protein content upstream, e.g., by altering expression of protein(s) ("secondary effects").

[0044] An "active site" of a protein refers to an area on the surface of a protein, e.g., an enzyme molecule or surface membrane receptor, to which a binding molecule, e.g. substrate or reciprocal ligand, is bound and results in a change in the protein and/or ligand . For a receptor, the conformation may change, the protein may become susceptible to phosphorylation or dephosphorylation or other processing. For the most part, the active site will be the site(s) of an enzyme where the substrate and/or a cofactor bind, where the substrate and cofactor undergo a catalytic reaction; where two proteins form a complex, e.g. the site at which a G protein binds to a surface membrane receptor, two kringle structures bind, sites at which transcription factors bind to other proteins; or sites at which proteins bind to specific nucleic acid sequences, etc.

[0045] In referring to affinity for a tABP to a target protein, one is concerned with the on-rate of the tABP with the target protein, since there is a negligible off- rate, where the tABP covalently bonds to the target protein. One can determine relative on-rates between tABPs by having less than a stoichiometric amount of the target protein as compared to the total amount of one or more tABPs and then measuring the relative amounts of the conjugates for each of the tABPs. In this way one can obtain a measure of the relative activity of each of the tABPs toward the active target protein, which for the purposes of this invention may be considered the affinity, if not the binding affinity, of the tABP for the target protein.

[0046] Exemplary target proteins include enzymes, such as oxidoreductases, hydrolases, ligases, isomerases, transferases, and lyases (and including such enzymes or enzyme groups as serine hydrolases, metallo-hydrolases, dehydrogenases, e.g. alcohol and aldehyde dehydrogenases, and nucleotide triphosphate (NT)-dependent enzymes), although, the invention envisions tABPs which recognize any protein, e.g., enzyme, family. Other target proteins include proteins that bind to each other or to nucleic acids, such as transcription factors, kringle structure containing proteins, nucleic acid binding proteins, G-protein binding receptors, cAMP binding proteins, etc.

[0047] Structure of tABPs

[0048] The tABPs of the present invention comprise at a minimum a warhead, and a linker moiety to a solid phase. In the APBs of the present invention, at least one covalent bond in the tABP or between the tABP and the solid phase is cleavable in order to permit release of tABP-bound active target proteins from the solid phase. Such tABPs also optionally comprise a ligand, as defined hereinafter, linked to the remainder of the tABP by a linker moiety. [0049] Activity-based probes have been described generally, for example, in the following: PCT Application No. PCT/US99/19415, WO 00/11208, entitled "Rapid Quantitative Analysis of Proteins or Protein Function in Complex Mixtures"; PCT Application No. PCT/USOO/34187, WO 01/77684, entitled "Proteomic Analysis"; PCT Application No. PCT/USOO/34167, WO 01/77668, entitled "Proteomic

Analysis"; U.S. Provisional Application No. 60/266,687, entitled, "Activity Based Probe Analysis," filed February 5, 2001; PCT Application No. PCT/US02/03808, WO 02/063271, entitled "Activity Based Probe Analysis", filed February 5, 2002; each of which is hereby incorporated by reference in their entirety, including all tabes, figures, and claims.

[0050] As will be described hereinafter, each of the warhead, the linker moiety ("L"), and the ligand ("X") may be independently selected to provide different target specificities. Each of these components of a tABP is described in additional detail below.

[0051] The term "warhead" as used herein refers to the portion of a tABP that is directed to and binds with an active site of an active target protein. The warhead comprises a functional group ("F") and an optional affinity moiety ("R"). Functional group (F) refers to one or more chemical groups within a tABP that specifically and covalently bond to the active site of a protein. The functional group may, by its very structure, be directed to the active site of a target protein. Alternatively, a separate affinity moiety (R) may be provided. Affinity moiety (R) refers to a chemical group, which may be a single atom, that is conjugated to the functional group or associated with the linker moiety that provides enhanced binding affinity for protein targets and/or changes the binding profile of the warhead. The affinity moiety is preferably less than 1 kilodalton in mass.

[0052] The term "linker moiety" refers to a bond or chain of atoms used to link one moiety to another, serving as a covalent linkage between two or more moieties. [0053] The term "ligand" as used herein refers to a molecule that can be used to detect and/or capture the tABP in combination with any other moieties that are bound strongly to the ligand, so as to be retained in the process of the reaction of the functional group with the target active protein. The ligand may be added to the warhead-linker moiety combination after reaction with the target protein, to form the complete tABP. For this purpose, the warhead-linker moiety combination will include a chemically reactive functionality, normally not found in proteins, that will react with a reciprocal functionality on the ligand, e.g. vic.-diols with boronic acid, photoactivated groups, such as diazo bisulfites, etc. The warhead-linker moiety is then reacted with the ligand to complete the tABP.

[0054] The term "solid phase" as used herein refers to a wide variety of materials including solids, semi-solids, gels, films, membranes, meshes, felts, composites, particles, and the like typically used by those of skill in the art to sequester molecules. The solid phase can be non-porous or porous. Suitable solid phases include those developed and/or used as solid phases in solid phase binding assays. See, e.g., chapter 9 of Immunoassay, E. P. Diamandis and T. K. Christopoulos eds., Academic Press: New York, 1996, hereby incorporated by reference. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. See, e.g., Leon et al., Bioorg. Med. Chem. Lett. 8: 2997 (1998); Kessler et al., Agnew. Chem. hit. Ed. 40: 165 (2001); Smith et al., J. Comb. Med. 1: 326 (1999); Orain et al., Tetrahedron Lett. 42: 515 (2001); Papanikos et al., J. Am. Chem. Soc. 123: 2176 (2001); Gottschling et al., Bioorg. And Medicinal Chem. Lett. 11: 2997 (2001).

[0055] Methods for linking molecules to solid phases are well known and include methods used for immobilizing reagents on solid phases for solid phase binding assays or for affinity chromatography (see, e.g., Diamandis and Christopoulos cited above, and Hermanson, Greg T., Immobilized Affinity Ligand Techniques, Academic Press: San Diego, 1992, hereby incorporated by reference). Surfaces such as those described above may be modified to provide linkage sites, for example by bromoacetylation, silation, addition of amino groups using nitric acid, and attachment of dendrimers and/or star polymers. This list is not meant to be limiting, and any method known to those of skill in the art may be employed. The only limitation on the method and location of the linkage is that the solid phase should not interfere with binding of a tABP to its active target protein(s).

[0056] ABPs can be immobilized on different regions of one or more solid phases to form a patterned array. Such a patterned array having two or more regions comprising ABPs that differ in structure and/or reactivities from each other could be used to simultaneously measure the presence, amount, or activity of a plurality of active target proteins .

[0057] It is preferred that at least one bond within the tABP be cleavable so that active target proteins bound by the tABP, or peptides generated therefrom, may be released. In particularly preferred embodiments, at least a portion of the tABP remains bound to the active target protein. The cleavable bond may be within the warhead portion, the linker portion, and/or between the tABP and the solid phase. Preferably, that portion of the tABP that remains bound following cleavage of the tABP comprises the ligand portion of the tABP. Methods for introducing cleavable linkages into molecules are well known to those of skill in the art. See, e.g., U.S. Patent No. 6,310,244; U.S. Patent No. 6,075,166; and 6,046,005. Typically, such linkages are cleavable by acid or base hydrolysis, reduction of a disulfide bond, incubation with hydroxylamine; incubation in sodium periodate; exposure to light (i.e., photocleavable linkages); and/or enzymatic cleavage. The tABP-protein or tABP-peptide conjugates may also be cleaved from the solid phase using mass spectrometry ionization techniques such as SELDI, MALDI, etc.

[0058] The tABP will have an affinity for an active site, which may be specific for a particular active site or generally shared by a plurality of related proteins. The affinity may be affected by the choice of the functional group, the linker moiety, the binding moiety, the ligand, or a combination thereof. As described hereinafter, one or more tABPs may be designed that exhibit specificity for a single target protein, or that exhibit specificity for a plurality of targets that may be structurally or functionally related.

[0059] The tABPs of the subject invention may be illustrated by the following formula:

[R* (F - L) - X]-[solid phase]

where * indicates that R may be optionally present, and L, if present, may be bound to either F, L or both F and L, and where the solid phase may be covalently linked to any one of R, F,L, or X either before or following binding of the tABP to an active target protein.

[0060] Exemplary Fs as used in a tABP of the invention include an alkylating agent, acylating agent, ketone, aldehyde, sulphonate or a phosphorylating agent. Examples of particular Fs include, but are not limited to fluorophosphonyl, fiuorophosphoryl, fluorosulfonyl, alpha-haloketones or aldehydes or their ketals or acetals, respectively, alpha-haloacyls, nitriles, sulfonated alkyl or aryl thiols, iodoacetylamide group, maleimides, sulfonyl halides and esters, isocyanates, isothiocyanantes, tetrafluorophenyl esters, N-hydroxysuccinimidyl esters, acid halides, acid anhydrides, unsaturated carbonyls, alkynes, hydroxamates, alpha- halomethyll ydroxamates, aziridines, epoxides, or arsenates and their oxides. Sulfonyl groups may include sulfonates, sulfates, sulfinates, sulfamates, etc., in effect, any reactive functionality having a sulfur group bonded to two oxygen atoms.

Epoxides may include aliphatic, aralkyl, cycloaliphatic and spiro epoxides, the latter exemplified by fumagillin, which is specific for metalloproteases.

[0061] The linker moiety L, which potentially can be as short as a covalent bond, is preferred to be other than a bond. Since in many cases, the synthetic strategy will be able to include a functionalized site for linking, the functionality can be taken advantage of in choosing the linking moiety. The choice of linker moiety has been shown to alter the specificity of an ABP. See, e.g., Kidd et ah, Biochemistry (2001) 40: 4005-15. For example, an alkylene linker moiety and a linker moiety comprising a repeating alkyleneoxy structure (polyethylene glycols, or"PEG"), have distinct specificities and provide distinct protein profiles. Thus, one of skill in the art can select the linker moiety of the tABP in order to provide additional specificity of the tABP for a particular protein or protein class.

[0062] Linker moieties include among others, ethers, polyethers, diamines, ether diamines, polyether diamines, amides, polyamides, polythioethers, disulfides, silyl ethers, alkyl or alkenyl chains (straight chain or branched and portions of which may be cyclic) aryl, diaryl or alkyl-aryl groups, having from 0 to 3 sites of aliphatic unsaturation. While normally amino acids and oligopeptides are not preferred, when used they will normally employ amino acids of from 2 - 3 carbon atoms, i.e. glycine and alanine. Aryl groups in linker moieties can contain one or more heteroatoms (e.g., N, O or S atoms). The linker moieties, when other than a bond, will have from about 1 to 60 atoms, usually 1 to 30 atoms, where the atoms include C, N, O, S, P, etc., particularly C, N and O, and will generally have from about 1 to 12 carbon atoms and from about 0 to 8, usually 0 to 6 heteroatoms. The number of atoms referred to above are exclusive of hydrogen in referring to the number of atoms in a group, unless indicated otherwise.

[0063] Linker moieties may be varied widely depending on their function, including alkyleneoxy and polyalkyleneoxy groups, where alkylene is of from 2 - 3 carbon atoms, methylene and polymethylene, polyamide, polyester, and the like, where individual monomers will generally be of from 1 to 6, more usually 1 to 4 carbon atoms. The oligomers will generally have from about 1 to 10, more usually 1 to 8 monomeric units. The monomeric units may be amino acids, both naturally occurring and synthetic, oligonucleotides, both naturally occurring and synthetic, condensation polymer monomeric units and combinations thereof.

[0064] In various embodiments, linker moieties may also be elaborated following binding of functional group F to an active target protein. In these embodiments, additional material may be added to a warhead-linker moiety combination after reaction with the target protein, to form the complete tABP. For this purpose, the warhead-partial linker moiety combination will include a chemically reactive group, normally not found in proteins, that will react with a reciprocal functionality on the remainder of the linker moiety, which may be attached to the solid phase and/or the ligand, e.g. viccinal-diols with boronic acid, photoactivated groups, such as diazo, azide with an alkene or alkyne, o-alkyl hydroxylamine with a ketone or aldehyde, etc. The warhead-partial linker moiety is then reacted with the additional material to complete the tABP.

[0065] The ligand portion permits capture of the conjugate of the target protein and the probe. The ligand may be displaced from the capture reagent by addition of a displacing ligand, which may be free ligand or a derivative of the ligand, or by changing solvent (e.g., solvent type or pH) or temperature or the linker may be cleaved chemically, enzymatically, thermally or photochemically to release the isolated materials (see discussion of the linker moiety, below).

[0066] Examples of ligands, X, include, but are not limited to, detectable labels such as fluorescent moieties and electrochemical labels, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, a polypeptide, a metal chelate, and or a saccharide. Examples of ligands and their capture reagents also include but are not limited to: dethiobiotin or structurally modified biotin-based reagents, including deiminobiotin, which bind to proteins of the avidin/streptavidin family, which may, for example, be used in the forms of strepavidin-Agarose, oligomeric-avidin-Agarose, or monomeric-avidin-Agarose; any vicinal diols, such as 1,2-dihydroxyethane (HO-CH₂-CH₂-OH), and other 1,2- dihyroxyalkanes including those of cyclic alkanes, e.g., 1,2-dihydroxycyclohexane which bind to an alkyl or aryl boronic acid or boronic acid esters, such as phenyl- B(OH)₂ or hexyl-B(OEthyl) which may be attached via the alkyl or aryl group to a solid support material, such as Agarose; maltose which binds to maltose binding protein (as well as any other sugar/sugar binding protein pair or more generally to any ligand/ligand binding protein pairs that has properties discussed above); a hapten, such as the dinitrophenyl group, to which an antibody can be generated; a ligand which binds to a transition metal, for example, an oligomeric histidine will bind to Ni(II), the transition metal capture reagent may be used in the form of a resin bound chelated transition metal, such as nitrilotriacetic acid-chelated Ni(II) or iminodiacetic acid-chelated Ni(II); glutathione which binds to glutathione-S-transferase. For the most part, the ligands will be haptens that bind to a naturally occurring receptor, e.g. biotin and avidin, or an antibody or will be a detectable label, that is also a hapten.

[0067] One may use chemical affinity resins, e.g. metal chelates, to allow for digestion of proteins on the solid phase resin and facilitate automation. One example of this is the use of immobilized nickel (II) chelates to purify peptides that have six consecutive histidine residues (His-6 tag) (as described in the I vitrogen product brochureProBond ™ Resin (Purification) Catalog nos. R801-01, R801-15 Version D 000913 28-0076), which could be adapted to include non-peptidic chemical linkage coupling a series of imidazole-containing moieties. Alternative chemical attachments include phenyldiboronic acids (described in Bergseid, M. et al. Biotechniques (2000) 29(5), 1126-1133), and disulfide reagents (described in Daniel, SM et al., Biotechniques (1998) 24(3), 484-489). Additionally, chemical affinity tags that are useful in combinatorial synthesis could be adapted for modified peptide purification (reviewed in Porco, JA (2000) Comb. Chem. High Throughput Screening 3(2) 93-102

[0068] The term "fluorescent moiety" refers to a ligand that can be excited by electromagnetic radiation, and that emits electromagnetic radiation in response in an amount sufficient to be detected in an assay. The skilled artisan will understand that a fluorescent moiety absorbs and emits over a number of wavelengths, referred to as an "absorbance spectrum" and an "emission spectrum." A fluorescent moiety will exhibit a peak emission wavelength that is a longer wavelength than its peak absorbance wavelength. The term "peak" refers to the highest point in the absorbance or emission spectrum.

[0069] The fluorescent moiety may be varied widely depending upon the protocol to be used, the number of different probes employed in the same assay, whether a single or plurality of lanes are used in the electrophoresis, the availability of excitation and detection devices, and the like. For the most part, the fluorescent moieties that are employed as ligands will absorb in the ultraviolet, infrared, and/or most preferably in the visible range and emit in the ultraviolet, infrared, and/or most preferably in the visible range. Absorption will generally be in the range of about 250 to 750 nm and emission will generally be in the range of about 350 to 800nm. Illustrative fluorescent moieties include xanthene dyes, naphthylamine dyes, coumarins, cyanine dyes and metal chelate dyes, such as fluorescein, rhodamine, rosamine, the BODIPY dyes (FL, TMR, and TR), dansyl, lanthanide cryptates, erbium, terbium and ruthenium chelates, e.g. squarates, and the like. Additionally, in certain embodiments, one or more fluorescent moieties can be energy transfer dyes such as those described in Waggoner et al., U.S. Patent no. 6,008,373. The literature amply describes methods for linking fluorescent moieties through a wide variety of linker moieties to other groups. The fluorescent moieties that find use will normally be under 2kDal, usually under lkDal.

[0070] Preferred fluorescent moieties can include elaborated conjugated pyran molecules, including xanthenes. Such molecules include eosin, erythrosin, fluorescein, Oregon green, and various commercially available Alexa Fluor ® dyes (Molecular Probes, h e). Structural examples of such dyes include:

[0071] Particularly preferred fluorescent moieties are the rhodamine dyes.

These molecules typically have the general structure:

[0072] Where X is -CO₂H, or -SO₃H; Y is -H, -CH₃, or together with R forms a six-membered ring; Z is -H or together with R forms a six-membered ring; and R is -H, -CH₃, -CH₂CH₃, or together with Y or Z forms a six-membered ring. Rhodamine molecules such as tetramethylrhodamine, 5-carboxytetramethyhhodamine, 6- carboxytetramethyhhodamine, carboxyrhodamine-6G, rhodamine-B sulfonyl chloride, rhodamine-red-X, and carboxy-X-rhodamine are well known to those of skill in the art. See, e.g., Handbook of Fluorescent Probes and Research Products, Molecular Probes, Inc., 2001, which is hereby incorporated by reference in its entirety. Advantageous properties of rhodamines include high quantum yields, low sensitivity of fluorescence over a pH range of from about pH 3 to about pH 8, advantageous water solubility, good photostabihty, and absorption of light in the visible spectrum. Particularly preferred fiuorescers are 5- carboxytetramethylrhodamine and 6-carboxytetramethylrhodamine.

[0073] Other preferred fluorescent moieties include the

BODIPY dyes, which are elaborations of a 4-bora-3a,4a-diaza--s^,-indacene structure. Exemplary structures are provided below:

[0074] Yet other preferred fluorescent moieties include the cyanine dyes, conjugated structures comprising a polymethine chain terminating in nitrogen atoms. Typically, the nitrogens are themselves part of a conjugated heterocycle. An exemplary structures is provided below:

[0075] Also of interest for use as ligands are matched dyes as described in

U.S. Patent No. 6,127,134, which is hereby incorporated by reference in its entirety, including all tables, figures, and claims, which is concerned with labeling proteins with dyes that have different emissions, but have little or no effect on relative migration of labeled proteins in an electrophoretic separation. Of particular interest are the cyanine dyes disclosed therein, being selected in '134 because of their positive charge, which matches the lysine to which the cyanine dyes bind, h addition there is the opportunity to vary the polyene spacer between cyclic ends, while keeping the molecular weight about the same with the introduction of an alkyl group in the shorter polyene chain dye to offset the longer polyene. Also described are the BODIPY dyes, which lack a charge. The advantage of having two dyes that similarly affect the migration of the protein would be present when comparing the native and inactived samples, although this would require that in the inactivated sample at least a portion of the protein is monosubstituted. [0076] In each of the foregoing examples of preferred fluorescent moieties, carboxyl groups can provide convenient attachment sites for linker moieties, hi the particularly preferred 5- and 6-carboxyrhodamine molecules, the 5- or 6- carboxyl is particularly preferred as an attachment site:

[0077] In general, any affinity label-capture reagent commonly used for affinity enrichment, which meets the suitability criteria discussed above, can be used in the method of the invention. Biotin and biotin-based affinity tags are particularly illustrated herein. Of particular interest are structurally modified biotins, such as deiminobiotin or dethiobiotin, which will elute from avidin or streptavidin (strept/avidin) columns with biotin or under solvent conditions compatible with ESI- MS analysis, such as dilute acids containing 10-20% organic solvent. For example, deiminobiotin tagged compounds will elute in solvents below about pH 4.

[0078] Design of tABPs and libraries of tABPs

[0079] tABPs may be designed and synthesized using combinatorial chemistry and/or rational design methods. A detailed description of a tABP design strategy, in which a fluorescent moiety can act as a ligand, is provided in PCT Application No. PCT/US02/03808, WO 02/063271, entitled "Activity Based Probe Analysis", filed February 5, 2002, PCT Application No. PCT/US00/34187, WO 01/77684, entitled "Proteomic Analysis," and PCT Application No. PCT/US00/34167, WO 01/77668, entitled "Proteomic Analysis," each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims. As described therein, goals of a design strategy are to provide tABPs that are able to react covalently with a targeted group of active proteins, while minimizing non-specific labeling.

[0080] One strategy that may be utilized to design tABPs is to first identify a potentially reactive amino acid that is conserved at the sequence level in the region that is targeted for tABP labeling, and to select an appropriate functional group for attachment to an appropriate affinity moiety (e.g., an adenosine analogue for ATP binding proteins). Potential reactive amino acids include serine, threonine, tyrosine, lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, methionine, and cysteine.

[0081 ] One can also consider the composition of the linker moiety between the warhead moiety and the ligand of the tABP, as this can affect the selectivity and specificity of the resulting tABPs. Linker moieties may be either obtained commercially (see, e.g., Pierce Chemical Company Catalog and Handbook 1994-95, pages O-90 through O-l 10, which is hereby incorporated by reference) or synthesized as needed. A library of molecules comprising, for example, linker chemistries exhibiting varying lengths, hydrophobicities, etc., maybe constructed. Moreover, the library of tABPs can also be expanded by varying identity of the ligand (e.g., using a variety of different fluorescent moieties), and/or the location of linker moiety attachment point on the ligand (e.g., 5-TMR linkage vs. 6-TMR linkage), as these can also affect the selectivity and specificity of the resulting tABPs.

[0082] The linker may also be designed to provide the ability to attach a solid phase, a ligand, or both, following reaction of the functional group with a target protein. Using a modular "click chemistry" approach for example, a warhead-first linker moiety combination may be provided with a chemically reactive group, normally not found in proteins, that will react with a reciprocal functionality on a second linker moiety which may be attached to the solid phase and/or the ligand. This reaction further elaborates the first linker moiety, forming the completed tABP. Click chemistry methods are well known in the art. See, e.g., Kolb et al., Agnew Chem. Int. Ed. Engl. 40: 2004-21 (2001); Seo et al, J. Org. Chem. 68: 609-12 (2003). These methods may be particularly useful when the presence of a solid phase and/or ligand interfere with or alter the binding of a tABP with a target protein.

[0083] Examples of suitable reciprocal functionalities include viccinal-diols with boronic acid, photoactivated groups, such as diazo, azide with an alkene or alkyne, o-alkyl hydroxylamine with a ketone or aldehyde, etc. Fig. 3 describes the general approach of the react-elaborate scheme, showing that the general approach is applicable to both solid phase-based and non-solid phase-based activity based probes. In this figure, a warhead-first linker moiety combination is reacted with a target protein; further elaboration of the first linker can provide a covalent linkage to a detectable moiety (e.g., TAMRA), a capturable moiety (e.g., TAMRA or Biotin), or a solid phase (depicted as a sphere).

[0084] Fig. 4 schematically describes the use of one exemplary "react- elaborate" scheme using an azide/alkyne cycloaddition, in which the functional group/first linker moiety is reacted with a target protein, and then is further elaborated to form the complete tABP. In part (A), the first linker moiety comprises a linkage to a ligand, and is subsequently coupled to a solid phase using the azide/alkyne cycloaddition to form the complete tABP; in part (B), a functional group is reacted with a target protein, and then is coupled to a solid phase/ligand portion to form the complete tABP; and in part C, a functional group/solid phase portion is reacted with a target protein, and then is coupled to a ligand to form the complete tABP.

[0085] Fig. 5 provides exemplary functional group/first linker moiety constructs, as well as exemplary second linker moiety/ligand constructs for use in azide/alkyne cycloaddition click chemistry. The skilled artisan will understand that all or a portion of the polyoxyethylene groups in the first linker moiety may be replaced with other suitable groups as described herein, including alkyl and amide-linked (e.g., amino acid) groups. Likewise, the alkyl portions of the second linker moiety may be similarly replaced, e.g, with polyoxyethylene groups or amide-linked groups. The use of a bifunctional structure, such as a lysine (providing both an α-amino and ε-amino group), within a linker moiety can allow that linker moiety to couple both a solid phase and a ligand, two ligands, etc.

[0086] Fig. 6 provides a second exemplary "react-elaborate" scheme using a reaction known in the art as a Staudinger Ligation. See, e.g., Saxon and Bertozzi, Science 287: 2007-10 (2000). Additional reaction mechanisms suitable in the methods described herein will be apparent to those of skill in the art.

[0087] hi the case of a combinatorial library, as indicated above, numerous variations as to the structure of the tABP can be prepared. These various members may then be screened with a complex protein mixture to determine which members of the library are inactive with inactivated target protein(s), but react with active target protein(s). In carrying out the subject methodology, one or a plurality of tABPs may be added to a complex protein sample as described herein.

[0088] In an alternative design strategy, a functional group may be selected that imparts an element of "chemical specificity" to the tABP. In these embodiments, the requirement for an affinity moiety to achieve adequate probe selectivity can be eliminated. The incorporation of an affinity moiety into a tABP can affect the number of proteins targeted by a probe. Thus, depending on the number of target proteins of interest and the similarity of active site binding selectivity, the artisan can choose to include the affinity moiety or not, as required. For example, a fluorophosphonate reactive group provides a classical affinity label for serine hydrolases that selectively reacts with the activated serine nucleophile of catalytic triad and other serine hydrolase classes.

[0089] While the number of tABPs that can be used is theoretically unlimited, preferably not more than about 20 are used. Since the sources of the protein mixture will vary widely and one biomolecule may have an influence on the activity of the tABP, as well as on the reactivity of a protein, the mixture may be subject to dilution, fractionation, precipitation, extraction, dialysis, chromatography or other processing to obtain the desired composition. For the most part, the composition will not be significantly modified, maintaining substantially the composition obtained from the source. In some instances the pH may be modified, solvents added, or the like, to enhance the reaction of the active target proteins with the tABP(s) or change the tABP profile as to the active target proteins.

[0090] Analysis of Complex Protein Mixtures

[0091 ] The methods of the present invention can be divided generally into two classes, referred to herein for convenience as "screening" methods and "identification" methods.

[0092] "Screening" refers to methods in which one or more complex protein mixtures suspected of containing one or more target proteins are mixed with one or more tABPs, whereby active target proteins present in the complex protein mixture(s) are labeled. The proteins are subsequently digested using one or more proteases to generate tABP-labeled peptides. The tABPs of the present invention preferably react with and bind to a single residue on an active target protein, and preferably a single tABP-labeled peptide is generated from each labeled active target protein. A signal is then generated from the tABP-labeled peptide(s), which can be correlated to the presence or amount of labeled active target proteins in the original complex protein mixture, preferably following a separation method (e.g., slab gel or capillary electrophoresis, or liquid chromatography).

[0093] hi contrast, "identification" refers to methods in which the molecular weight and/or the sequence of one or more tABP-labeled peptides, generated as described in the preceding paragraph, are determined by mass specfroscopy ("MS"). In these methods, a capture (sequestration) step is typically performed to purify tABP- labeled peptide(s) prior to MS analysis.

[0094] The terms "mass spectrometry" or "MS" as used herein refer to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z." In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z"). See, e.g., U.S. Patent Nos. 6,204,500, entitled "Mass Spectrometry From Surfaces;" 6,107,623, entitled "Methods and Apparatus for Tandem Mass Spectrometry;" 6,268,144, entitled "DNA Diagnostics Based On Mass Spectrometry;" 6,124,137, entitled "Surface-Enhanced Photolabile Attachment And Release For Desorption And Detection Of Analytes;" Wright et al., "Proteinchip surface enhanced laser desorption/ionization (SELDI) mass spectrometry: a novel protein biochip technology for detection of prostate cancer biomarkers in complex protein mixtures," Prostate Cancer andProstatic Diseases 2: 264-76 (1999); and Merchant and Weinberger, "Recent advancements in surface- enhanced laser desorption/ionization-time of flight-mass spectrometry," Electrophoresis 21 : 1164-67 (2000), each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims. Molecules (e.g., peptides) in a test sample can be ionized by any method known to the skilled artisan. These methods include, but are not limited to, electron ionization, chemical ionization, fast atom bombardment, field desorption, and matrix-assisted laser desorption ionization ("MALDI"), surface enhanced laser desorption ionization ("SELDI"), photon ionization, electrospray, and inductively coupled plasma.

[0095] As used herein, reference to a "screening" method is not intended to imply that a given peptide, and its source protein, cannot be identified in such methods. For example, a particular peptide may exhibit a characteristic migration rate in a separation method that can be used to identify the peptide. Alternatively, several separation methods and/or conditions may be employed, and the separation profile of migration patterns generated for a given peptide can be used to identify the peptide. Instead, the distinction between a screening method and an identification method is the use of MS to directly measure molecular weight in identification methods. Additionally, the skilled artisan will understand that a single complex protein mixture may be analyzed by both screening and identification methods, as exemplified in Figure 2.

[0096] The terms "separation" and "separating" as used herein refer to methods that enrich the concentration of a molecule of interest in a particular location or container relative to other molecules originally present. For example, gel electrophoresis enriches the concentration of molecules that migrate at a particular rate relative to other molecules originally present that migrate at different rates. Numerous additional analytical procedures are known to the artisan for separating and analyzing complex protein mixtures (e.g., chromatographic methods such as HPLC, FPLC, ion exchange, size exclusion; mass spectrometry; differential centrifugation). The term separating does not indicate that a desired component is obtained in a "pure" form; only that the desired component has increased in abundance relative to the other components present. For example, a complex protein mixture may be separated by electrophoresis, whereby a desired component is separated into one or more bands or spots present on a gel. Other, undesired, components may be present in the same band or spot; nonetheless, the desired component will have increased in abundance in that band or spot. A separation profile obtained from a separation procedure can be expressed as an elution time or retention time using a chromatography column, a migration distance, Rf, migration time, or elution time using an electophoresis apparatus, or any other expression commonly used by one of skill in the art to distinguish between separated components.

[0097] A particular type of separating method is referred to herein as

"capture" or "sequestration." These methods enrich the concentration of molecules capable of being sequestered (e.g., by binding to one or more receptors) relative to other molecules not so capable (e.g., removed by washing out molecules that do not bind to a receptor). As described herein, the tABPs of the present invention provide a sequestration platform by their covalent linkage to a solid phase.

[0098] h addition, one or more molecules may also be sequestered by being bound to a receptor that is itself bound to a surface. Following removal of unbound molecules, the sequestered molecules may be released from the receptor(s) for further processing. As used herein with respect to receptors, the phrases "specifically binds" and "specific binding" refers to a molecule or molecular complex (e.g., an antibody or binding fragment thereof) that binds to its intended ligand(s) (e.g., a cognate antigen) with at least a 2-fold greater affinity compared to the binding of the receptor to molecules other than the intended ligand(s). In preferred embodiments, such a receptor binds to binding partners with at least a 5 -fold, more preferably at least a 10- fold, even more preferably at least a 100-fold, and most preferably at least a 1000-fold greater affinity compared to non-partner binding. In addition to antibody-antigen pairs, suitable receptor-ligand pairs include but are not limited to receptor-hormone pairs, avidin-biotin pairs, streptavidin-biotin pairs, and metal-chelate pairs.

[0099] Labeling of active target proteins by tABPs

[00100] In carrying out both screening and identification methods, one or a plurality of tABPs will be added to a complex protein mixture as described herein, where the tABPs will react with the active target proteins present. The complex protein mixtures may come from different sources and be used for different purposes. This may include a relatively pure sample of the enzyme to determine the activity in relation to total protein of the sample. The sample may be a single cell or a mixture of cells, a neoplastic sample or other biopsy or tissue comprising a single cell type or a mixture of cell types, such as tissue from an organ, e.g. heart, lung, esophagus, kidney, brain, blood, etc., diseased tissue or healthy tissue, etc. The cells may be prokaryotic or eukaryotic, vertebrate or non-vertebrate, particularly mammalian and more particularly human. The cells or tissues, or lysates thereof, may be prepared in a variety of ways, including fractionation, using chromatography, centrifugation, precipitation, fractionation, fluorescence activated cell sorting, dilution, dialysis, concentration, etc. The sample will usually be treated so as to preserve the activity of the target protein(s), so that the manner of treatment will be mild, ambient or lower temperatures will be used, particularly below 37°C, and other denaturing conditions will be avoided, such as organic solvents, detergents or high salts.

[0100] Generally, the amount of each tABP will be sufficient to react with all of the active target protein for the tABP expected to be in the proteome. Typically the amount of each tABP is present in excess over its target proteins on a molar basis. After incubating the reaction mixture, generally for a time for the reaction to go substantially to completion, usually not more than about 2h, generally for about 0.1 - 60min, at a temperature in the range of about 20 - 40°C, the reaction may be quenched. Since the extent of the reaction will increase with time, the longer the time, the more cross-reactivity may be anticipated. A preferred time will be employed to provide the most favorable results, that is, the greatest level of reaction with the target proteins with the least cross-reactivity.

[0101] To enhance the accuracy of the quantitation, internal standards can be employed, where known proteins are reacted with one or more tABPs to provide one or more conjugates of known composition. These internal standards can be used to account for sample losses during processing, account for variations in protein digest efficiencies, and/or account for variations in relative migration times in separation procedures. A predetermined amount of these tABP-labeled standards may be added to a sample, so that the sample will have a known concentration of the standard. The internal standard is then subjected to the same processes as the component of the sample.

[0102] Where the procedure is substantially reproducible, an internal standard may be a protein that is also present in the sample being analyzed. One would expect to obtain a predictable signal from the standard, and any increase over this amount can be attributed to active target protein present in the sample. Alternatively, the internal standard may be selected to be different from any conjugate in the sample, hi particularly preferred embodiments, one or more internal standards can employ ligands (e.g., fluorescent moieties) that are differentially detectable in comparison to those used for labeling the complex protein mixture. This can be particularly advantageous in separation methods, such as electrophoresis or chromatography, as standard signals can be easily distinguished from signals obtained from the labeled active target proteins.

[0103] One or more internal standards may also be selected that provide, for example, a proteolysis site known to react slowly with the protease(s) being employed, and preferably selected to react more slowly than one or more proteins of interest present in the complex protein mixture. Such standards can be used to monitor the course of hydrolysis.

[0104] Digesting labeled active target proteins [0105] Following tABP labeling of active target proteins, protein digestion may be used to produce tABP-labeled peptides. The digestion may be performed while the proteins are in solution or when the conjugates are sequestered, e.g., when bound to a solid support. Digestion preferably employs only one protease; however, two or more, usually not more than three, proteases may be used. The proteases may themselves be in solution or bound to a surface. The proteases may be combined in the same reaction mixture, or the sample may be divided into aliquots and each of the aliquots treated with a different protease. Digestion may also occur before binding to the conjugate to a support and/or a after the conjugates are bound to a solid support. Enzymes that find use include, but are not limited to, trypsin, chymotrypsin, bromelain, papain, carboxypeptidase A, B and Y, proteinase A and K, chymopapain, plasmin, subtilisin, clostripain etc.

[0106] In particularly preferred embodiments, additional steps can be used to reduce the complexity of the analysis to be performed. For example, the complex protein mixture can be denatured following labeling, e.g., by the addition of urea, guanidinium salts, detergents, organic solvents, etc., in order to reduce or eliminate unwanted proteolysis from endogenous proteases present in the mixture. Additionally, cysteine residues can be reduced and alkylated to maintain the homogeneity of cysteine-containing peptides and to prevent refolding of endogenous proteases following removal of the denaturant. Moreover, proteases can be combined with additional enzymes, such as glycosidases, phpsphatases, sulfatases, etc., that can act to remove post-translational modifications from proteins. Examples of such posttranslational modifications include, but are not limited to, glycosylations, phosphorylations, sulfations, prenylations, methylations, amidations, and myristolations. Such steps can be mixed and matched by the skilled artisan, depending on the requirements of a particular analysis.

[0107] Prior to digestion, a buffer exchange step may be employed, e.g., by gel filtration, dialysis, etc. This step may be used to remove excess tABPs, to remove denaturant, and/or to provide suitable buffer conditions for digestion. In particularly preferred embodiments, buffer exchange is performed by gravity flow gel filtration. [0108] Digestion will be carried out in an aqueous buffered medium, generally at a pH in the range of about 4 to 10, depending on the requirements of the protease.

The concentration of the protease will generally be in the range of about 6 10^" M to about 6 x 10^"6 M, more preferably in the range of about 1.8 x 10^"8 M to about 2 x 10^"7 M, and most preferably about 6 x 10^"7 M (e.g., 150 ng / 10 μL). The term "about" in this context means +/- 10% of a givem measurement. The time for the digestion will be sufficient to go to at least substantial completion, so that at least substantially all of the protein will have been digested. Digests may be performed at a temperature that is compatible with the protease(s) employed, preferably from 20°C to 40°C, most preferably about 37°C. Where the digestion takes place in solution, the protease may be quenched by any convenient means, including heating or acidification of the sample. Alternatively, quenching can be achieved by sequestering the fragment conjugates with a receptor for the ligand bound to a surface, or by addition of a protease inhibitor (e.g., E64, DIFP, PMSF, etc.). Where the proteins are bound to a surface, the proteases may be washed away before the bound digested protein is released.

[0109] Release of tABP-labeled peptides from the solid phase

[0110] Sequestration relies on to the use of a solid support that can be easily manipulated during wash steps. The support may be beads, including paramagnetic beads, prepared from various materials, such as Bioglas, polystyrene, polyacrylate, polymethylmethacrylate, polyethylene, polysaccharides, such as Agarose, cellulose, amylose, etc., polyurethane, and the like. When beads are employed, the beads will generally have a cross-dimension in the range of about 5 to lOOμ. Instead of beads, one may use solid supports, such as slides, the walls of vessels, e.g. microtiter well walls, capillaries, etc. There is an extensive literature of receptor bound supports that is readily applicable to this invention, since the sequestering step is conventional.

[0111] Following sequestration, tABP-labeled proteins and/or peptides may then be released by cleavage of the linkage between the tABP and the solid phase. The particular method of release will depend upon the chemistry employed in the linkage. Examples of linkages that are cleavable using reducing agents (e.g., DTT), hydroxylamine, sodium periodate, and base hydrolysis are disclosed in the Pierce Catalog and Handbook, pages T-155 - T-200, Pierce Chemical Company, 1994, which is hereby incorporated by reference. See also, Leon et al., Bioorg. Med. Chem. Lett. 8: 2997 (1998); Kessler et al, Agnew. Chem. Int. Ed. 40: 165 (2001), each of which is hereby incorporated by reference. Exemplary cleavable linkages are provided hereinafter

[0112] "Screening" analysis methods

[0113] In screening methods, a signal is generated from the various tABP- labeled proteins and/or peptides obtained from the original complex protein mixture. If only a single tABP-labeled protein or peptide is associated with a particular signal (e.g., if each tABP reacts with a single target protein and each tABP is distinguishable) a signal may be generated without further separation. More typically, however, one or more separation methods are employed to separate various tABP- labeled proteins or peptides prior to signal generation.

[0114] These separation methods include, but are not limited to, liquid chromatography, particularly reverse phase liquid chromatography, where the components are separated by their differences in hydrophobicity, or electrophoresis, particularly slab gel or capillary electrophoresis. The electrophoresis may involve one- or two-dimensional electrophoresis, may be in a gel, may use a capillary or may use a channel in a microfluidic device. See e.g., Opiteck, et al., Anal. Chem. (1998) 258:349-61; U.S. Patent Nos. 4,415,655; 4,481,094; 4,865,707; and 4,946,794; Laemmli, UK, Nature (1970) 227, 680-685; and Sambrook, J.; MacCallum, P. & Russell, D. (2001) "Molecular Cloning: A Laboratory Manual." 3^rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY.. Liquid chromatography may use a combination of size exclusion liquid chromatography followed by RP-HPLC or only RP-HPLC. The conditions employed are conventional for liquid chromatographic separation of proteins and peptides and commercial equipment and materials are available. See, e.g., U.S. Patent Nos. 5,041,538 and 5,290,920 and W091/15228, as exemplary. A suitable eluant can include a water/acetonitrile gradient, optionally containing 0.1% trifluoroacetic acid; 0.1 % trifluoroacetic acid; or 0.1% formic acid. The conjugates can be monitored by their fluorescence and may be isolated in wells for further investigation. A separation profile in such methods may be sufficient information to identify the peptide and, therefore, the protein.

[0115] For screening, a particularly preferred separation/signal generation method is capillary electrophoresis with laser-induced fluorescence ("CE-LIF"). Capillary electrophoresis methods are well known to the skilled artisan. See, e.g., Kasicka, "Recent advances in capillary electrophoresis of Peptides," Electrophoresis (2001) 22:4139-62; Sanchez and Smith, "Capillary electrophoresis," Methods Enzymol. (1997) 289:469-78; Xu, "Capillary electrophoresis," Anal. Chem. (1995) 67(12):463R-473R; Landers, James (Ed.) Handbook of Capillary Electrophoresis, 2nd Ed. CRC Press, 1997. The resolution of CE peptide separations can be up to 10-fold higher in comparison to reverse phase HPLC, and single molecule detection can be achieved when coupled to LIF. Additionally, multichannel detectors and multicapillary (e.g., 96 capillary) instruments permit multiplexed, high throughput analyses. An exemplary configuration of a CE-LIF multichannel detection instrument is shown in Figure 1.

[0116] A signal can then be generated from the tABP-labeled protein(s) and/or peptide(s) present, and, if appropriate, the intensity compared from two or more samples. Detection and measurement can b>e achieved with a CCD, photomultiplier, etc., where the information may be transferred to a data processor for analysis. The different components of the samples and their relative amounts as measured by the intensity of their emissions can be analyzed by the data processor and a profile obtained that compares the number of components and the ratios between two or more samples, and or results can be compared to other profiles for comparison. In this way, one obtains the characteristics of the complex protein mixture as it is affected by changes in the cells due to differentiation, maturation, cell type and changes in the cellular environment.

[0117] "Identification" analysis methods [0118] As discussed above, where the migration rates in the separation stages of a screening method provide the necessary identification of the tABP-labeled protein(s) and/or peptide(s) no further analysis is required. However, where further confirmation of the results from the earlier analysis is desired or the earlier results do not provide certainty as to the identification and amount of a particular component, an identification method using mass spectrometry (MS) can be employed. See, for example, WO 00/11208. The use of mass spectrometry will be described below. Such identification methods potentially provide greater information, but requires greater sample size in comparison to, for example, capillary electrohoresis, and has a lower throughput.

[0119] In identification methods, chromato graphic and/or electrophoretic separation methods as described herein may be used to simplify the mixtures introduced into the mass spectrometer, allowing for a more accurate analysis. These separation methods may be employed prior to, following, or in lieu of sequestration of the tABP-labeled peptides described above. The use of fluorescent moieties as tABP ligands can permit the use of an online fluorescence detector to trigger ESI-MS data collection or fraction collection for subsequent analysis, e.g., providing sample on a MALDI plate. In this way, only fractions and bands that contain tABP-labeled peptides will be selected for further processing, thereby avoiding using the MS with certain fractions.

[0120] In particularly preferred embodiments, the identification methods described herein can be combined with one or more separation methods to develop a "separation profile" that can be used to identify tABP-labeled proteins and/or peptides without the need for MS analysis, these methods, a sample (e.g., material from a chromatography column) is divided into at least two portions; one portion is used for MS analysis, and the other portion(s) are used for one or more separation methods (e.g., a single CE run, or two or more CE runs using different separation conditions). The peptide identification obtained from the MS analysis can be assigned to the observed separation profile (e.g., the elution time of the peptide observed in the CE run(s)). Observation of this separation profile in subsequent samples can then be correlated to the peptide known to exhibit that separation profile. [0121] The identification methods described herein may also utilize tABPs that differ isotopically in order to enhance the information obtained from MS procedures. For example, using automated multistage MS, the mass spectrometer may be operated in a dual mode in which it alternates in successive scans between measuring the relative quantities of peptides obtained from the prior fractionation and recording the sequence information of the peptides. Peptides may be quantified by measuring in the MS mode the relative signal intensities for pairs of peptide ions of identical sequence that are tagged with the isotopically light or heavy forms of the reagent, respectively, and which therefore differ in mass by the mass differential encoded with the tABP. Peptide sequence information may be automatically generated by selecting peptide ions of a particular mass-to-charge (m/z) ratio for collision-induced dissociation (CID) in the mass spectrometer operating in the MSⁿ mode. (Link, et al., (1997) Electrophoresis 18:1314-34; Gygi, et al., (1999) idid 20:310-9; and Gygi et al., (1999) Mol. Cell. Biol. 19:1720-30). The resulting CID spectra may be then automatically correlated with sequence databases to identify the protein from which the sequenced peptide originated. Combination of the results generated by MS and MS" analyses of affinity tagged and differentially labeled peptide samples allows the determination of the relative quantities as well as the sequence identities of the components of protein mixtures.

[0122] Protein identification by MSⁿ may be accomplished by correlating the sequence contained in the CID mass spectrum with one or more sequence databases, e.g., using computer searching algorithms (Eng. et al. (1994) J. Am. Soc. Mass Spectrom. 5:976-89; Mann, et al., (1994) Anal. Chem. 66:4390-99; Qin, et al., (1997) ibid 69:3995-4001; Clauser, et al., (1995) Proc. Natl. Acad. Sci. USA 92:5072-76). Pairs of identical peptides tagged with the light and heavy affinity tagged reagents, respectively (or in analysis of more than two samples, sets of identical tagged peptides in which each set member is differentially isotopically labeled) are chemically identical and therefore serve as mutual internal standards for accurate quantitation. The MS measurement readily differentiates between peptides originating from different samples, representing different cell states or other parameter, because of the difference between isotopically distinct reagents attached to the peptides. The ratios between the intensities of the differing weight components of these pairs or sets of peaks provide an accurate measure of the relative abundance of the peptides and the correlative proteins because the MS intensity response to a given peptide is independent of the isotopic composition of the reagents. The use of isotopically labeled internal standards is standard practice in quantitative mass spectrometry (De Leenheer, et al., (1992) Mass Spectrom. Rev. 11:249-307).

[0123] The tABP-labeled peptides may provide specific fragmentation patterns in MS procedures. In this manner, the fragmentation pattern of the tABP- labeled peptides will aid in the identification of the proteins, or identifying which peptide(s) from a protein are labeled by a tABP. As an added advantage, where the tABP is positively charged, it will enhance the signal due to its easier ionization.

[0124] Isotopically distinguishable tABPs are useful when using MS detection for either the second dimension of separation, as in MALDI/TOF or when doing identification as in MS". Convenient isotopic labels are ²H, ¹³C, ¹⁵N, ¹⁷0, ¹⁸O or ³⁴S. The use of the isotopically labeled tABPs also allows for the use of isotopically differing internal standards. The internal standard tABP will typically be otherwise identical to the sample tABP.

[0125] The above procedures allow for analysis of complex protein mixtures such as proteomes. The analysis can be associated with screening of candidate compounds to determine the effect of the compound on the regulation of the target proteins, identification of the pathways that the candidate compound affects and the cellular response to the candidate compound. In this way the effectiveness of drugs may be analyzed, cross-reactivity determined, toxicity evaluated and other effects on cell physiology determined. Besides candidate compounds, the subject analysis may be used with natural products, environmental sample screening, and the like, hi each case one can identify specific proteins that are affected by the environment of the cells and compare the effect of the environment with changes observed with other environments. The subject methods can be used in high throughput primary or secondary screening, where candidate compounds are evaluated for their efficacy and O 03/079014

cross-reactivity, their influence on physiologic pathways and their interactions with other drugs that may be taken for the same indication.

[0126] Kits can be provided that combine one or more tABPs for specific and/or related groups of proteins, with the proteolytic enzymes, in solution, but usually either as a lyophilized or bound to a support. Also included can be solid supports for the sequestration of the conjugates, both intact and fragment, where the supports may be any solid support, such as particles and beads of from about 5 to lOOμ, the walls of vessels, such as the microtiter wells of microtiter plates, capillaries, etc., to which the receptor is bound. The solid supports may be treated with an innocuous protein, such as serum albumin, to occupy hot spots and inhibit nonspecific binding. Pre-labeled internal standards may also be supplied.

[0127] The following examples are offered by way of illustration and not by way of limitation.

[0128] Example 1 : Tethering of ABP-labeled active target proteins

[0129] Figure 1 provides a schematic description of two alternative methods by which active target proteins can become tethered to a solid support via a cleavable linker. In Figure 1(a), an ABP is attached to a solid phase via a cleavable linker. A complex protein mixture (e.g., a proteome) is treated with the tethered probe and active proteins from targeted families are covalently bound to the support. Proteins from other families that do not react with the probe are in solution and are washed away. Cleavage of the covalently bound protein family yields soluble proteins that are covalently modified at the active site.

[0130] In an alternative method shown in Figure 1(b), an ABP is contacted in solution with a complex protein mixture (e.g., a proteome). Following binding of active proteins from targeted families, the ABP-bound active target proteins are captured by covalent binding of the ABP to the support. Proteins from other families that do not react with the probe are in solution and are washed away. Cleavage of the covalently bound protein family again yields soluble proteins that are covalently modified at the active site. In these methods, a pair of complementary reactive groups must be provided on the solid phase and the ABP employed. Preferably, the complementary reactive groups do not react to an appreciable extent with reactive groups present in components of the complex protein mixture.

[0131] Figure 1 (c) provides a specific example of the method described in Figure 1(a), using a serine hydrolase-directed fluorophosphonate ABP attached to a photocleavable linker. Similarly, Figure 1(d) provides a specific example of the method described in Figure 1(a) using a serine hydrolase-directed fluorophosphonate ABP attached to an acid-cleavable linker. In this example, the ABP also comprises a fluorescent ligand. As noted in Figure 1(d), treatment with trypsin while the proteins are tethered to the support will yield a soluble mixture of non-active site labeled fragments that can either be identified or removed by washing. After removal of the soluble tryptic fragments, cleavage of the remaining active site fragments enables separate identification of the two components.

[0132] Figure 1(e) provides a specific example of the method described in figure 1(b), again using using a serine hydrolase-directed fluorophosphonate ABP attached to a photocleavable linker. While this example is drawn to a azide/nitrile pair, forming a tetrazole, the skilled artisan will understand that other reactive pairs, such as aryl iodide/alkyne coupling catalyzed by an aryl phosphine; aryl iodide/alkene coupling catalyzed by palladium; aryl chloride, bromide, or iodide/aryl boronic acid coupling catalyzed by palladium; boronic acid/diol coupling; and/or boronic acid/amino diol coupling, would also provide suitable chemistry for this method.

[0133] Example 2: Analysis of multiple samples

[0134] Figure 2 provides a schematic description of two alternative methods by which two samples may be compared using the methods of the present invention, using ABP(s) attached to a solid phase via a cleavable linker as an exemplary platform for the analysis. As described in figure 1, complex protein mixtures (e.g., proteomes) are treated with the tethered probe and active proteins from targeted families are covalently bound to the support. Proteins from other families that do not react with the probe are in solution and are washed away. Cleavage of the covalently bound protein family yields soluble proteins that are covalently modified at the active site. In Figure 2(a), distinguishable labels (Label- 1 and Label-2) are attached to the sequestered proteins from the two samples, while in Figure 2(b), the tABPs are themselves distinguishable by the attached ligands (LI and L2).

[0135] hi Figures 2(c) and 2(d), the use of isotopic labels in these methods to assist in mass spec identification are shown. In Figure 2(c), isotopically distinguishable labels are incorporated into the tethered serine hydrolase, while in Figure 2(d), the tABPs are isotopically distinguishable. One result of the method described in Figure 2(c) is that multiple labeled peptide fragments may be distinguished by mass spec analysis rather than just the active site fragment, as the isotopic label is not restricted to a single site in each serine hydrolase. This can provide greater assurance that protein identities based upon mass spec sequence data are accurate.

[0136] Example 3: Synthesis of 5-N-(l 1 '-Azido-3 ',6',9'-trioxoundecanyl)- amino- 1 -fluoro-2,4-dinitro-benzene

[0138] To a stirred solution of 1 -amino- 11 -azido-3, 6,9-trioxoundecane (61.7 mg, 0.282 mmole) in dichloromethane (3 ml) at room temperature were added 1,5- difluoro-2,4-dinitrobenzene (68.95 mg, 0.338 mmole) and N,N-diisopropylethylamine (98 μL, 0.516 mmol). The resulting mixture was stirred at the temperature overnight and subsequently purified by preparative HPLC to give the title compound as a yellow oil (58.4 mg, 52% yield): 1H ΝMR (400MHz, CDC1₃) δ 9.13 (d, 1H, J = 8.4 Hz), 8.80 (br. s, 1H), 6.67 (d, 1H, J = 13.2 Hz), 3.83 (t, 2H, J = 4.8 Hz), 3.6-3.7 (m, 10H), 3.54 (q, 2H, J = 5.2 Hz); 3.38 (t, 2H, J = 5.2 Hz); API-ES-MS: 424 ([M + Νa]⁺); 403 ([M + H]⁺).

[0139] Example 4: Synthesis of 4-N-(ll '-Azido-3',6',9'-trioxoundecanyl)- amino-2,6-dichloro- 1,3,5 -triazine

[0141] To a stirred solution of 1-anιino-l 1 -azido-3, 6,9-trioxoundecane

(74.6mg, 0.34 mmole) in dichloromethane (4 ml) at room temperature were added cyanuric chloride (75.3 mg, 0.408 mmole) and NN-diisopropylethylamine (118 μL, 0.621 mmol). The resulting mixture was stirred at the temperature overnight and purified by preparative HPLC to give the title compound as a colorless oil (34.5mg, 28% yield): 1H ΝMR (400MHz, CDC1₃) δ 6.67 (br. s, IH), 3.6-3.7 (m, 14H), 3.40 (t, 2H, J = 5.2 Hz); API-ES-MS: 403 ([M + H]⁺).

[0142] Example 5 : Synthesis of N-(l 1 '-Azido-3 ',6',9'-trioxoundecanyl)-2- chloroacetylamide

[0144] To a stirred solution of 1 -amino- 1 l-azido-3,6,9-trioxoundecane (68.2 mg, 0.311 mmole) in dichloromethane (4 ml) at 4°C were added 2-chloroacetyl chloride (42.2 mg, 0.373 mmole) and NN-diisopropylethylamine (70 μL, 0.368 mmol). The resulting mixture was allowed to warm up to room temperature overnight and purified by preparative HPLC to give the title compound as a colorless oil (43 mg, 47% yield): 1H ΝMR (400MHz, CDC1₃) δ 6.99 (br. s, IH), 4.05 (s, 2H), 3.6-3.7 (m, 10H), 3.59 (t, 2H, J = 5.2 Hz), 3.50 (q, 2H, J - 5.2 Hz), 3.38 (t, 2H, J = 5.2 Hz); API-ES-MS: 295 ([M + H]⁺). [0145] Example 6: Synthesis of 5-(and -6)-Carbonyltetramethylrhodamine

-N-(l 1 '-azido-3 ',6',9'-trioxo-undecanyl)amide

[0147] To a stirred solution of 1 -amino- 11 -azido-3, 6,9-trioxoundecane (6.7 mg, 0.03 mmole) in dichloromethane (0.5 ml) at room temperature were added 5-(and -6)-carboxytetramethylrhodamine, succinimidyl ester (9.3 mg, 0.017 mmole) in DMF (0.5 mL) and NN-diisopropylethylamine (20 μL, 0.105 mmol). The resulting mixture was stirred overnight at this temperature and after removal of the solvent purified by preparative HPLC to give the title compound as a reddish powder (8.5 mg, 76% yield): 1H ΝMR (400MHz, DMSO-d₆) 6 8.96 (dd, IH), 8.54 (br. s, IH), 8.25 (dd, IH, J = 6.8 Hz), 8.15 (d, IH), 8.01 (br. s, IH), 7.73 (s, IH), 7.42 (d, IH), 6.68 (s, 3H), 3.3- 3.6 (m, 24H), 3.07 (br. s, 4H); API-ES-MS: 631 ([M + H]⁺).

[0148] Example 7: Synthesis of O-(ω-Fluoromonoethylphosphono-decyl)-

N-propargyl-carbamate

[0150] To a stirred solution of propargyl amine (8.9 mg, 0.161 mmole) and

Et₃Ν (20 μl, 0.14 mmole) in dichloromethane (500 μl) was added a solution of O-(ω- fluoromonoethylphosphono-decyl)carbonic acid succinimidyl ester (10 mg, 0.025 mmole) in dichloromethane (300 μl) at room temperature. The resulting mixture was stirred for 3 hours at this temperature and then purified by flash chromatography on a small C18 silica cartridge (Alltech), eluting with 50% ethyl acetate in hexanes to give the title compound as a light yellow oil (8.7 mg, 51%): 1H NMR (400MHz, CDC1₃) δ 7.69 (t, IH), 4.88 (br. s, IH), 4.27 (m, 2H), 4.08 (t, 2H), 3.97 (s, 2H), 2.23 (t, IH, J = 2.4 Hz), 1.92 (m, 2H), 1.6-1.7 (m, 2H), 1.2-1.4 (m, 12H), 1.12 (t, 3H, J = 7.2 Hz); ³¹P NMR (162 MHz,CDCl₃) δ 34.48 (s, IP), 29.88 (s, IP). API-ES-MS: 350 ([M + H]⁺).

[0151] Example 8 : Synthesis of O-(ω-Fluoromonoethylphosphono- butynoyl)-N-(6-heptynoyl)- 3,5-dioxooctyldiamine:

[0153] To a stirred solution of N-(ω-fluoromonoethylphosphono-butynoyl -

3,5-dioxooctyldiamine (32.5mg, O.lmmole) and Et₃Ν (20 μl, 0.14 mmole) in dichloromethane (500 μl) was added a solution of 6-heptynoic acid succinimidyl ester (24 mg, 0.11 mmole) in dichloromethane (300 μl) at room temperature. The resulting mixture was stirred for 3 hours at this temperature and after removal of the solvent purified by prep. HPLC to give the title compound as a colorless oil (33.6mg, 77% yield): 1H NMR (400MHz, CDC1₃) δ 7.95 (br. s, IH), 7.87 (br. s, IH), 4.15 (m, 2H), 3.48 (s, 2H), 3.35-3.38 (m, 8H), 3.14-3.18 (m, 2H), 2.73-2.75(m, 2H), 1.93-2.22 (m, 6H), 1.92 (m, 2H), 1.67-1.75 (m, IH), 1.50-1.59 (m, 2H), 1.35-1.46 (m, 2H), 1.26 (t, 3H, J = 7.2 Hz); ³¹P NMR (162 MHz,CDCl₃) δ 36.63 (s, 0.5P), 30.10 (s, 0.5P). API-ES-MS: 437 ([M + H]⁺).

[0154] Example 9: Synthesis of N-[5-(and -6)-

Carbonyltetramethyhhodamine] -N'-(6-Heptynoyl)- 1 ,6-hexanediamine

[0156] A solution of 6-heptynoic acid (12.6 mg, 0.1 mmole), N- hydroxysuccinimide (12.7mg, 0.11 mmol), and 1,3-diisopropylcarboddiimide (13.9 mg, 0.11 mmol) in dichloromethane (0.5 ml) was stirred at room temperature for 6 hours before N-[5-(and -6)-carbonyltetramethyhhodamine]-l,6-hexanediamine (10 mg, 0.018 mmol) in DMF (0.5 mL) and triethylamine (20 μL, 0.14 mmol) were added. The resulting mixture was stirred overnight and after removal of the solvent purified by preparative HPLC to give the title compound as a reddish powder (6.6 mg, 58% yield): 1H ΝMR (400MHz, DMSO-d₆) δ 8.88 (dd, 0.5H), 8.73 (dd, 0.5H), 8.66 (d, 0.5H), 8.27 (d, IH), 7.73-7.79 (m, IH), 7.86 (d, 0.5H), 7.54 (d, 0.5 H), 7.0-7.05 (m, 3.5H), 6.95 (s, 2H), 3.25-3.56 (m, 16H), 2.72 (m, 2H), 2.1 (dq, 2H, J = 14.8, 2.4 Hz ), 2.03 (m, 2H), 1.3-1.58 (m, 12H); API-ES-MS: 637 ([M + H]⁺).

[0157] Example 10: Synthesis of N-[5-(and -6)-

Carbonyltetramethylrhodamine]-N'-(6-Heptynoyl)-3,5-dioxooctyldiamine

[0159] A solution of 6-heptynoic acid (12.6 mg, 0.1 mmole), N- hydroxysuccinimide (12.7mg, 0.11 mmol), and 1,3-diisopropylcarboddiimide (13.9 mg, 0.11 mmol) in dichloromethane (0.5 ml) was stirred at room temperature for 6 hours before N-[5-(and -6)-carbonylteframethyhhodamine]-3,5-dioxooctyldiamine (15 mg, 0.027 mmol) in DMF (1 mL) and triethylamine (20 μL, 0.14 mmol) were added. The resulting mixture was stirred overnight and after removal of the solvent purified by preparative HPLC to give the title compound as a reddish powder (7.5 mg, 42% yield): 1H NMR (400MHz, DMSO-d₆) δ 8.96 (dd, 0.5H), 8.83 (dd, 0.5H), 8.68 (d, 0.5H), 8.30 (d, 1H), 8.23 (d, 0.5H), 7.86 (d, 0.5H), 7.81-7.85 (m, IH), 7.57 (d, 0.5 H), 7.0-7.03 (m, 3H), 6.95 (s, 2H), 3.25-3.58 (m, 22H), 3.17 (q, IH), 3.10 (q, IH), 2.78 (m, IH), 2.66-2.72 (m, 2H), 2.19 (dt, IH), 2.03-2.13 (m, 2H), 1.3-1.58 (m, 4H); API-ES-MS: 669 ([M + H]⁺).

[0160] Example 11 : Synthesis of N-5-(and -6)-

Carbonyltetramethylrhodamine]-N'-(l-methyl-2-diphenylphosphinoterephthalate)-

3,5-dioxooctyldiamine

[0162] To a stirred solution of 1 -methyl-2-diphenylphosphinoterephthalate

(26.2 mg, 0.072 mmole), 1-hydroxybenzotriazole (10.7mg, 0.079 mmol), and 1,3- diisopropylcarboddiimide (11.8 mg, 0.09 mmol) in DMF (1 ml) were added N-[5-(and -6)-carbonyltetramethylrhodamine]-3,5-dioxooctyldiamine (44 mg, 0.079 mmol) in DMF (1 mL) and triethylamine (20 μL, 0.14 mmol). The resulting mixture was stirred overnight and after removal of the solvent purified by preparative HPLC to give the title compound as a reddish powder (27.1 mg, 41% yield): 1H ΝMR (400MHz, DMSO-d₆) δ 8.96 (dd, 0.5H), 8.81 (dd, 0.5H), 8.69 (d, 0.5H), 8.61 (dd, 0.5H), 8.55 (dd, 0.5H), 8.30 (d, IH), 8.23 (dd, 0.5H), 7.99 (m, IH), 7.84-7.90 (m, 1.5H), 7.56 (d, 0.5H), 7.35-7.41 (m, 6H), 7.15-7.20 (m, 3H), 6.92-7.05 (m, 5H), 3.63 (s, 3H), 3.25-3.58 (m, 22H), 3.17 (q, IH), 3.10 (q, IH), 2.78 (m, IH), 2.66-2.72 (m, 2H), 2.19 (dt, IH), 2.03-2.13 (m, 2H), 1.3-1.58 (m, 4H); ); ³¹P ΝMR (162 MHz,CDCl₃) δ -4.15 (s, 0.5P), -4.12 (s, 0.5P). API-ES-MS: 906 ([M + H]⁺). [0163] Example 12: "Click Reaction" in Solution

[0164] Figure 7 exemplifies a typical "click reaction" for performance of the methods described herein. In this example, the molecule from Example 8 in DMSO was added to a solution of protein and/or proteome samples at 2-10 mg/mL so that the final concentration of the probe is 2-10 μM. After incubation at room temperature for 1 hr or 6 hr at 4 °C, to the solution were added ligation reagent (20-50 μM) such as the TAMRA azide in DMSO, tris(2-carboxyethyl)phosphine (TCEP) (0.5-2 mM) in water, and copper sulfate (0.5-2 mM) in water. After gently shaking for 1 hr at room temperature, the mixture was subjected to standard biochemical assays such as SDS/PAGE to monitor the "click reaction". In a typical experiment, 5 μM of probe: FP-PEG3-Alkyne was reacted in Hepes buffer, pH 8.0 with Mouse Liver Proteome (at 5 mg/mL) at room temperature for lhr., followed by "click reaction" with 50 μM of the molecule in Example 6 and ImM of TCEP and CuSO₄.

[0165] 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 invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, h case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0166] While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

Claims

WHAT IS CLAIMED IS:

1. A method of processing a complex protein mixture, comprising contacting said complex protein mixture with one or more activity based probes that specifically bind to one or more active target proteins in said complex protein mixture, wherein said activity based probes are covalently bound to a solid phase, or are subsequently contacted with a solid phase that covalently bind said activity based probes, whereby said active target proteins bound to said activity based probes are sequestered on said solid phase; removing one or more components from said complex protein mixture that are not sequestered on said solid phase; and releasing sequestered active target proteins by cleaving one or more covalent bonds in said activity based probes or between said activity based probes and said solid phase.

2. A method of processing a complex protein mixture, comprising contacting said complex protein mixture with one or more activity based probes that specifically bind to one or more active target proteins in said complex protein mixture, wherein said activity based probes are covalently bound to a solid phase, or are subsequently contacted with a solid phase that covalently bind said activity based probes, whereby said active target proteins bound to said activity based probes are sequestered on said solid phase; removing one or more components from said complex protein mixture that are not sequestered on said solid phase; and releasing sequestered active target proteins by cleaving one or more covalent bonds in said activity based probes or between said activity based probes and said solid phase, wherein prior to or following said releasing step, said sequestered active target proteins are proteolyzed to generate one or more peptides bound to said activity based probes.

3. A method of processing two or more complex protein mixtures, comprising: contacting a first complex protein mixture with a first set of one or more activity based probes that specifically bind to one or more active target proteins in said first complex protein mixture, and contacting a second complex protein mixture with a second set of one or more activity based probes that specifically bind to one or more active target proteins in said second complex protein mixture, wherein said activity based probes are covalently bound to a solid phase, or are subsequently contacted with a solid phase that covalently bind said activity based probes, whereby active target proteins in said first and second complex protein mixtures bound to said activity based probes are sequestered on said solid phase, and wherein corresponding activity based probes in said first and second sets comprise a label by which said corresponding probes may be distinguished from one another; removing one or more components from said complex protein mixture that are not sequestered on said solid phase; and releasing sequestered active target proteins by cleaving one or more covalent bonds in said activity based probes or between said activity based probes and said solid phase.

4. A method of processing two or more complex protein mixtures, comprising: contacting a first complex protein mixture with a first set of one or more activity based probes that specifically bind to one or more active target proteins in said first complex protein mixture, and contacting a second complex protein mixture with a second set of one or more activity based probes that specifically bind to one or more active target proteins in said second complex protein mixture, wherein said activity based probes are covalently bound to a solid phase, or are subsequently contacted with a solid phase that covalently bind said activity based probes, whereby active target proteins in said first and second complex protein mixtures bound to said activity based probes are sequestered on said solid phase, and wherein corresponding activity based probes in said first and second sets comprise a label by which said corresponding probes may be distinguished from one another; removing one or more components from said complex protein mixture that are not sequestered on said solid phase; and releasing sequestered active target proteins by cleaving one or more covalent bonds in said activity based probes or between said activity based probes and said solid phase wherein prior to or following said releasing step, said sequestered active target proteins are proteolyzed to generate one or more peptides bound to said activity based probes.

5. A method of processing two or more complex protein mixtures, comprising: contacting a first complex protein mixture with a first set of one or more activity based probes that specifically bind to one or more active target proteins in said first complex protein mixture, and contacting a second complex protein mixture with a second set of one or more activity based probes that specifically bind to one or more active target proteins in said second complex protein mixture, wherein said activity based probes are covalently bound to a solid phase, or are subsequently contacted with a solid phase that covalently bind said activity based probes, whereby active target proteins in said first and second complex protein mixtures bound to said activity based probes are sequestered on said solid phase; removing one or more components from said complex protein mixture that are not sequestered on said solid phase; labeling active target proteins in said first and second complex protein mixtures bound to said activity based probes sequestered on said solid phase, whereby active target proteins from said first and second complex protein mixtures may be distinguished from one another; and releasing sequestered active target proteins by cleaving one or more covalent bonds in said activity based probes or between said activity based probes and said solid phase.

6. A method of processing two or more complex protein mixtures, comprising: contacting a first complex protein mixture with a first set of one or more activity based probes that specifically bind to one or more active target proteins in said first complex protein mixture, and contacting a second complex protein mixture with a second set of one or more activity based probes that specifically bind to one or more active target proteins in said second complex protein mixture, wherein said activity based probes are covalently bound to a solid phase, or are subsequently contacted with a solid phase that covalently bind said activity based probes, whereby active target proteins in said first and second complex protein mixtures bound to said activity based probes are sequestered on said solid phase; removing one or more components from said complex protein mixture that are not sequestered on said solid phase; labeling active target proteins in said first and second complex protein mixtures bound to said activity based probes sequestered on said solid phase, whereby active target proteins from said first and second complex protein mixtures may be distinguished from one another; and releasing sequestered active target proteins by cleaving one or more covalent bonds in said activity based probes or between said activity based probes and said solid phase wherein prior to or following said releasing step, said sequestered active target proteins are proteolyzed to generate one or more peptides bound to said activity based probes.

7. A method according to claim 3 or 4, wherein said corresponding activity based probes in said first and second sets comprise an isotopic label distinguishable by mass spectrometry.

8. A method according to claim 3 or 4, wherein said corresponding activity based probes in said first and second sets comprise a fluorescent label distinguishable optically.

9. A method according to claim 5 or 6, wherein said active target proteins are labeled with an isotopic label distinguishable by mass spectrometry.

10. A method according to claim 5 or 6, wherein said active target proteins are labeled with a fluorescent label distinguishable optically.

11. A method according to any one of claims 1-6, wherein said complex protein mixture(s) are proteomes.

12. A method of analyzing a complex protein mixture, comprising: processing a sample by the method of claim 1; and determining the presence, amount, activity, or identity of said released active target proteins.

13. A method according to claim 12, wherein the presence, amount, activity, or identity of said released active target proteins is determined by separating said released active target proteins into one or more components, and generating a signal from one or more active target proteins bound to one of said activity based probes.

14. A method of analyzing a complex protein mixture, comprising: processing a sample by the method of claim 2; and determining the presence, amount, activity, or identity of one or more of said peptides bound to said activity based probes.

15. A method according to claim 14, wherein the presence, amount, activity, or identity of said peptides bound to said activity based probes is determined by separating said peptides bound to said activity based probes into one or more components, and generating a signal from one or more peptides bound to one of said activity based probes.

16. A composition comprising: one or more activity based probes covalently bound to a solid phase.

17. A composition according to claim 16, further comprising a protein or peptide derived from a complex protein mixture covalently bound to one or more of said activity based probes.

18. A composition according to claim 16, wherein said solid phase comprises a plurality of different activity based probes boxmd at discrete locations on said solid phase.