WO2008022355A2 - Procédés et réactifs de détection de groupes phosphomonoester - Google Patents

Procédés et réactifs de détection de groupes phosphomonoester Download PDF

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Publication number
WO2008022355A2
WO2008022355A2 PCT/US2007/076343 US2007076343W WO2008022355A2 WO 2008022355 A2 WO2008022355 A2 WO 2008022355A2 US 2007076343 W US2007076343 W US 2007076343W WO 2008022355 A2 WO2008022355 A2 WO 2008022355A2
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Prior art keywords
group
composition
phospho
sample
anion
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PCT/US2007/076343
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English (en)
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WO2008022355A3 (fr
Inventor
Wayne F. Patton
Thomas E. Miller
Linan Song
William Henry Connors
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Perkinelmer Las, Inc.
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Publication of WO2008022355A2 publication Critical patent/WO2008022355A2/fr
Publication of WO2008022355A3 publication Critical patent/WO2008022355A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F3/00—Compounds containing elements of Groups 2 or 12 of the Periodic Table
    • C07F3/003—Compounds containing elements of Groups 2 or 12 of the Periodic Table without C-Metal linkages
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms

Definitions

  • compositions that include a chelating moiety that can selectively binding to a phosphomonoester group and a sufficient concentration of counter-anions to deter non-specific binding of the chelating moiety.
  • the compositions can be used to selectively detect a phosphomonoester group, e.g., on a target molecule.
  • compositions described herein are useful in a variety of applications including, but not limited to: (i) detecting the presence or absence of a phosphorylated molecule (e.g., in a sample, in a gel, or on a membrane); (ii) determining the substrate specificity of one or more phospho-transfer enzymatic activities; (iii) identifying compounds capable of modifying one or more phospho-transfer activities; (iv) optimizing reaction conditions for one or more phospho-transfer activities, or (v) identifying one or more phospho-transfer activities (e.g., identify an unknown kinase activity in a sample).
  • compositions described herein are useful in, e.g., methods for determining whether or not a subject has, or is at risk of developing, a disease characterized by aberrant function or expression of a phospho- transfer activity or methods for identifying compounds useful for treating such disorders.
  • the disclosure provides a composition
  • a composition comprising: a dinuclear metal chelating moiety; a divalent metal cation capable of simultaneously binding to the dinuclear metal chelating moiety and a phosphomonoester group on a target molecule (e.g., a phosphorylated target molecule); and a counter-anion that binds the divalent metal cation with an affinity lower than the affinity with which the phosphomonoester group on a target molecule (e.g., a phosphorylated target molecule) binds the divalent metal cation.
  • a target molecule e.g., a phosphorylated target molecule
  • the disclosure provides a composition comprising a ternary complex comprising: a dinuclear metal chelating moiety; a divalent metal cation capable of simultaneously binding to the dinuclear metal chelating moiety and a phosphomonoester group; and a counter-anion, wherein the counter-anion binds the divalent metal cation with an affinity lower than the affinity with which the phosphomonoester group binds the divalent metal cation.
  • the disclosure features a composition
  • a composition comprising: (1) a dinuclear chelating moiety (e.g., a dinuclear metal chelating moiety); (2) divalent cations (e.g., divalent metal cations); and (3) a counter-anion.
  • the three components are present at a concentration such that at least 80, 90, 95, 98, or 99.5% of the chelating moieties in the composition are in a ternary complex with the divalent cations and the counter-anion.
  • the dinuclear chelating moiety is capable of binding to a phosphomonoester group, e.g., on a phosphorylated molecule.
  • the counter-anion binds to the chelating moiety at a site such that it competes with the phosphomonoester group for binding to the complex.
  • the counter-anion can bind the divalent metal cation with an affinity that is at least equal to the affinity with which a carboxylate anion binds to the divalent metal cation. In some embodiments of any of the compositions, the counter-anion can bind the divalent metal cation with an affinity that is greater than the affinity with which a carboxylate anion binds to the divalent metal cation.
  • the dinuclear metal chelating moiety can comprise the structure of formula (I):
  • each Ri, R 2 , R 3 , and R 4 are, independently, an H; an alkyl group having 1 to 16 carbon atoms; an acyl group; a carboxyalkyl group; an acylalkyl group; a carbamoylalkyl group; a cyanoalkyl group; a hydroxyalkyl group; an aminoalkyl group; or a haloalkyl group having 1 to 16 carbon atoms and 1 to 5 halogens; a carboxyl group; a carbamoyl group; a hydroxyl group; a cyano group; an amino group; or a halogen group, each n and m are independently 0 or 1; and Ar is aryl.
  • Each n and m can be 0 and/or 1.
  • Each of Ri, R 2 , R3, and R 4 can be H.
  • the Ar can be, e.g., anthracene, , or a phenyl.
  • each of Ri, R 2 , R3, and R 4 can be H; n and m can each be 1 ; and Ar can be
  • the dinuclear metal chelating moiety can comprise the structure of formula (II):
  • each R 1 is independently H, Ci-C 6 alkyl, or arylalkyl
  • R 2 is H, Ci-C 6 alkyl, or arylalkyl
  • each R 3 is independently a nitrogen containing moiety comprising a nitrogen and a linker.
  • the nitrogen can be a component in a heteroaryl ring.
  • Each R 1 can be H and/or R 2 can be H. In some embodiments, each R 1 is H and R 2 is H.
  • Each R can be the same nitrogen containing moiety comprising a nitrogen containing heteroaryl ring and a linker or R can comprise a fused nitrogen containing
  • the linker of R can be S f v or
  • Each R 3 can contain, or be, a pyridyl.
  • each R 3 can be R R , wherein each R 4 is independently H or C1-C6 alkyl.
  • each R 4 can be methyl.
  • the dinuclear metal chelating moiety can comprise the structure of formula (III):
  • each R 1 is independently H, Ci-C 6 alkyl, or arylalkyl
  • R 2 is H, Ci-C 6 alkyl, or arylalkyl; each R is independently a nitrogen containing moiety comprising a nitrogen containing ring and a linker; and each R 4 is independently H, Ci-C 6 alkyl or a nitrogen containing moiety comprising a nitrogen containing ring and a linker.
  • Each R 1 and R 2 can be H. In some embodiments, each R 1 is H and R 2 is H.
  • Each R 3 can be the same nitrogen containing moiety comprising a nitrogen
  • Each R 3 can be, e.g.,
  • Each R can be the same nitrogen containing moiety comprising a nitrogen containing ring and a linker and each R 4 is H.
  • each R 3 is and each R 4 is H.
  • R 3 and R 4 for each occurrence, are the same nitrogen containing moiety comprising a nitrogen containing ring and a linker.
  • R 3 and R 4 for each occurrence, are the same nitrogen containing moiety comprising a nitrogen containing ring and a linker.
  • R 4 for each occurrence, can be
  • the dinuclear metal chelating complex can have the structure of formula (IV) (a representative chelate for forming an alkoxide-bridged M(II)2 hexaazacryptate complex):
  • the dinuclear metal chelating moiety can have the structure of formula (V), a representative chelate for forming a naphthyridine -based M(II)2 complex:
  • the dinuclear metal chelating moiety can have the structure of formula (VI):
  • the dinuclear metal chelating moiety can have the structure of formula (VI), wherein R1-R5, n, m, and p are as follows:
  • R is an unsulfonated (or non-sulfonated) directly detectable label.
  • R can be selected from the group consisting of
  • the dinuclear metal chelating moiety can have the structure of formula (VI), wherein R1-R5, n, m, and p are as follows:
  • the composition can contain, or be, the metal cation is Zn 2+ and can have the following formula:
  • the dinuclear metal chelating moiety can be detectably labeled.
  • the detectable label can be a fluorescent label, a luminescent label, a chemoluminescent label, a radioactive label, or a nano-sized precious metal such as gold or silver.
  • the fluorescent label is selected from the group consisting of FITC, TRITC, coumarin, or a ruthenium-based organic dye.
  • the dinuclear metal chelating moiety can be fluorescent.
  • the counter- anion can be a dye comprising a hydroxyl moiety.
  • the dye can be selected from the group consisting of a polyhydroxybenzenesulfonephthalein dye and a polyhydroxybenzenephthalein dye.
  • the dye can be selected from the group consisting of pyrocatechol red, pyrogallol red, bromopyrogallol red, pyrogallol phthalein, and combinations of any of the foregoing.
  • the counter-anion can be a catechol-based dye.
  • the counter-anion can be, or contain, a 2,3,7-trihydroxy-9R-6-isoxanthone, wherein the R group at the C9 position is selected from the group consisting of a C1-C8 alkyl group which may be cyclic or acyclic, linear or branched, optionally substituted with a group selected from OH, halo, -SO 3 H, and COOH; and an aryl group optionally substituted with a group selected from OH, halo, SO3H, and COOH.
  • the aryl group can be a phenyl.
  • the aryl group can be substituted with one or more Br, with one or more SO 3 H, with one or more COOH, or with one of more of any combination of the foregoing.
  • the counter-anion can be, or contain, a 3,4,5-trihydroxy-9R-6-isoxanthone, wherein the R group at the C9 position is selected from the group consisting of a C1-C8 alkyl group which may be cyclic or acyclic, linear or branched, optionally substituted with a group selected from OH, halo, -SO 3 H, and COOH; and an aryl group optionally substituted with a group selected from OH, halo, SO3H, and COOH; and wherein the isoxanthone moiety is optionally substituted with one or more groups selected from OH and halo.
  • the isoxanthone moiety can be substituted with one or more Br.
  • the aryl group can be a phenyl and the phenyl can be substituted with SO 3 H or COOH, or both.
  • the R group can be 2-sulfophenyl or 2-carboxyphenyl.
  • the isoxanthone moiety can be substituted with one or more (e.g., two, three, four, or more) halo groups (e.g., Br).
  • the Br groups can occur at the 2,7 positions or the 2,5 positions of the isoxanthone.
  • the counter- anion can be selected from the group consisting of Pyrogallol red (3,4,5-trihydroxy-9-(2'- sulfophenyl)-6-isoxanthone) and Gallein (4,5-dihydroxyfluoresceine).
  • the counter- anion is selected from the group consisting of BPR (3,4,5-trihydroxy-2,7-dibromo-9-(2'- sulfophenyl)-6-isoxanthone) and BG (dibromogallein).
  • the counter- anion is selected from the group consisting of Bromopyrogallol Red, Pyrocatechol Violet, Pyrogallol Phtalein, Pyrogallol Red, Quercetin dihydrate (3,3',4',5,7- pentahydroxyflavone), Piceatannol (3,4,3',5'-Tetrahydroxy-trans-stilbene), and Tiron (4,5-dihydroxy-l,3-benzenedisulfonic acid).
  • the counter- anion in complex with the dinuclear metal chelating moiety can have a different detectable signal than the free counter-anion.
  • the divalent metal cation can be Zn(II) or any other divalent metal cations described herein.
  • the composition can contain at least two divalent metal cations (e.g., at least two of the same divalent metal cations or at least two different divalent metal cations).
  • the dinuclear metal chelating moiety and the counter-anion are not covalently joined.
  • the disclosure provides a method for forming a ternary complex, the method comprising: contacting (i) a dinuclear metal chelating moiety with (ii) a salt comprising a divalent metal cation and (ii) a counter-anion under conditions where (i), (ii), and (iii) form a ternary complex, wherein the divalent metal cation is capable of simultaneously binding to the dinuclear metal chelating moiety and a phosphorylated target molecule and wherein the counter-anion binds the divalent metal cation with an affinity lower than the affinity with which the phosphorylated target molecule binds the divalent metal cation.
  • the method can also include the step of determining the amount of ternary complex formed. Also featured is a composition formed from the method.
  • the disclosure features a method of detecting a phosphomonoester group on a target molecule.
  • the method includes forming a mixture that includes the target molecule and a dinuclear chelating moiety is capable of binding to a phosphomonoester group under conditions in which non-specific binding of the chelating moiety is reduced.
  • the mixture includes a sufficient concentration of counter-anions that are capable of binding to the chelating moiety at a site in competition with the phosphomonoester group for binding to the chelating moiety.
  • the mixture can be formed in any appropriate manner.
  • a composition containing the chelating moiety and the counter-anion can be added to a sample containing the target molecule.
  • the counter-anion can be added to a sample containing the target molecule first, and the chelating moiety is added subsequently.
  • the counter-anion can be present in the mixture at a concentration of between 1-50 or 5-200 micromolar.
  • the disclosure features a method for detecting the presence or absence of a phosphomonoester group, the method comprising: contacting a target molecule with a binding agent capable of selectively binding to a phosphomonoester group under conditions in which binding of the phosphomonoester group to the binding agent competes with binding of a counter-anion to the binding agent; and detecting binding of the binding agent to the target molecule as an indication that the target molecule comprises a phosphomonoester group.
  • the binding agent can be any of the compositions described above and the phosphomonoester-selective binding agent portion (e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions) of the composition binds to a phosphomonoester group, if present, on the target molecule.
  • the method can further include the step of after contacting the target molecule with any of the compositions described above, separating the target molecule from the unbound composition.
  • the disclosure features a method for detecting the presence or absence of a phosphomonoester group, the method comprising: contacting a target molecule with any of the compositions described above; and detecting binding of the dinuclear metal-chelating moiety (in complex with at least one (e.g., one or two) divalent metal cation) to the target molecule as an indication that the target molecule comprises a phosphomonoester group.
  • the method can further include the step of after contacting the target molecule with any of the compositions described above, separating the target molecule from the unbound composition.
  • the target molecule can be phosphorylated.
  • the dinuclear metal chelating moiety can be detectably labeled (e.g., fluorescently labeled).
  • the detecting can comprise determining the presence or absence of the detectable label. In some embodiments, the detecting can comprise determining the presence or amount of free detectable counter- anion as an indication that the dinuclear metal-chelating moiety bound to the target molecule. In some embodiments of any of the above methods, the counter-anion can be selected from the group consisting of Bromopyrogallol Red, Pyrocatechol Violet, Pyrogallol Phtalein, Pyrogallol Red, Quercetin dehydrate, Piceatannol, and Tiron.
  • the counter-anion in complex with the dinuclear metal chelating moiety can have a different detectable signal than the free counter-anion.
  • the disclosure features a method for detecting the presence or absence of a target molecule comprising a phosphomonoester group (e.g., phosphorylated target molecule) in a sample, the method comprising: separating a target molecule from a sample using a separation matrix; contacting the target molecule with a binding agent capable of selectively binding to a phosphomonoester group under conditions in which binding of the phosphomonoester group to the binding agent competes with binding of a counter-anion to the binding agent; and detecting binding of the binding agent to the target molecule as an indication that the target molecule is phosphorylated.
  • a target molecule comprising a phosphomonoester group (e.g., phosphorylated target molecule) in a sample
  • the method comprising: separating a target molecule from a sample using a separation matrix; contacting the target molecule with a binding agent capable of selectively binding to a phosphomonoester group under conditions in which binding of the phosphomon
  • the binding agent can be any of the compositions described above and the phosphomonoester-selective binding agent portion (e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions) of the composition binds to a phosphomonoester group, if present, on the target molecule.
  • the phosphomonoester-selective binding agent portion e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions
  • the disclosure features a method for detecting the presence or absence of a a target molecule comprising a phosphomonoester group (e.g., phosphorylated target molecule) in a sample.
  • the method can include the steps of: separating a target molecule from a sample using a separation matrix; contacting the target molecule with any of the compositions described above; and detecting binding of the dinuclear metal-chelating moiety to the target molecule as an indication that the target molecule is phosphorylated.
  • the separation matrix can be, e.g., a polyacrylamide gel, a thin layer chromatography plate, a size exclusion chromatography matrix, a capillary electrophoresis matrix, or an agarose gel matrix.
  • more than one (e.g., two, three, four, five, 10, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 1000, or 2000 or more) target molecule(s) can be separated from the sample.
  • the presence or absence of more than one phosphorylated target molecule can be detected.
  • the above methods can further include the step of isolating the target molecule from the separation matrix and/or analyzing the isolated target molecule.
  • the analyzing can include mass spectrometry.
  • separating can include resolving the target molecules in two dimensions.
  • the methods can further include the step of determining the total amount of target molecules in the sample.
  • the target molecules can be proteins.
  • the determining can include contacting the separation matrix with a total protein stain such as an organic dye, silver stain, a radiolabel, a reverse stain, a fluorescent stain, and a chemiluminescent stain.
  • the protein stain can be silver nitrate (a silver stain) or Coomassie blue.
  • the disclosure features a method for detecting the presence or absence of a phospho-transfer activity in a sample.
  • the method includes the steps of: contacting a phospho-transfer substrate with a sample; contacting the phospho-transfer substrate with a binding agent capable of selectively binding to a phosphomonoester group under conditions in which binding of the phosphomonoester group to the binding agent competes with binding of a counter-anion to the binding agent; and detecting binding of the binding agent to the phospho-transfer substrate as an indication that the phospho-transfer substrate comprises a phosphomonoester group.
  • the binding agent can be any of the compositions described above and the phosphomonoester-selective binding agent portion (e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions) of the composition binds to a phosphomonoester group, if present, on the phospho-transfer substrate.
  • the method can further include the step of after contacting the phospho-transfer substrate with any of the compositions described above, separating the phospho-transfer substrate from the unbound composition.
  • the disclosure features a method for detecting the presence or absence of a phospho-transfer activity in a sample.
  • the method includes the steps of: contacting a phospho-transfer substrate with a sample; contacting the phospho-transfer substrate with any of the compositions described above; and detecting binding of the dinuclear metal chelating moiety to the target molecule.
  • the method can further include the step of after contacting the phospho-transfer substrate with any of the compositions described above, separating the phospho-transfer substrate from the unbound composition.
  • the above methods can further include the step of prior to detecting, separating the phospho-transfer substrate from the sample using a separation matrix.
  • the separation matrix can be any of those described herein.
  • the phospho-transfer activity is a kinase activity (e.g., a tyrosine kinase, a threonine/serine kinase, a saccharide kinase, or a lipid kinase activity.
  • the kinase activity can be, e.g., the activity of Ca2+/Calmodulin Dependent Protein Kinase II.
  • the phospho-transfer activity can be a phosphatase activity (e.g., a tyrosine-specific phosphatase, a threonine/serine specific phosphatase, a dual-specificity phosphatase, a saccharide phosphatase, a histidine-specific phosphatase, or a lipid phosphatase activity.
  • the phosphatase activity can be the activity PP2A.
  • the phospho- transfer activity is a phosphodiesterase activity or a nucleotide cyclase activity.
  • the phospho-transfer substrate can comprise a polypeptide, a nucleic acid, a nucleotide (e.g., ATP or GTP), a cyclic nucleotide (cAMP or cGMP), a saccharide, or a lipid.
  • the phospho-transfer substrate can be attached to a solid support such as a multi-well assay plate, an array (e.g., a protein array), or a particle (e.g., an encoded particle).
  • the particle can be encoded and the phospho-transfer substrate identifiable by a particle code.
  • the binding of the dinuclear metal chelating moiety (in complex with one or more divalent metal cations) to the target molecule indicates the presence of a kinase activity in the sample. In some embodiments of the above methods, a reduction or absence of binding of the dinuclear metal chelating moiety (in complex with one or more divalent metal cations) to the target molecule indicates the presence of a phosphatase activity in the sample.
  • more than one phospho-transfer substrate is contacted with the sample.
  • the dinuclear metal chelating moiety can be detectably labeled (e.g., fluorescently labeled). In some embodiments of any of the above methods, the detecting can comprise determining the amount or presence of the detectable label bound to the phospho-transfer substrate. In some embodiments, the detecting can comprise determining the presence or amount of free detectable counter-anion as an indication that the dinuclear metal- chelating moiety bound to the phospho-transfer substrate.
  • the counter-anion can be selected from the group consisting of Bromopyrogallol Red, Pyrocatechol Violet, Pyrogallol Phtalein, Pyrogallol Red, Quercetin dehydrate, Piceatannol, and Tiron.
  • the counter-anion in complex with the dinuclear metal chelating moiety can have a different detectable signal than the free counter-anion.
  • the disclosure features a method for detecting the presence or absence of more than one phospho-transfer activity in a sample.
  • the method includes the steps of: contacting at least two phospho-transfer substrates with a sample; contacting the at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine or more) phospho-transfer substrates with a binding agent capable of selectively binding to a phosphomonoester group under conditions in which binding of the phosphomonoester group to the binding agent competes with binding of a counter-anion to the binding agent; and detecting binding of the dinuclear metal chelating moiety to at least one of the at least two phospho-transfer substrates.
  • the binding agent can be any of the compositions described above and the phosphomonoester-selective binding agent portion (e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions) of the composition binds to a phosphomonoester group, if present, on the phospho-transfer substrate.
  • the phosphomonoester-selective binding agent portion e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions
  • the disclosure features a method for detecting the presence or absence of more than one phospho-transfer activity in a sample, the method comprising: contacting at least two phospho-transfer substrates with a sample; contacting the at least two phospho-transfer substrates with any of the compositions described above; and detecting binding of the dinuclear metal chelating moiety to at least one of the at least two phospho-transfer substrates.
  • the phospho-transfer activity can be, e.g., a kinase activity, a phosphatase activity, a phosphodiesterase activity, or a nucleotide cyclase activity.
  • the disclosure features a method for detecting the presence or absence of a phospho-transfer activity in a sample.
  • the method includes the steps of: contacting at least one encoded particle with a sample, wherein the at least one encoded particle has an attached phospho-transfer substrate, the phospho-transfer substrate being identifiable by a particle code; contacting the at least one encoded particle with any of the compositions described above; and detecting binding of the dinuclear metal chelating moiety to at least one encoded particle.
  • the phospho-transfer activity can be, e.g., a kinase activity, a phosphatase activity, a phosphodiesterase activity, or a nucleotide cyclase activity.
  • the above methods can further include the step of after contacting the at least one encoded particle and the sample, separating the at least one encoded particle from the sample and/or separating the at least one encoded particle from the unbound dinuclear metal chelating moiety.
  • the above methods can further include the step of immobilizing the phospho-transfer substrate on at least one of the encoded particles.
  • the contacting at least one encoded particle with a sample and contacting the at least one encoded particle with the composition occur at the same time.
  • the dinuclear metal chelating moiety can be detectably labeled (e.g., fluorescently labeled).
  • the detecting comprises determining the presence or amount of the binding of the detectable dinuclear metal chelating moiety to at least one encoded particle contacted with the sample as compared to the presence or amount of binding of the detectable dinuclear metal chelating moiety to the at least one encoded particle not contacted with the sample or determining the presence or amount of free detectable counter-anion as an indication that the dinuclear metal-chelating moiety bound to the target molecule.
  • the disclosure features a method for detecting the presence of one or more phospho-transfer activities in a sample.
  • the method can include the steps of: contacting a mixture of encoded particles with a sample, wherein the mixture of encoded particles comprises one or more pluralities of encoded particles, each plurality of particles having a different attached phospho-transfer substrate, the phospho-transfer substrate being identifiable by a particle code; contacting the mixture with a binding agent capable of selectively binding to a phosphomonoester group under conditions in which binding of the phosphomonoester group to the binding agent competes with binding of a counter-anion to the binding agent; and detecting binding of the binding agent to at least one of the encoded particles.
  • the binding agent can be any of the compositions described above and the phosphomonoester-selective binding agent portion (e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions) of the composition binds to a phosphomonoester group, if present, on the phospho-transfer substrate.
  • the phospho-transfer substrate can be any of those described herein.
  • the disclosure features a method for detecting the presence of one or more phospho-transfer activities in a sample, which method includes the steps of: contacting a mixture of encoded particles with a sample, wherein the mixture of encoded particles comprises one or more pluralities of encoded particles, each plurality of particles having a different attached phospho-transfer substrate, the phospho-transfer substrate being identifiable by a particle code; contacting the mixture with any of the compositions described above; and detecting binding of the dinuclear metal chelating moiety (in complex with at least one (e.g., one or two) divalent metal cations) to at least one of the encoded particles.
  • the phospho-transfer substrate can be any of those described herein.
  • the dinuclear metal chelating moiety can be detectably labeled.
  • the detecting can comprise (i) determining the presence or amount of the binding of the detectably labeled dinuclear metal chelating moiety to at least one encoded particle contacted with the sample as compared to the presence or amount of binding of the detectably labeled dinuclear metal chelating moiety to at least one encoded particle not contacted with the sample or (ii) determining the presence or amount of free detectable counter-anion as an indication that the dinuclear metal-chelating moiety bound to the target molecule.
  • the counter-anion can be selected from the group consisting of Bromopyrogallol Red, Pyrocatechol Violet, Pyrogallol Phtalein, Pyrogallol Red, Quercetin dehydrate, Piceatannol, and Tiron.
  • the above methods can include the step of: (i) immobilizing the phospho-transfer substrate on the encoded particles; (ii) after contacting the mixture of encoded particles with the sample, separating the mixture of encoded particles from sample; (iii) after contacting the mixture of encoded particles with the composition, separating the encoded particles from the unbound dinuclear metal chelating moiety; and /or (iv) measuring the amount of binding of the dinuclear metal chelating moiety to at least one of the phospho-transfer substrates attached to the encoded particles.
  • the sample can be a biological sample (e.g., blood, semen, sweat, urine, a tissue sample, cerebral-spinal fluid, or lacrimal fluid).
  • the methods can also include the step of obtaining a biological sample from a subject.
  • the subject can be, e.g., a mammal such as a human.
  • the sample can be a lysate prepared from a cell.
  • the methods can then further include the step of preparing a lysate from the cell.
  • an increase in the amount of the dinuclear metal chelating moiety bound to the phospho-transfer substrate contacted with the sample as compared to the amount bound to the phospho-transfer substrate not contacted with the sample indicates the presence of a phospho-transfer activity in the sample.
  • a decrease in the amount of the dinuclear metal chelating moiety bound to the phospho-transfer substrate contacted with the sample as compared to the amount bound to the phospho-transfer substrate not contacted with the sample indicates the presence of a phospho-transfer activity in the sample.
  • the encoded particles can be magnetic.
  • the mixture of encoded particles can comprise, e.g., more than one plurality of particles.
  • the mixture can contain, or be, at least two pluralities comprising different phospho-transfer substrates, each substrate comprising a recognition site for a different phospho-transfer activity.
  • the mixture of encoded particles can be contacted with more than one sample in parallel.
  • the detecting can comprise flow cytometry.
  • the disclosure provides a method for identifying a compound that modulates the activity of a phospho-transfer enzyme.
  • the method can include the steps of: contacting a phospho-transfer substrate with a phospho-transfer enzyme in the presence of a compound; contacting the phospho-transfer substrate with a binding agent capable of selectively binding to a phosphomonoester group under conditions in which binding of the phosphomonoester group to the binding agent competes with binding of a counter-anion to the binding agent; and determining whether the compound modulates the activity of the phospho-transfer enzyme, wherein a difference in the amount of binding of the binding agent to the substrate in the presence of the compound as compared to the amount of binding in the absence of the compound indicates that the compound modulates the activity of the phospho-transfer enzyme.
  • the compound can inhibit or stimulate the activity of the phospho-transfer enzyme.
  • the disclosure features a method for identifying a compound that modulates the activity of a phospho-transfer enzyme.
  • the method can include the steps of: contacting a phospho-transfer substrate with a phospho-transfer enzyme in the presence of a compound; contacting the phospho-transfer substrate with any of the compositions described above; and determining whether the compound modulates the activity of the phospho-transfer enzyme, wherein a difference in the amount of binding of the dinuclear metal chelating moiety to the substrate in the presence of the compound as compared to the amount of binding in the absence of the compound indicates that the compound modulates the activity of the phospho-transfer enzyme.
  • the compound can inhibit or stimulate the activity of the phospho-transfer enzyme.
  • the binding agent can be any of the compositions described above and the phosphomonoester-selective binding agent portion (e.g., the dinuclear metal chelating moiety in complex with at least one (e.g., one or two) metal cations such as Zn(II) ions) of the composition binds to a phosphomonoester group, if present, on the phospho-transfer substrate.
  • the phospho- transfer substrate can be any of those described herein.
  • the phospho-transfer substrate and phospho-transfer enzyme can be contacted (in the presence of a compound) in a cell.
  • the method can also after contacting a phospho-transfer substrate with a phospho-transfer enzyme in the presence of a compound in the cell, isolating the phospho-transfer substrate from the cell.
  • the phospho-transfer substrate can be any of those described herein.
  • the phospho-transfer substrate and phospho-transfer enzyme are contacted (in the presence of a compound) in a subject.
  • the method can include the step of after contacting a phospho- transfer substrate with a phospho-transfer enzyme in the presence of a compound in the subject, isolating the phospho-transfer substrate from the subject.
  • the subject can be, e.g., a mammal such as a human.
  • the subject can have a cancer.
  • the above methods can further include the step of prior to determining whether the compound modulates the activity of the phospho-transfer enzyme, separating the phospho-transfer substrate from the phospho-transfer enzyme (or the cell lysate containing the phospho-transfer enzyme) using a separation matrix.
  • the separation matrix can be any of those described herein.
  • the composition containing the dinuclear metal chelating moiety (and at least one divalent metal cation) and the counter-anion can be added to a sample containing the target molecule or phospho-transfer substrate.
  • the counter-anion can be added to a sample containing the target molecule or phospho-transfer substrate first, and the chelating moiety added subsequently.
  • the counter-anion can be present in the sample at a concentration of between 1-50 or 5-200 micromolar.
  • the counter-anion can be a quencher and the dinuclear metal chelating moiety can contain a detectable label (e.g., a fluorophore), wherein the counter-anion quencher, when in complex with the dinuclear chelating moiety (and at least one (e.g., one or two) divalent metal cations), quenches the signal produced from the detectable label and wherein displacement of the counter-anion quencher upon the binding of the dinuclear metal chelating moiety (in complex with at least one divalent metal ion) to a phosphomonoester group results in the detectable label producing a detectable signal.
  • a detectable label e.g., a fluorophore
  • the counter-anion can be a quencher and the the dinuclear chelating moiety itself is detectable (e.g., fluoroscent), wherein the counter-anion quencher, when in complex with the dinuclear chelating moiety (and at least one (e.g., one or two) divalent metal cations), quenches the signal produced from the dinuclear chelating moiety (and at least one (e.g., one or two) divalent metal cations) and wherein displacement of the counter-anion quencher upon the dinuclear chelating moiety (and at least one (e.g., one or two) divalent metal cations) to a phosphomonoester group results in the detectable signal (e.g., fluorescent signal) produced from the dinuclear metal chelating moiety.
  • the detectable signal e.g., fluorescent signal
  • the phospho- transfer substrate or the target molecule is not a free phosphate.
  • the disclosure provides a kit comprising any of the compositions described above and instructions for detecting the binding of the dinuclear metal chelating moiety.
  • the kit can also include at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight or more) control molecule(s).
  • the at least one control molecule can be a polypeptide.
  • the at least one control molecule can comprise a phosphomonoester (e.g., the control molecule can be phosphorylated).
  • the disclosure features a kit comprising: (i) a dinuclear metal chelating moiety; (ii) a salt comprising a divalent metal cation; (iii) a salt comprising a counter-anion, wherein the divalent metal cation is capable of simultaneously binding the dinuclear metal chelating moiety and the counter-anion and wherein the counter anion binds the divalent metal cation with an affinity lower than the affinity with which the phosphorylated target molecule binds the divalent metal cation; and (iv) instructions for forming a ternary complex of (i), (ii), and (iii).
  • the disclosure features a kit, which contains mixture of encoded particles, the mixture comprising one or more pluralities of encoded particles, each plurality of particles having a different attached phospho-transfer substrate, the phospho-transfer substrate being identifiable by a particle code; and any of the compositions described above.
  • the kit can optionally include instructions for detecting a phospho-transfer substrate (e.g., a substrate containing a phosphomonester group).
  • At least some of the encoded particles can be magnetic.
  • the kit can contain more than one plurality of encoded particles.
  • the kit can contain at least two pluralities of encoded particles, each containing different nucleic acid particle codes.
  • the kits can also, optionally, include one or more of the following: a centrifuge (e.g., a microcentrifuge); one or more polyacrylamide gels (or mini-gels); gel-electrophoresis buffers; a protein dye such as SYPRO® Ruby protein gel stain (PerkinElmer), silver stain (silver nitrate), or colloidal Coomassie blue; fluorescent (or, e.g., luminescent or radiolabeled) gel imaging system and/or computer software for analyzing the gels; one or more reagents for preparing a cell lysate from a cell sample and/or fractionating a cell lysate (e.g., the Cellular Protein Fractionation Kit (PerkinElmer catalogue No. PRDIOIAOOIKT); and/
  • Phosphomonoester-selective binding agent refers to a complex comprising a dinuclear chelating moiety and one or more divalent metal cations.
  • Fig. 1 is a schematic representation depicting the fluorescent emission profile of the l,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato Zn(II) 2 complex in 25 mM Piperazine-l,4-bis(2-ethanesulfonic acid) (PIPES) buffer, pH 7.0.
  • PPES Piperazine-l,4-bis(2-ethanesulfonic acid)
  • Fig. 2 is a schematic representation depicting the emission profile of a non- limiting directly detectably labeled phosphomonoester-selective binding agent disclosed herein, namely ruthenium Zn Phos-tagTM, in the absence (left panel) or presence (right panel) Of HPO 4 2" .
  • Fig. 3 is a schematic representation depicting the fluorescence resonance energy transfer (FRET) profiles of PIPES buffer only (purple line), ruthenium dye only (green line), a ruthenium labeled Phos-tagTM zinc complex (pink line) and a biotin labeled dinuclear zinc complex (brown line).
  • FRET fluorescence resonance energy transfer
  • Fig. 4 is a schematic representation depicting the nonspecific interaction of thiohydrazone dinuclear zinc complex with carboxylate groups on proteins. "R” represents the protein in this schematic.
  • the C-terminal carboxyl group and the carboxyl groups of glutamate and aspartate residues are targets of this type of nonspecific interaction.
  • Fig. 5 is a schematic representation depicting the nonspecific interaction of alkoxide-bridged hexaazacryptate dinuclear zinc complex with carboxylate groups on proteins. "R” represents the protein in this schematic. In some embodiments, the C- terminal carboxyl group and the carboxyl groups of glutamate and aspartate residues are targets of this type of nonspecific interaction.
  • Fig. 6 is a schematic representation depicting the nonspecific interaction of 1,3- bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dinuclear zinc complex with carboxylate groups on proteins.
  • R represents the protein in this schematic.
  • the C-terminal carboxyl group and the carboxyl groups of glutamate and aspartate residues are targets of this type of nonspecific interaction.
  • Fig. 7 is a schematic depicting the chemical synthesis of Phos-tagTM-TRITC, a TRITC labeled phosphomonoester-selective binding agent.
  • Fig. 8 is a schematic depicting the chemical synthesis of Phos-tagTM-FITC, a FITC labeled phosphomonoester-selective binding agent.
  • Fig. 9 is a schematic depicting the chemical synthesis of Phos-tagTM -Coumarin, a coumarin labeled phosphomonoester-selective binding agent.
  • Fig. 10 is a schematic depicting the chemical synthesis of Phos-tagTM-Ruthenium Dye, a ruthenium dye labeled phosphomonoester-selective binding agent.
  • Fig. 11 is a photograph of an SDS-PAGE gel depicting the electrophoretically resolved proteins stained with SYPRO® Ruby dye (left lane), a TRITC-labeled phosphomonoester-selective binding agent(middle lane), and a ruthenium labeled phosphomonoester-selective binding agent (right lane).
  • SYPRO® Ruby dye a TRITC-labeled phosphomonoester-selective binding agent
  • ruthenium labeled phosphomonoester-selective binding agent right lane.
  • ovalbumin is phosphorylated.
  • Fig. 12 is (top half) a schematic depicting a phosphomonoester-selective binding agent, Phos-tagTM bound to Zn2+, bound to various anions including phosphate, sulfate, carboxylate, chlorine, and the competition of phosphate and sulfate anions for binding to Phos-TagTM bound to Zn2+; and (bottom half) is a pair of photographs of a PVDF membrane and a gel showing the specific binding by TRITC-labeled Zn Phos-tagTM (i.e., Phos-tagTM 540) of electrophoretically resolved ovalbumin.
  • TRITC-labeled Zn Phos-tagTM i.e., Phos-tagTM 540
  • Fig. 13 is a pair of photographs of 2-D electrophoretic gels, wherein 100 ⁇ g of protein from rat liver cytosol was resolved and stained with TRITC-labeled Zn Phos- tagTM (left) and Ruthenium labeled Zn Phos-tagTM (right).
  • Fig. 14 is the representation of the 2-D gel of Fig. 13 (left), where five circled spots are excised and subjected to further analysis.
  • Fig. 15 is a representation of the mass spectra of the phosphorylated peptide isolated from a tryptic digest of ovalbumin using Ti ⁇ 2-coated magnetic beads.
  • Fig. 16 is a representation of the mass spectra of the phosphorylated peptide isolated from the trypsin digest of ovalbumin.
  • Fig. 17 is a representation of the MS/MS spectra of the fragment ion from parent mass ion 2088.9 Da.
  • the peptide MW 1088.9 is EVVGSAEAGVD AASVSEEFR (SEQ ID NO:1); phosphorylated at the first S (G-pS-A).
  • Fig. 18 is a schematic showing the non-covalent interaction of a Pyrocatechol Violet counter-anion (upper left) with a dinuclear zinc Phos-tagTM, (upper right, above arrow) to form a Pyrocatechol Violet/Phos-tagTM complex (lower left).
  • the Pyrocatechol Violet counter-anion complex with the dinuclear zinc Phos-tagTM i.e., two Zn2+ cations bound to Phos-tagTM
  • Phosphomonoester moieties can displace the Pyrocatechol Violet counter-anion quencher (thus freeing it), and unmask the fluorescence of the dinuclear zinc Phos-tagTM complexed (non-covalently) to the phosphomonoester group (lower left).
  • Fig. 19 is a schematic representation illustrating the displacement of Pyrocatechol violet from Zn Phos-tagTM by a phosphomonoester residue.
  • the complex comprising a pyrocatechol violet molecule non-covalently bound to Zn Phos-tagTM interacts with HPO42- to result in the HPO 4 non-covalently bound to the Zn Phos-tagTM and the pyrocatechol violet released as a free molecule.
  • Fig. 20 is a representation of a graph showing the change in UV/Visible absorbance spectrum obtained by adding Zn Phos-tagTM to pyrocatechol violet.
  • the right panel is a photograph showing the visible color change of pyrocatechol violet alone (left) and pyrocatechol violet in the presence of Zn Phos-tagTM (right).
  • Fig. 21 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with pyrocatechol violet and exposed to 250 ⁇ M of acetate anions, chlorine anions, phosphate anions, and no anion.
  • Fig. 22 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with pyrocatechol violet and exposed to 200 ⁇ M of phosphoserine peptide (pink line), phosphothreonine peptide, phosphotyrosine peptide and an unphosphorylated peptide.
  • Fig. 23 is a graph showing the relative binding of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM) to phosphoserine peptide, phosphothreonine peptide, phosphotyrosine peptide and an unphosphorylated peptide.
  • Fig. 24 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with pyrocatechol violet and exposed to a phosphothreonine peptide having the sequence "RRREEETEEEAA"(SEQ ID NO:2), wherein the threonine is phosphorylated ("pThr"), shown at the bottom of the figure at 10 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, or in the presence of no peptide.
  • Fig. 25 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with pyrocatechol violet and exposed to a phosphoserine peptide having the sequence "RRREEESEEEAA"(SEQ ID NO:3), wherein the serine is phosphorylated (“pSer”), shown at the bottom of the figure at 10 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M or in the presence of no peptide.
  • Fig. 26 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with pyrocatechol violet and exposed to a phosphotyrosine peptide having the sequence "KEPQYEEIPIYL"(SEQ ID NO:4), wherein the tyrosine is phosphorylated (“pTyr”), shown at the bottom of the figure at 10 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M or in the presence of no peptide.
  • Fig. 27 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM (a non- limiting phosphomonoester-selective binding agent of the invention) bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with pyrocatechol violet and exposed to 3mg/ml of ovalbumin, alpha or beta casein, bovine serum albumin (BSA), and no protein.
  • Phos-tagTM a non- limiting phosphomonoester-selective binding agent of the invention
  • Fig. 28 is a photograph showing the visible color of tubes containing, from left to right, 50 ⁇ M pyrocatechol violet alone; 50 ⁇ M pyrocatechol-Zn Phos-tagTM complex; 100 ⁇ M phosphopeptide plus 50 ⁇ M pyrocatechol-Zn Phos-tagTM complex; 100 ⁇ M unphosphorylated peptide plus 50 ⁇ M pyrocatechol-Zn Phos-tagTM complex; 1.5 mg/ml ovalbumin (a phosphoprotein) plus 50 ⁇ M pyrocatechol-Zn Phos-tagTM complex; and 1.5 mg/ml bovine serum albumin (an unphosphorylated protein) plus 50 ⁇ M pyrocatechol-Zn Phos-tagTM complex.
  • 50 ⁇ M pyrocatechol violet alone
  • 50 ⁇ M pyrocatechol-Zn Phos-tagTM complex 100 ⁇ M phosphopeptide plus 50 ⁇ M pyrocatechol-Zn Phos-tagTM complex
  • Fig. 29 is a representation of a UV/Visible absorbance spectrum of Piceatannol (100 ⁇ M in HEPES buffer; top left), Tiron (lOO ⁇ M in HEPES buffer; top right), and Quercetin (100 ⁇ M in a 50:50 HEPES/acetonitrile buffer; bottom).
  • Fig. 30 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with Quercetin (top left), Tiron (top right), or Piceatannol (bottom) in the presence and absence of 400 ⁇ M HPO 4 2" .
  • the excitation wavelength used for Quercetin, Tiron, and Piceatannol were 370 nm, 290 nm, and 340 nm, respectively.
  • Fig. 31 is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with Quercetin (left) or Tiron (right) at a concentration of 100 ⁇ M (in 10 mM HEPES, pH 7) and exposed to phosphorylated peptides: pY (at a concentration of 100 ⁇ M); pT (at a concentration of 100 ⁇ M); pS (at a concentration of 30 ⁇ M), and P2 (a non- phosphorylated peptide at a concentration of 100 ⁇ M).
  • Zn Phos-tagTM is non-covalently complexed with Quercetin (left) or Tiron (right) at a concentration of 100 ⁇ M (in 10 mM HEPES, pH 7) and exposed to phosphorylated peptides: pY (at a concentration of 100 ⁇ M); p
  • Zn Phos-tagTM is a representation of a UV/Visible absorbance spectrum of Phos-tagTM bound to Zn2+ (Zn Phos-tagTM), where the Zn Phos-tagTM is non-covalently complexed with Tiron (left) or Quercetin (right) at a concentration of 100 ⁇ M (in 10 rnM HEPES, pH 7) and exposed to various proteins - bovine serum albumin (BSA), ⁇ -casein, and ovalbumin - each at a concentration of 3 mg/ml.
  • BSA bovine serum albumin
  • Fig. 33 is a representation of a UV- Visible absorbance spectrum of a phenylfluorone (PF) solution (50 ⁇ M), the sensing mixture of Phos-tagTM Mn complex and PF at 1 :2: 1 ratio (50 ⁇ M), and the Phos-tagTM-Mn-PF complex (50 ⁇ M) in the presence of phosphate anions at concentrations of 50 ⁇ M in a reaction buffer of 10 mM Borate containing 0.6 ⁇ M cetylpyridinium chloride and 2 mM Triton X-100 at pH 9.7.
  • PF phenylfluorone
  • Fig. 34 is a representation of a UV- Visible absorbance spectrum of Bromopyrogallol red (BPR) solution (20 ⁇ M), the sensing mixture of Phos-tag Zn complex and BPR at 1 :2:1 ratio (20 ⁇ M), and the Phos-tag-Zn-BPR complex (20 ⁇ M) in the presence of phosphate anions at concentrations of 100 ⁇ M in a reaction buffer of 10 mM HEPES, pH 7.
  • BPR Bromopyrogallol red
  • Fig. 35 is a series of photographs of polyacrylamide gels stained with counter- anion -phosphomonoester binding agent complexes described herein or phosphomonoester-selective binding agents without counter anions.
  • Figs. 36A-36C are a series of fluorescence intensity traces of polyacrylamide gel lanes containing phosphorylated polypeptides and stained with either counter-anion - phosphomonoester binding agent complexes described herein or phosphomonoester- selective binding agents without counter anions.
  • Fig. 37 is a schematic diagram showing a number of peptide or nucleic acid substrates attached to a single bead, where each substrate attached to the same bead has the identical amino acid or nucleic acid sequence.
  • peptide substrates are attached to the bead prior to incubation with a kinase (shown as widened figure eight structures) in a phosphorylation reaction mixture containing free phosphate (e.g., ATP; free phosphate shown as "P").
  • a kinase shown as widened figure eight structures
  • a phosphorylation reaction mixture containing free phosphate (e.g., ATP; free phosphate shown as "P").
  • Fig. 38 is a schematic diagram showing the bead-immobilized substrates following removal of the kinase and unbound phosphate.
  • Fig. 39 is a schematic diagram showing the bead-immobilized substrates bound to a detectable phosphomonoester-selective binding agent of the disclosure.
  • Fig. 40 is a schematic diagram showing two different beads, where the substrates bound to one bead have a different sequence than the substrates bound to a second bead.
  • the first substrate is depicted with diamonds
  • the second substrate is depicted with hexagons.
  • Fig. 41 is a schematic diagram showing the recognition of the phosphorylated residues on the substrates attached to both beads by the detectable phosphomonoester- selective binding agent of the disclosure.
  • Fig. 42 is a schematic diagram showing a non- limiting embodiment of the disclosure in which two different peptide sequences are incubated in a phosphorylation reaction mixture simultaneously with two different protein kinases, each of which specifically recognizes its phospho-acceptor site on the respective peptides, (shown as differently patterned widened figure 8's). Note that the two peptide sequences are covalently attached to two different single stranded nucleic acid sequences, such that each molecule has the identical amino acid and nucleic acid sequence.
  • Fig. 43 is a schematic diagram showing the phosphorylation patterns of Peptide 2 (diamonds; attached to nucleic acid sequence 2) and Peptide 1 (hexagons; attached to nucleic acid sequence 1) following incubation with the protein kinases in a phosphorylation reaction mixture.
  • Fig. 44 is a schematic diagram showing the attachment of the peptides of Fig. 39 to one of two beads.
  • Each bead contains multiple sequences that will hybridize to the single stranded nucleic acid sequence on one of the two peptides.
  • all of the Peptide 1 sequences bind to one bead, while all of the Peptide 2 sequences bind to another bead.
  • Fig. 45 shows the specific binding of the phosphorylated phosphoacceptor site on the peptides with the detectable phosphomonoester-selective binding agent of the disclosure.
  • the beads which can be distinguished from each other, can be analyzed to determine how much detectable phosphomonoester-selective binding agent is bound to each, indicating how much each respective peptide substrate is phosphorylated.
  • phosphomonoester groups e.g., phosphomonoester groups of proteins, nucleic acids, lipids, or small molecules (e.g., a phosphorylated molecule).
  • small molecules e.g., a phosphorylated molecule
  • the disclosure features dinuclear metal chelating moieties supplied with an appropriate milieu of counter anions and divalent cations (e.g., Zn(II)) that bind to the dinuclear metal chelating moieties in solution, which are useful for selectively detecting phosphomonoester groups such as those present on phosphorylated molecules (e.g., phosphoproteins, phosphopeptides, phosphorylated lipids, or phosphorylated nucleic acids).
  • phosphomonoester groups such as those present on phosphorylated molecules (e.g., phosphoproteins, phosphopeptides, phosphorylated lipids, or phosphorylated nucleic acids).
  • This counter anion-pairing detection technique employs counter-anions that bind with lower affinity to the dinuclear metal chelate complex than a phosphomonoester-containing moiety but with, e.g., equal to or higher affinity than a carboxylate-containing moiety, such as a glutamate or aspartate residue.
  • Carboxylic acids are organic acids characterized by the presence of a carboxyl group (-COOH). In general, the salts and anions of carboxylic acids are referred to as carboxylates.
  • the counter-anion and the dinuclear metal chelating moiety/divalent metal complex reduces nonspecific interaction with molecules such as proteins and peptides and with certain matrices commonly employed to separate or display proteins and peptides, such as polyacrylamide, polyvinylidene difluoride (PVDF) membrane, planar microarray surfaces, polymeric beads and thin-layer chromatography matrices.
  • the reagents allow for highly selective detection of, e.g., phosphomonoester group containing molecules such as phosphorylated proteins or phosphorylated peptides.
  • a binding agent that specifically binds to a phosphomonoester group can be a composition comprising: a dinuclear metal chelating moiety; a divalent metal cation capable of simultaneously binding to the dinuclear metal chelating moiety and a phosphorylated target molecule; and a counter-anion that binds the divalent metal cation (in complex with the dinuclear metal chelating moiety) with an affinity lower than the affinity with which the phosphorylated target molecule binds the divalent metal cation.
  • the counter-anion can be capable of binding to the divalent metal cation with an affinity that is at least equal to, or greater than, the affinity with which a carboxylate anion binds to the divalent metal cation.
  • a "phosphomonoester-selective binding agent" (a complex comprising a dinuclear metal chelating moiety and a divalent metal such as Zn(II)) can bind to a phosphomonoester residue with a dissociation constant (kD) of about 500 nM, or about 100 nM, or about 50 nM, or about 25 nM, or about 2.5 nM.
  • a phosphomonoester-selective binding agent can bind to a phosphomonoester group with a kD of less than 100 (e.g., less than 50, less than 25, less than 20, less than 10, less than 5, less than 1, less than 0.750) ⁇ M.
  • the phosphomonoester-selective binding agent is bound to a cation (e.g., a zinc cation), wherein the cation-phosphomonoester-selective binding agent is able to bind to phosphate monoester residues.
  • a phosphomonoester-selective binding agent is also termed a dinuclear or binuclear metal chelating moiety.
  • the chelating moiety is selected from the group consisting of ⁇ -hydroxo- ⁇ -phenolato dinuclear chelates, hexaazacryptate dinuclear chelates, or alkoxide-bridged dinuclear chelates.
  • the dinuclear metal chelating moiety can comprise the structure of formula (I):
  • each R 1 , R 2 , R 3 , and R 4 are, independently, an H; an alkyl group having 1 to 16 carbon atoms; an acyl group; a carboxyalkyl group; an acylalkyl group; a carbamoylalkyl group; a cyanoalkyl group; a hydroxyalkyl group; an aminoalkyl group; or a haloalkyl group having 1 to 16 carbon atoms and 1 to 5 halogens; a carboxyl group; a carbamoyl group; a hydroxyl group; a cyano group; an amino group; or a halogen group, each n and m are independently 0 or 1 ; and
  • Ar is aryl
  • Each n and m can be 0 and/or 1.
  • Each of Ri, R 2 , R3, and R 4 can be H.
  • the Ar can be, e.g., anthracene, , or phenyl.
  • each of Ri, R 2 , R 3 , and R 4 can be H; n and m can each be 1 ; and Ar can be
  • the dinuclear metal chelating moiety can comprise the structure of formula (II):
  • each R 1 is independently H, Ci-C 6 alkyl, or arylalkyl
  • R 2 is H, Ci-C 6 alkyl, or arylalkyl
  • each R is independently a nitrogen containing moiety comprising a nitrogen and a linker.
  • the nitrogen can be a component in a heteroaryl ring.
  • Each R 1 can be H and/or R 2 can be H. In some embodiments, each R 1 is H and R 2 is H.
  • Each R 3 can be the same nitrogen containing moiety comprising a nitrogen containing heteroaryl ring and a linker or R 3 can comprise a fused nitrogen containing
  • the linker of R J can be Each R 3 can contain, or be, a pyridyl.
  • each R 3 can be R R , wherein each R 4 is independently H or C1-C6 alkyl.
  • each R 4 can be methyl.
  • the dinuclear metal chelating moiety can comprise the structure of formula (III):
  • each R 1 is independently H, Ci-C 6 alkyl, or arylalkyl
  • R 2 is H, Ci-C 6 alkyl, or arylalkyl; each R 3 is independently a nitrogen containing moiety comprising a nitrogen containing ring and a linker; and each R 4 is independently H, Ci-C 6 alkyl or a nitrogen containing moiety comprising a nitrogen containing ring and a linker.
  • Each R 1 and R 2 can be H. In some embodiments, each R 1 is H and R 2 is H.
  • Each R can be the same nitrogen containing moiety comprising a nitrogen
  • Each R 3 can be, e.g., or is
  • Each R can be the same nitrogen containing moiety comprising a nitrogen containing ring and a linker and each R 4 is H. In some embodiments, each R 3 is and each R 4 is H.
  • R 3 and R 4 are the same nitrogen containing moiety comprising a nitrogen containing ring and a linker.
  • R 3 and R 4 for each occurrence, are the same nitrogen containing moiety comprising a nitrogen containing ring and a linker.
  • R 4 for each occurrence, can be
  • the dinuclear metal chelating complex can have the structure of formula (IV) (a representative chelate for forming an alkoxide-bridged M(II)2 hexaazacryptate complex):
  • the dinuclear metal chelating moiety can have the structure of formula (V), a representative chelate for forming a naphthyridine-based M(II)2 complex:
  • the dinuclear metal chelating moiety can have the structure of formula (VI):
  • Ri-R 4 are as in formula (I); n and m as in formula (I); R 5 can be OH, Cl- C3 alkyl, or halo; and p can be 0 to 4.
  • the composition can contain, or be, the metal cation is Zn 2+ and can have the following formula:
  • the dinuclear metal chelating moiety can have the structure ula (VI), wherein R1-R5, n, m, and p are as follows:
  • R is an unsulfonated (or non-sulfonated) directly detectable label.
  • R can be selected from the group consisting of
  • the dinuclear metal chelating moiety can have the structure ula (VI), wherein R 1 -R 5 , n, m, and p are as follows:
  • the counter-anion can be selected from the group consisting of SO 4 2" , S 2 O 8 2" , SO 3 2" , SO 2 2" ), S 2 O 8 2 ,S 2 O 3 2 ,SeO 4 2 and SeO 3 2" .
  • the counter-anion can be selected from the group consisting of monocarboxylate anion and dicarboxylate anion.
  • the monocarboxylate anion can be an isonicotinate anion and the dicarboxylate anion can be a terephthalate anion.
  • the counter-anion can be a hydroxyl-containing dye.
  • the dye can be one selected from the group consisting of a polyhydroxybenzenesulfonephthalein dye, a polyhydroxybenzenephthalein dye, pyrocatechol violet (3,3',4-trihydroxyfuchsone-2"-sulfonic acid; catechol sulfonephthalein), pyrogallol red (3,4,5-trihydroxy-9-(2'-sulfophenyl)- 6-isoxanthone, pyrogallolsulfophthalein), bromopyrogallol red (3,4,5-trihydroxy-2,7- dibromo-9-(2'-sulfophenyl)-6-isoxanthone, dibromopyrogallolsulfophthalein), galleon (galleinphthalein, pyrogallophthalein,4,5-dihydroxyfluoresceine), Quercetin dihydrate (3,3',4',5,7
  • the counter-anion can contain a catechol group.
  • Many molecules contain a catechol group.
  • the hormones epinephrine and norepinephrine both contain catechols.
  • the neurotransmitter dopamine also contains a catechol group.
  • Catechols are known to coordinate to two transition metal cations in phenoxo- bridged dinuclear metal complexes at neutral pH value.
  • catechol-containing compounds like pyrocatechol violet and pyrogallol red are non-limiting counter-anions for the phosphomonoester detection systems described herein.
  • pyrocatechol violet the strong violet color formed upon complexation of pyrocatechol violet with dinuclear transition metal complexes should serve an additional benefit, quenching of the inherent fluorescence of the dinuclear zinc complexes. In electrophoresis gel staining applications, this quencher counter-anion approach would generate a detection system that would not require no post-staining wash step whatsoever. Thus, only the dinuclear complexes directly bound to phosphomonoester-containing molecules would fluoresce under this situation.
  • Ruthenium can be coupled to a chelate comprising a catechol group, making it easier for the ruthenium to form a complex with a phosphomonoester-selective binding agent.
  • Phos-tagTM a non- limiting phosphomonoester-selective binding agent of the invention, can be bound to a ruthenium complex (containing a catechol moiety) to form a complex.
  • conjugation of an innately fluorescent phosphomonoester-selective binding agent of the invention e.g., a l,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2- olato Zn(II)2 complex (Zn Phos-tag) or a 2, 6-diformyl-p-cresol-bis[N-methyl-3- thiohydrazone] Zn(II)2 complex
  • certain ruthenium complexes such as bis(2,2'- bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester label, or a bis(hexafluorophosphate), bis(2,2'-bipyridine)-4,4'-dicarboxybipyridine-ruthenium di(N- succinimidyl ester) bis(hexafluorophosphate) label
  • ruthenium complexes such as bis(2,2'- bipyridine
  • This trimetallic phosphomonoester-binding agent can be induced to fluoresce at about 610 nm using a wide range of excitation wavelengths, including mid-wavelength range ultraviolet, long-wavelength range ultraviolet and visible-wavelength light (-450 nm).
  • the innately fluorescent Phos-tagTM may excite the ruthenium in a Fluorescent Resonance Energy Transfer (FRET) interaction to cause the entire complex to emit at approximately 610 nm.
  • FRET Fluorescent Resonance Energy Transfer
  • the phosphomonoester may compete the ruthenium off the Phos-tagTM, allowing the ruthenium to emit by itself and the Phos-tagTM to emit by itself, which may result in emissions at both 425 nm (approximately) and 610 (approximately). Consequently, these types of phosphomonoester-selective binding agents of the invention can be analyzed on a wide variety of commercially available imaging platforms.
  • the counter-anion is a catechol-based dye.
  • the catechol-based dye can be a 2,3,7-trihydroxy-9R-6-isoxanthone, wherein the R group at the C9 position is selected from the group consisting of: a C1-C8 alkyl group which may be cyclic or acyclic, linear or branched, optionally substituted with a group selected from OH, halo, -SO 3 H, and COOH; and an aryl group optionally substituted with a group selected from OH, halo, SO3H, and COOH.
  • the aryl group can be a phenyl.
  • the aryl group can be substituted with one or more Br, SO 3 H, COOH, or combinations of any of the foregoing.
  • the counter-anion can be a 3,4,5-trihydroxy-9R-6- isoxanthone, wherein the R group at the C9 position is selected from the group consisting of a C1-C8 alkyl group which may be cyclic or acyclic, linear or branched, optionally substituted with a group selected from OH, halo, - SO 3 H, and COOH; and an aryl group optionally substituted with a group selected from OH, halo, SO3H, and COOH; and wherein the isoxanthone moiety is optionally substituted with one or more groups selected from OH and halo.
  • the isoxanthone moiety can be substituted with one or more halo groups such as Br.
  • the isoxanthone moiety can be substituted with 2 Br groups such as Br groups at the 2,7 positions or the 2,5 positions of the isoxanthone.
  • the aryl group can be a phenyl.
  • the phenyl can substituted with SO 3 H or COOH, or both.
  • the R group can be 2-sulfophenyl or 2-carboxyphenyl.
  • the counter-anion can be selected from the group consisting of Pyrogallol red (3,4,5-trihydroxy-9-(2'-sulfophenyl)-6-isoxanthone) and Gallein (4,5-dihydroxyfluoresceine).
  • the counter-anion can be selected from the group consisting of BPR (3,4,5-trihydroxy-2,7-dibromo-9-(2'- sulfophenyl)-6-isoxanthone) and BG (dibromogallein). Additional counter-anions are described in, e.g., Ivanov et al. (2006) J. Anal. Chem. 61(11): 1040-1062, the disclosure of which is incorporated herein by reference in its entirety.
  • the counter-anion in complex with the dinuclear metal chelating moiety has a different detectable signal than the free counter-anion.
  • Divalent cations include, e.g., Co (II), Cu (II), Cd (II), Mn (II), Ni (II), Fe (II) and Zn (II).
  • the divalent cation also include hetero-dinuclear complexes such as, e.g., Cu(II)-Cd(II), Fe(II)-Zn(II) complexes.
  • the dinuclear metal chelating moiety and divalent metal cation can be at a 1 :2 ratio in the complex. In some embodiments, the dinuclear metal chelating moiety is bound to two divalent cations.
  • the same dinuclear metal chelating moiety is bound to two divalent cations, where the two divalent cations are the same (e.g., two Zn2+ cations with a single phospho-monoester-selective binding agent (see, e.g., Zn Phos-tagTM, a non- limiting phosphomonoester-selective binding agent of the invention, in the upper right of Fig. 18)).
  • the dissociation constant (KD) of the binding of the above-structure to a phosphate monoester residue is about 25 nM.
  • At least 40% at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more
  • the dinuclear metal chelating moieties can be in complex with counter-anions.
  • the counter-anion can be a quencher and the phosphomonoester-selective binding agent can contain a detectable label (e.g., a fluorophore), wherein the quencher quenches the signal produced from the detectable label when in proximity.
  • a detectable label e.g., a fluorophore
  • liberation of the counter-anion quencher upon the binding of the phosphomonoester-selective binding agent ot a phosphomonoester group would allow the detectable label to produce a detectable signal.
  • the counter-anion can be a quencher and the phosphomonoester-selective binding agent can itself be detectable (e.g., fluoroscent), wherein the quencher quenches the signal produced from the phosphomonoester-selective binding agent when the two compounds are complexed.
  • the counter-anion quencher upon the binding of the phosphomonoester-selective binding agent to a phosphomonoester group would allow for the fluorescent signal to be produced from the phosphomonoester-selective binding agent.
  • any of the binding agents described herein can be detectably labeled.
  • any of the phosphomonoester-selective binding agents described herein can contain a detectable label.
  • the phosphomonoester-selective binding agents can be directly or indirectly labeled.
  • a directly detectably labeled phosphomonoester-selective binding agent can be detected without use of a secondary molecule that produces a signal.
  • biotinylated or streptavidin-bound binding agents are examples of indirectly detectably labeled binding agents when the biotin or streptavidin interacts with a secondary molecule, namely labeled streptavidin or labeled biotin, respectively, to be detected.
  • the detectable label coupled to the binding agent described herein can be non-sulfonated. Accordingly, use of the non-sulfonated detectable label described herein can result in reduced background when using the directly labeled phosphomonoester-binding molecule described herein to bind to phosphomonoester residues on molecules of interest.
  • unsulfonated or non- sulfonated is meant that the detectable label does not contain a
  • a directly detectable label used in the technology described herein can be coupled to the binding agent (e.g., the dinuclear metal chelating moiety) at any location on the binding agent provided that the resulting compound (i.e., the binding agent coupled to a non-sulfonated directly detectable label) retains its ability to selectively bind to phosphomonoester residues.
  • the binding agent e.g., the dinuclear metal chelating moiety
  • the directly detectable labels used in the technology described herein are luminescent labels.
  • Non- limiting luminescent labels that can be conjugated to the phosphomonoester-selective binding agent to form directly detectable labels useful in the technology described herein include a fluorescein - 5 - isothiocyanate (5 -FITC) label, a fluorescein - 6 - isothiocyanate label (6-FITC), a tetratmethylrhodamine-5(6)- isothiocyanate (TRITC) label, a carboxyfluorescein diacetate succinimidyl ester label, a 5 - (and - 6) - carboxytetramethylrhodamine, succinimidyl ester label, a 6 - carboxytetramethylrhodamine, succinimidyl ester label, a 6 - carboxytetramethylrhodamine, succinimidyl ester label, a 6 - carboxytetra
  • Unsulfonated versions of cyanine dyes and squaraine dyes are also appropriate.
  • Various nonreactive fluorophores such as oxazines and thiazines (Azure B, Nile Blue, Oxazine 750) can be coupled to the phosphomonoester-selective binding agent by means of a bifunctional cross-linking agent, such as a water soluble carbodiimide.
  • the directly detectable label is the phosphomonoester- binding agent itself.
  • l,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato Zn(II)2 complexes Phos-tagTM
  • 2, 6-diformyl-p-cresol-bis[N-methyl-3- thiohydrazone] Zn(II)2 complexes are ultraviolet light-excitable fluorophores that emit in the vicinity of 450-480 nm.
  • Fig. 1 shows the fluorescence emission profile of the 1,3- bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato Zn(II)2 complex.
  • conjugation of an innately fluorescent phosphomonoester-binding agent to certain ruthenium complexes such as bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine- ruthenium N-succinimidyl ester label, or a bis(hexafluorophosphate), bis(2,2'-bipyridine)- 4,4'-dicarboxybipyridine-ruthenium di(N-succinimidyl ester) bis(hexafluorophosphate) label generates an especially preferred reporter for the detection of phosphomonoester- containing species.
  • This trimetallic phosphomonoester-binding agent can be induced to fluoresce at about 610 nm using a wide range of excitation wavelengths, including mid- wavelength range ultraviolet, long-wavelength range ultraviolet and visible-wavelength light (-450 nm).
  • excitation wavelengths including mid- wavelength range ultraviolet, long-wavelength range ultraviolet and visible-wavelength light (-450 nm).
  • FRET fluorescence resonance energy transfer
  • Fig. 2 shows excitation spectra of: PIPES buffer (pH 7.0), Bis(2,2'-bipyridine)-4'- methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester label, Bis(2,2'-bipyridine)- 4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester label that has been conjugated to N,N,N ' -Tri(2-pyridylmethyl)-N ' -[5 -N"-(2-aminoethyl)carbamoyl-2- pyridylmethyl]-l,3-diaminopropane-2-ol according to example 4 and loaded with Zn 2+ , and a biotin-conjugate of N,N,N'-Tri(2-pyridylmethyl)-N'-[5-N"-(2- aminoethyl)carbamoyl-2-pyr
  • the biotin-modified phosphomonoester-binding agent displays a strong fluorescence emission maximum centered at about 425 nm upon excitation at 340 nm. However, no 425 nm emission is observed with the ruthenium dye conjugate of the phosphomonoester-selective binding agent. Instead, fluorescence resonance energy transfer occurs (FRET) and emission is observed only at about 610 nm.
  • FRET fluorescence resonance energy transfer
  • the directly detectable labels of the technology described herein are chemiluminescent.
  • Luminarin 1 and Luminarin 2 are examples of N-hydroxysuccinimidyl esters of chemiluminescent substrates that can readily be coupled to the phosphomonoester-selective binding agent.
  • Certain fluorophores, such as fluorescein isothiocyanate (FITC) can be oxidized by agents such as NaClO in alkaline solution and in the presence of certain cationic surfactants, such as cetyltrimethylammonium bromide, to generate a chemiluminescent signal.
  • FITC fluorescein isothiocyanate
  • the phosphomonoester-selective binding agent coupled to FITC is a suitable chemiluminescent detector of phosphoproteins.
  • Fluorophores such as dansyl chloride, A- chloro-7-nitro-2, 1 ,3-benzontinezole (NBD-Cl), 4-fluoro-7-nitro-2, 1 ,3-benzontinezole (NBD-F), naphthalene-2,3-dialdehyde (NDA) and o-phthaldialdehyde (OPA) can be coupled to the phosphomonoester-selective binding agent.
  • the derivatized product can be induced to bind to a phosphomonoester-containing molecule, such as a phosphoprotein within a polyacrylamide gel, and then reacted with certain oxylates and hydroperoxide to generate a chemiluminescent signal.
  • 2-methoxy-2,4-diphenyl-3(2H furanone (MDPF) can be coupled to the phosphomonoester-binding agent, induced to bind to a phosphomonoester-containing molecule and detected by chemiluminescence thru incubation with bis-(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide.
  • acylhydrazides such as isoluminol
  • acridinium derivatives such as isoluminol
  • dioxethanes such as isoxethanes
  • coelenterazines such as peroxyoxalic derivatives
  • peroxyoxalic derivatives can be coupled to the phosphomonester binding agent in order to generate a chemiluminescent reporter of phosphorylation.
  • the chemiluminescent-labeled phosphomonoester selective-binding agent of the technology described herein is very sensitive and produces low background.
  • the directly detectable labels of the technology described herein are assayed by resonance light scattering or surface-enhanced Raman scattering.
  • nano-sized gold or silver particles are coupled to the phosphomonoester- selective binding agent.
  • the scattered light has a clear (not cloudy) color that depends on composition and particle size. This scattered light can be used as the signal for ultrasensitive molecule detection.
  • gold particles as detection labels for phosphorylated molecules is that their light producing power is equivalent to more than 500,000 fluorescein molecules, they can be detected at concentrations as low as 10-15 M using a simple illuminator, they do not photobleach and the color of scattered light can be changed by changing particle size or composition.
  • Fig. 2 shows excitation spectra of: PIPES buffer (pH 7.0), Bis(2,2'-bipyridine)-4'- methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester label, Bis(2,2'-bipyridine)- 4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester label that has been conjugated to N,N,N ' -Tri(2-pyridylmethyl)-N ' -[5 -N"-(2-aminoethyl)carbamoyl-2- pyridylmethyl]-l,3-diaminopropane-2-ol according to example 4 and loaded with Zn 2+ , and a biotin-conjugate of N,N,N'-Tri(2-pyridylmethyl)-N'-[5-N"-(2- aminoethyl)carbamoyl-2-pyr
  • the biotin-modified phosphomonoester-binding agent displays a strong fluorescence emission maximum centered at about 425 nm upon excitation at 340 nm. However, no 425 nm emission is observed with the ruthenium dye conjugate of the phosphomonoester-selective binding agent. Instead, fluorescence resonance energy transfer occurs (FRET) and emission is observed only at about 610 nm.
  • FRET fluorescence resonance energy transfer
  • the directly detectable labels of the technology described herein are chemiluminescent.
  • Luminarin 1 and Luminarin 2 are examples of N-hydroxysuccinimidyl esters of chemiluminescent substrates that can readily be coupled to the phosphomonoester-selective binding agent.
  • Certain fluorophores, such as fluorescein isothiocyanate (FITC) can be oxidized by agents such as NaClO in alkaline solution and in the presence of certain cationic surfactants, such as cetyltrimethylammonium bromide, to generate a chemiluminescent signal.
  • FITC fluorescein isothiocyanate
  • the phosphomonoester-selective binding agent coupled to FITC is a suitable chemiluminescent detector of phosphoproteins.
  • Fluorophores such as dansyl chloride, 4- chloro-7-nitro-2, 1 ,3-benzontinezole (NBD-Cl), 4-fluoro-7-nitro-2, 1 ,3-benzontinezole (NBD-F), naphthalene-2,3-dialdehyde (NDA) and o-phthaldialdehyde (OPA) can be coupled to the phosphomonoester-selective binding agent.
  • the derivatized product can be induced to bind to a phosphomonoester-containing molecule, such as a phosphoprotein within a polyacrylamide gel, and then reacted with certain oxylates and hydroperoxide to generate a chemiluminescent signal.
  • 2-methoxy-2,4-diphenyl-3(2H furanone (MDPF) can be coupled to the phosphomonoester-binding agent, induced to bind to a phosphomonoester-containing molecule and detected by chemiluminescence thru incubation with bis-(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide.
  • acylhydrazides such as isoluminol
  • acridinium derivatives such as isoluminol
  • dioxethanes such as isoxethanes
  • coelenterazines such as peroxyoxalic derivatives
  • peroxyoxalic derivatives can be coupled to the phosphomonester binding agent in order to generate a chemiluminescent reporter of phosphorylation.
  • the chemiluminescent-labeled phosphomonoester selective-binding agent of the technology described herein is very sensitive and produces low background.
  • a phosphomonoester-selective binding agent can become coupled to a phosphorylated target molecule thru the presence of a reactive group on the phosphomonoester-selective binding agent that will form a covalent bond when brought within proximity to the phosphorylated target molecule.
  • a reactive group as used herein refers to a chemical moiety that becomes chemically active by exposure to an appropriate wavelength, typically a UV wavelength. Once activated the reactive group is capable of forming a covalent bond with a proximal moiety on a biological or non-biological component.
  • the phosphomonoester- selective binding agent can contain a photoactivatable group that can form a covalent bond with a phosphorylated target molecule when brought within proximity by the formation of the ternery complex and activated by an appropriate wavelength of light.
  • Photoactivatable groups include, but are not limited to, benzophenones, aryl azides and diazirines.
  • Additional non-limiting directly detectable labels of the technology described herein include luminescent quantum dots, optically active metal naonoparticles, molecular beacons, fluorescent dendrimers, fluorescently-labeled latex nanospheres, and fluorescent aptamers.
  • non- limiting examples of phosphomonoester-selective binding agents of the invention are described in Koike et al., U.S. Patent Publication No. 2005-0038258 published Feb. 17, 2005, Koike et al., U.S. Patent Publication No. 2004- 0198712 published Oct. 7, 2004; Koike et al., European Patent Publication No. 1614706 published Jan. 11, 2006; Koike et al., European Patent Publication No. 1602923 published Dec. 7, 2005; Yashiro et al. (1995) J. Chem. Soc. Commun. 17: 1793-1794; and Yamaguchi et al. (2001) Chem. Commun.
  • the phosphomonoester-selective binding agents of the invention excludes antibodies, such as monoclonal antibodies, polyclonal antibodies, and antibody fragments.
  • the phosphomonoester-selective binding agent of the invention excludes calcium ion indicator dyes induced to bind to trivalent transition metal cations, such as Ga2+ or Fe2+, thru preparation in aqueous-based media with solution pH value of less than 6.5, as described in, e.g., Agnew et al. U.S. Patent No. 7,102,005, the disclosure of which is incorporated herein by reference in its entirety.
  • the method can include the steps of contacting (i) a dinuclear metal chelating moiety with (ii) a salt comprising divalent metal cation and (ii) a counter-anion under conditions where (i), (ii), and (iii) form a ternary complex, wherein the divalent metal cation is capable of simultaneously binding to the dinuclear metal chelating moiety and a phosphorylated target molecule and wherein the counter-anion binds the divalent metal cation with an affinity lower than the affinity with which the phosphorylated target molecule binds the divalent metal cation.
  • kits for detecting the presence or absence of phosphomonoester groups, e.g., a phosphomonoester group on a molecule.
  • the kit can contain any of the counter-anion-phosphomonoester-selective binding agent compositions described herein and, optionally, instructions for detecting the presence or absence of a phosphomonester group on a molecule using the compositions.
  • the kits can also include one or more control molecules (e.g., polypeptides or nucleic acids).
  • the control molecules can contain a phosphomonoester group (e.g., can be phosphorylated such as a phosphorylated polypeptide).
  • the kits can also include one or more wash buffers, fixing buffers, and/or "blocking buffers" for use in, e.g., polyacrylamide gel electrophoresis- coupled detection methods.
  • kits comprising:
  • a salt comprising a counter-anion, wherein the divalent metal cation is capable of simultaneously binding the dinuclear metal chelating moiety and the counter-anion and wherein the counter anion binds the divalent metal cation with an affinity lower than the affinity with which the phosphorylated target molecule binds the divalent metal cation;
  • kits for forming a ternary complex of (i), (ii), and (iii).
  • the kits can include any of the control molecules, instructions, or buffers described above.
  • compositions described can be used in a variety of methods, e.g., methods for detecting phosphomonoester groups such as those of phosphorylated molecules.
  • the methods described herein are also useful in detecting (and/or quantitating) phospho-transfer activity in a sample.
  • the methods and reagents described herein are useful, e.g., for evaluating the phosphorylation state of molecules that participate in signal transduction events.
  • compositions described herein are useful in, e.g., methods for determining whether or not a subject has, or is at risk of developing, a disease characterized by aberrant function or expression of a phospho- transfer activity or methods for identifying compounds useful for treating such disorders.
  • the disclosure features methods for detecting the presence or absence of a phosphomonoester group, e.g., the phosphomonoester group of a phosphorylated molecule.
  • the method can include the steps of contacting a target molecule with any of the binding agents that selectively bind to phosphomonoesters described herein and detecting binding of the dinuclear metal-chelating moiety/divalent metal complex to the target molecule as an indication that the target molecule comprises a phosphomonoester group.
  • a target molecule is contacted with a composition (a ternary complex) comprising a dinuclear metal chelating moiety; a divalent metal cation capable of simultaneously binding to the dinuclear metal chelating moiety and a phosphorylated target molecule; and a counter-anion that binds the divalent metal cation with an affinity lower than the affinity with which the phosphorylated target molecule binds the divalent metal cation.
  • a composition (a ternary complex) comprising a dinuclear metal chelating moiety; a divalent metal cation capable of simultaneously binding to the dinuclear metal chelating moiety and a phosphorylated target molecule; and a counter-anion that binds the divalent metal cation with an affinity lower than the affinity with which the phosphorylated target molecule binds the divalent metal cation.
  • the target molecule contains a phosphomonoester group (e.g., if the target molecule is phosphorylated)
  • the counter-anion is displaced and the dinuclear metal chelating moiety (in complex with the divalent metal cation; the phosphomonoester- selective binding agent) specifically binds to the phosphomonoester group.
  • the binding of the phosphomonoester-selective binding agent to the phosphomonoester group can be detected (or measured) by several mechanisms.
  • the dinuclear metal chelating moiety is detectably labeled, e.g., fluorescently labeled
  • the binding of the complex to the phosphomonoester group can be determined by detecting the detectable label.
  • Suitable detectable labels include, e.g., a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, allophycocyanin (APC), phycoerythrin, or any others described herein), a luminescent material (e.g., europium, terbium), a bioluminescent material (e.g., luciferase, luciferin, or aequorin), a radioactive material (e.g., 125 I, 131 1, 35 S, 32 P, 33 P, or 3 H), an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase), or an epitope tag specifically recognized by antibody that is detectably labeled.
  • a fluorescent material
  • the directly detectably labeled phosphomonoester-selective binding agents described herein can have lower background than indirectly detectably labeled agent.
  • the agent described herein can be added to a sample suspected of containing a phosphomonoester residue. After washing to remove the unbound agent, the sample can be read with a detector to determine if a detectably labeled phosphomonoester-selective binding agent has bound to a phosphomonoester residue in the sample.
  • identifying phosphomonoester residues with an indirectly detectably labeled agent can require multiple binding and rinsing steps. For example, the indirectly detectably labeled agent is first added to the sample suspected of containing the phosphomonoester residue.
  • the unbound agent is rinsed off by washing the sample, and the labeled secondary molecule is added, followed by a wash step to remove unbound labeled secondary molecule.
  • the sample can then be read with a detector to determine if a phosphomonoester residue is present in the sample.
  • the binding of the phosphomonoester-selective binding agent to a phosphomonoester group can be determined by detecting (and/or measuring) the amount of free counter-anion. That is, determining the amount of counter-anion displaced from the ternary complex upon the binding of the phosphomonoester-selective binding agent (dinuclear metal chelating moiety/divalent cation complex) to the phosphomonoester.
  • the counter-anion can be a quencher and the phosphomonoester-selective binding agent can contain a detectable label (e.g., a fluorophore), wherein the quencher quenches the signal produced from the detectable label when in proximity.
  • a detectable label e.g., a fluorophore
  • liberation of the counter-anion quencher upon the binding of the phosphomonoester-selective binding agent to a phosphomonoester group would allow the detectable label to produce a detectable signal.
  • the binding of the phosphomonoester-selective binding agent to a phosphomonoester group can be determined by detecting (and/or measuring) the amount of detectable label released from quenching by the counter-anion quencher.
  • a target molecule can be contacted with a composition (a ternary complex) comprising a dinuclear metal chelating moiety; a divalent metal cation; and a counter-anion that is a dye such as any of the catechol-based dyes described herein.
  • the dye can be one that produces one detectable signal (e.g., a color) when in complex with the dinuclear metal chelating moiety and the divalent metal ion, and a different detectable signal when not in complex (e.g., free).
  • the counter-anion catechol-based dye pyrocatechol violet generates a visible violet color when in complex with a phosphomonoester-selective binding agent but a yellow color when free from the complex (Example 8).
  • a phosphomonoester-selective binding agent/pyrocatechol complex can be added to a sample containing a phosphomonoester group (e.g., a phosphomonoester group of a phosphorylated molecule).
  • a phosphomonoester group e.g., a phosphomonoester group of a phosphorylated molecule.
  • the presence or amount of a yellow color in the solution indicates that the phosphomonoester-selective binding agent bound to a phosphomonoester group in the sample.
  • the disclosure also features methods for detecting the presence or absence of a phosphorylated target molecule in a sample.
  • the method can include the steps of separating a target molecule from a sample using a separation matrix; contacting a target molecule with any of the binding agents that selectively bind to phosphomonoester groups described herein; and detecting binding of the dinuclear metal-chelating moiety to the target molecule as an indication that the target molecule is phosphorylated.
  • the separation matrix can vary depending on the nature (e.g., size, charge, or hydrophobicity) of a target molecule to be separated.
  • the separation matrix is a cross-linked polymer gel whose composition and porosity is chosen based on the weight and composition of the molecule.
  • One non- limiting gel of the invention is made of different concentrations of acrylamide and a cross-linker (e.g. N 5 N'- methylenebisacrylamide) that are induced to polymerize using agents such as tetramethylethylenediamine (TEMED) and/or ammonium persulfate to result in a meshed network of polyacrylamide.
  • TEMED tetramethylethylenediamine
  • the pore size of the polyacrylamide gel can be altered by modifying the concentrations of the acrylamide and the cross-linker.
  • the acrylamide gel can include a denaturant such as sodium dodecyl sulfate or urea.
  • a suitable separation matrix include an agarose gel or a thin layer chromatography (TLC) plate. The latter can be useful, e.g., where a molecule is a lipid or small molecule (e.g., a hydrophobic small molecule).
  • the separation matrix can be an agarose gel, a capillary electrophoretic gel, or a size exclusion matrix (e.g., as used in high-performance liquid chromatography).
  • the target molecules can be separated in one dimension.
  • the target molecules can be separated in a second direction (e.g., isoelectric focusing or two-dimensional gel electrophoresis).
  • a mixture of target molecules can be separated by size in a first dimension and by charge (e.g., in a pH gradient) in a second dimension.
  • the target molecule e.g., a protein
  • Fixation can include contacting the molecule or the separation matrix containing the molecule with a fixing agent (or solution containing a fixing agent).
  • a fixing agent or solution containing a fixing agent
  • a polyacrylamide gel containing both phosphorylated and non-phosphorylated proteins can be contacted with a solution containing an alcohol such as isopropanol or methanol.
  • a fixed gel can, optionally, be dried.
  • one or more different polypeptides can be separated from a sample using polyacrylamide gel-electrophoresis.
  • the one or more different polypeptides can contain at least one polypeptide comprising a phosphomonoester group (e.g., a phosphorylated polypeptide).
  • the gel matrix can be fixed in a fixing solution (e.g., a solution containing 10% ethanol and/or 7% acetic acid or a methanol solution).
  • the gel matrix can then be washed and contacted with, e.g., any of the detectably labeled phosphomonoester/counter-anion complexes described herein (e.g., TRITC-, FITC-, or ruthenium-based organic dye-Phos-tagTM complexes).
  • detectably labeled phosphomonoester/counter-anion complexes described herein e.g., TRITC-, FITC-, or ruthenium-based organic dye-Phos-tagTM complexes.
  • unbound complex can be removed by washing or rinsing and the specific binding of a phosphomonoester-selective binding agent to a phosphomonoester group containing polypeptide is determined by detecting the detectable label.
  • the methods can also include the step of determining the total amount of molecules in a sample.
  • a gel matrix containing multiple different, separated polypeptides can be stained with a total protein stain to determine the total number and/or amount of proteins in the matrix.
  • Suitable protein stains include, e.g., organic dyes, silver stain, radiolabel, reverse stain, fluorescent stain, and chemiluminescent stain.
  • proteins can be colorimetric stained with silver or Coomassie blue dye; fluorescent labeled with SYPRO® Ruby (Invitrogen), Deep Purple (GE Healthcare), Flamingo (Bio-Rad), Krypton (Thermo-Fisher), LUCY (Sigma-Aldrich) or 5- hexadecanoylamino-fluorescein dyes; covalently labeled with fluorescent Cy2, Cy3, Cy5 or ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester- bis(hexafluorophosphate) dyes; or reverse stained with imidazole -zinc, methyl trichloroacetate, copper chloride, zinc chloride or potassium chloride.
  • the total protein stain can be used to determine the amount of phosphorylated forms of a target molecule as compared to the total amount of the target molecule in a sample.
  • SDS sodium dodecyl sulfate
  • the binding reaction is generally performed at a pH value of between about 5.5 and 8.5 in order to facilitate titration of the phosphomonoester group on the target protein or peptide to the dianionic form.
  • Monoanionic and neutral forms of the phosphomonoester group existing at more acidic pH value, bind with significantly lower affinity than the dianionic form, and in fact fail to displace the described counter-anion from the phosphomonoester-selective binding agent.
  • the phosphomonoester-selective binding agent can be used to detect phosphorylated molecules at concentrations of phosphomonoester-selective binding agent less than about 25 ⁇ M , such as less than about 2.5 ⁇ M , about 0.5.0 to about 2.0 ⁇ M , and about 1.0 to about 1.5 ⁇ M
  • the phosphomonoester-selective binding agents described herein can be used with a variety of media. Components of the media can be selected based on the properties of the particular phosphomonoester-selective binding agent, molecule and format of the assay.
  • the media can therefore have any of an acidic, neutral or basic pH.
  • detection of a phosphorylated molecule using a phosphomonoester-selective binding agent is carried out at neutral pH (e.g., about pH 6.5 to about pH 7.5).
  • the detected molecule can be isolated from the gel and analyzed.
  • the protein can be isolated and subjected to mass spectrometry to determine the location of the phosphomonoester group on the protein.
  • Methods of analyzing an target molecule containing a phosphomonoester group include, e.g., matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS)-based peptide mass fingerprinting or tandem mass spectrometry-based peptide sequencing and are described in, e.g., Schilling et al. (2006) J. Biol. Chem., Vol.
  • the molecule Prior to analyzing (e.g., mass spectrometric analysis of) the phosphomonoester group containing molecule, the molecule can be contacted with a protease, e.g., trypsin.
  • the molecule e.g., the protein
  • the molecule can be contacted with the protease prior to its isolation from the separation matrix (e.g., in-gel trypsinization) or after its separation from the matrix.
  • the molecules (e.g., the polypeptides) in a gel can be electrophoretically transferred to a membrane such as PVDF membrane.
  • a membrane such as PVDF membrane.
  • the membrane can be allowed to air dry and then soaked in an alcohol (e.g., ethanol or methanol) or an aqueous solution containing an alcohol.
  • the membrane can be "blocked.” That is, to reduce background and non-specific binding of detection reagents to the membrane, portions of the membrane not occupied by transferred protein can be contacted with a solution comprising a control polypeptide such as serum albumin.
  • the membrane can then be contacted with an anion-phosphomonoester binding complex described herein.
  • the membrane can be washed to remove unbound complex, and then binding of a phosphomonoester binding agent can be detected.
  • a CE apparatus generally consists of a sample vial, source and destination vials, a capillary, electrodes, a high-voltage power supply, a detector, and a data output and handling device.
  • the source vial, destination vial and capillary are filled with an electrolyte such as an aqueous buffer solution.
  • the capillary inlet is placed into a vial containing the sample and then returned to the source vial (sample is introduced into the capillary via capillary action, pressure, or siphoning).
  • sample is introduced into the capillary via capillary action, pressure, or siphoning.
  • the migration of the analytes is then initiated by an electric field that is applied between the source and destination vials and is supplied to the electrodes by the high- voltage power supply.
  • the analytes separate as they migrate due to their electrophoretic mobility and are detected near the outlet end of the capillary.
  • the output of the detector is sent to a data output and handling device such as an integrator or computer.
  • the sample can be contacted with a staining solution comprising any of the counter-anion-phosphomonoester-selective binding agent complexes described herein, e.g., counter-anion-detectably-labeled phosphomonoester- selective binding agent complexes (such as counter-anion-FITC-Phos-tagTM).
  • the detectably-labeled phosphomonoester-selective binding agent would thus bind to a phosphomonoester group, if present on a molecule in the sample, and as the molecules in the sample traversed the capillary, the labeled phosphomonoester-selective binding agent- bound molecules are detected by the detector.
  • capillary electrophoresis is described in, e.g., Lin et al. (2000) Anal. Chem. 72:5348-5355; and Skoog et al. Principles of Instrumental Analysis, 5th ed. Saunders college Publishing: Philadelphia, 1998, the disclosures of each of which are incorporated by reference in their entirety.
  • kinase activity such tyrosine kinase, threonine/serine kinase, saccharide kinase, or lipid kinase activity.
  • Exemplary kinases are set forth in the following Examples and include Ca2+/Calmodulin Dependent Protein Kinase II.
  • Phospho-transfer activity capable of removing a phosphate moiety from a substrate can be, e.g., a phosphatase activity.
  • Phosphatase activity can include, e.g., tyrosine-specif ⁇ c phosphatase, threonine/serine phosphatase, dual-specificity phosphatase, saccharide phosphatase, histidine-specif ⁇ c phosphatase, or lipid phosphatase activity.
  • An exemplary phosphatase for use in the methods described herein is Protein Phosphatase 2A (PP2A).
  • Phospho-transfer activities capable of modifying (e.g., cyclizing or decyclizing) a phosphate moiety on a substrate include, e.g., phosphodiesterase (PDE) and nucleotide cyclase activity.
  • PDE phosphodiesterase
  • nucleotide cyclase activity comprise a large group of enzymes organized into 11 distinct families based on biochemical and molecular properties.
  • PDEs include human Phosphodiesterase 3B, human Phosphodiesterase 1 IAl, human Phosphodiesterase 4A4, human Phosphodiesterase 4D3, and calf spleen Phosphodiesterase (Type II).
  • Phospho-transfer substrates can be used that are recognized with high affinity and specificity.
  • the substrate can be the cognate substrate of the enzyme, e.g., in vivo.
  • Peptide substrates can be short peptides, e.g., less than 15 or 10 amino acids, or can be contained in a larger protein, e.g., a full length protein or full length domain from a protein (such as a naturally occurring protein). Artificial substrates and mimetics of the cognate substrate can also be used.
  • substrates include the following: Forkhead transcription factors or p21WAF (for the kinase Akt); Androgen Receptor (for the phosphatase PP2A); AMP for the nucleotide cyclase adenylate cyclase); and cyclic GMP (cGMP; for the phosphodiesterase cGMP specific phosphodiesterase type 5)(see, e.g., Brunet et al. (1999) Cell 96:857-868; Li et al. (2002) J. Biol. Chem. 277:11352-11361; Yang et al. (2007) MoL Cell. Biol. 27(9):3390-3404; and Bakre et al.
  • Forkhead transcription factors or p21WAF for the kinase Akt
  • Androgen Receptor for the phosphatase PP2A
  • AMP for the nucleotide cyclase adenylate cyclas
  • Exemplary substrates include polypeptides (e.g., short peptide sequences, large macromolecules, or complexes of one or more polypeptides), nucleic acids (e.g., polymeric or monomeric DNA, RNA, or modified DNA or RNA), lipids, saccharides (e.g., mannose or glucose), small molecule compounds (e.g., steroids, vitamins), or phosphorylated forms of any of the aforementioned (e.g., phosphorylated polypeptides, nucleic acids, saccharides, lipids or small molecules).
  • the phosphorylated forms of the substrates can be cyclic phospho-forms such as cyclic nucleotide monophosphates (e.g., cAMP or cGMP).
  • the substrate is attached to a solid support via a heterologous moiety, such as a linker.
  • the heterologous moiety can be one that does not affect interaction between the substrate and the enzyme.
  • linker moieties can be used to attach any intact substrate to a support.
  • the linker portion, or substrate can contain a reactive group to facilitate chemical linkage to a support (see below).
  • the linker portion, or substrate can contain a moiety recognized by a binding partner (e.g., a first member of a binding pair) that is attached to, or is attachable to, a support. Suitable binding partners are described below.
  • the recognition site is a "phosphoacceptor site,” often, this is referred to as the phosphorylation consensus sequence.
  • Biological targets e.g., proteins and glycoproteins
  • a phosphoacceptor site for a kinase of biological targets thus typically includes a tyrosine, serine, or threonine residue surrounded by additional amino acid residues which help the kinase recognize its site.
  • Many phosphoacceptor sites are known (see, e.g., Kennelly, P. J., and Krebs, E. G.
  • a phosphatase recognition site includes a phosphorylated site, such as a phosphorylated serine, phosphorylated threonine, or phosphorylated tyrosine residue.
  • phosphatase recognition sites include Nterm-RRA(pT)VA-Cterm (where the "pT” is a phosphorylated threonine residue), which is recognized by several serine/threonine phosphatases such as the Protein Phosphatases 2 A, 2B and 2C, and N- term-END(pY)INASL-Cterm and Nterm-DADE(pY)LIPQQG-Cterm (where the "pY” are a phosphorylated tyrosine residues) which is recognized by many protein tyrosine phosphatases.
  • Nterm-RRA(pT)VA-Cterm where the "pT” is a phosphorylated threonine residue
  • serine/threonine phosphatases such as the Protein Phosphatases 2 A, 2B and 2C
  • N- term-END(pY)INASL-Cterm and Nterm-DADE(pY)LIPQQG-Cterm where the "pY” are
  • a target molecule or phospho-transfer substrate useful in a method described herein can be attached to a solid support, e.g., a porous or non-porous material that is insoluble.
  • the molecule or substrate can be associated with the support in variety of ways, e.g., covalently or non-covalently bound.
  • the molecule or substrate is generally attached such that it is accessible to the phospho-transfer activities in solution.
  • a support can be composed of a natural or synthetic material, an organic or inorganic material, such as a polymer, resin, metal or glass, and combinations thereof. Many suitable supports are known in the art and illustratively include, e.g., particles, such as Luminex®-type encoded particles, magnetic particles, and glass particles.
  • a support useful in a method described herein can have a variety of physical formats, which can include for example, a membrane, column, a hollow, solid, semisolid, pore or cavity containing particle such as a bead, a gel, a fiber, including a fiber optic material, a sheet, a matrix and sample receptacle.
  • sample receptacles include sample wells, tubes, capillaries, vials and any other vessel, groove or indentation capable of holding a sample, including those containing membranes, filters, matrices and the like.
  • a sample receptacle also can be contained on a multi-sample platform, such as a microplate, slide, microfluidics device, array substrate, mass spectrometry sample plate, and the like.
  • exemplary particles that can be used can have a variety of sizes and physical properties. Particles can be selected to have a variety of properties useful for particular experimental formats. For example, particles can be selected that remain suspended in a solution of desired viscosity or to readily precipitate in a solution of desired viscosity. Particles can be selected for ease of separation from sample constituents, for example, by including purification tags for separation with a suitable tag- binding material, paramagnetic properties for magnetic separation, and the like.
  • encoded particles are used.
  • Each particle includes a unique code (such as a bar code, luminescence code, fluorescence code, a nucleic acid code, and the like).
  • Encoding can be used to provide particles for evaluating different enzymatic activities in a single sample. Such methods can include contacting a sample to a mixture of encoded particles, and then contacting the encoded particles to detectably labeled tags for evaluating the enzymatic reacting.
  • the detectable tags can be used to detect the presence or amount of modified substrates (e.g., support-bound products, or free products).
  • the code is embedded (for example, within the interior of the particle) or otherwise attached to the particle in a manner that is stable through hybridization and analysis.
  • the code can be provided by any detectable means, such as by holographic encoding, by a fluorescence property, color, shape, size, weight, light emission, quantum dot emission and the like to identify particle and thus the capture probes immobilized thereto. Encoding can also be the ratio of two or more dyes in one particle that is different than the ratio present in another particle.
  • the particles may be encoded using optical, chemical, physical, or electronic tags. Examples of such coding technologies are optical bar codes fluorescent dyes, or other means.
  • the particle code is a nucleic acid, e.g., a single stranded nucleic acid.
  • a solid support such as a particle can be attached to a single-or double-stranded nucleic acid, each strand being about 10 nucleotides (e.g., about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, or about 150 or more nucleotides) in length.
  • nucleotides e.g., about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 35 nucleotides, about 40 nucleotides, about 50 nucleotides, about 75 nucleotides, about 100 nucleotides, or about 150 or more nucleotides
  • Different encoded particles can be used to evaluate a number of different enzymatic activities in parallel, so long as the encoding can be used to identify the intact substrate on a particular particle, and hence the activity being evaluated.
  • a sample can be contacted with a plurality of such coded particles.
  • the particle code is read as is the fluorescence associated with the particle from any probe used to evaluate modification of the intact substrate associated with the particles.
  • One exemplary platform utilizes mixtures of fluorescent dyes impregnated into polymer particles as the means to identify each member of a particle set to which a specific capture probe has been immobilized.
  • Another exemplary platform uses holographic barcodes to identify cylindrical glass particles.
  • Chandler et al. U.S. Patent No. 5,981,180 describes a particle-based system in which different particle types are encoded by mixtures of various proportions of two or more fluorescent dyes impregnated into polymer particles.
  • Soini U.S. Patent No. 5,028,545 describes a particle-based multiplexed assay system that employs time-resolved fluorescence for particle identification. Fulwyler (U.S. Patent No.
  • U.S. Patent No. 6,916,661 describes polymeric microparticles that are associated with nanoparticles that have dyes that provide a code for the particles.
  • the polymeric microparticles can have a diameter of less than one millimeter, e.g., a size ranging from about 0.1 to about 1,000 micrometers in diameter, e.g., 3-25 ⁇ m or about 6-12 ⁇ m.
  • the nanoparticles can have, e.g., a diameter from about 1 nanometer (nm) to about 100,000 nm in diameter, e.g., about 10 - 1,000 nm or 200 - 500 nm.
  • suitable solid-supports include, e.g., substrate arrays such as nucleic acid or peptide arrays. Like encoded particles, an important characteristic of an array is that all substrates (of a plurality of substrates) can be immobilized at predetermined positions such that each substrate can be identified by its position.
  • Exemplary arrays for use in detection methods described herein include, e.g., protein and nucleic acid arrays (see, e.g., U.S. Patent Nos. 6,197,599; 5,902,723; and 5,871,928; the disclosures of each of which are incorporated herein by reference in their entirety).
  • a target molecule or phospho-transfer substrate can be covalently or non- covalently bound to a support.
  • a variety of chemical reactions useful for covalently attaching a substrate to a support are well known to those skilled in the art (see, for example, Hartmann et al. (2002) J. Mater. Res. 17(2):473-478).
  • Illustrative examples of functional groups useful for covalent attachment of substrates to a support include alkyl, Si-OH, carboxy, carbonyl, hydroxyl, amide, amine, amino, ether, ester, epoxides, cyanate, isocyanate, thiocyanate, sulfhydryl, disulfide, oxide, diazo, iodine, sulfonic or similar groups having chemical or potential chemical reactivity.
  • Illustrative examples of binding partners useful for non-covalent attachment of substrates to a support include antibodies, antibody-like materials, and agents, e.g., that are capable of binding to antibodies such as, but not limited to, staphylococcal protein A or protein G.
  • the target molecule or phospho-transfer substrate can contain, or be, a nucleic acid that is complementary to the particle code nucleic acid.
  • a peptide substrate can be joined to a nucleic acid, which nucleic acid is complementary to a nucleic acid bound to a particle, such that the nucleic acids bind and tether the substrate to the particle.
  • a sample can be any composition.
  • the content of the sample can be known or unknown.
  • a sample contains or is suspected of containing one or more enzymes.
  • a sample can be derived from an organism or man-made source of enzyme.
  • a sample can be, e.g., one containing one or more enzymes in a known quantity or with a known activity.
  • a sample can be, for example, a specimen obtained from an individual or can be derived from such a specimen.
  • a sample can be a tissue section obtained by biopsy, or cells that are placed in or adapted to tissue culture.
  • a sample can also be, or contain, a biological fluid specimen such as urine, blood, plasma, serum, saliva, semen, sputum, cerebral spinal fluid, tears, mucus, sweat, milk, semen, and the like.
  • Biological samples can also be, or contain, fluid from ulcers or other surface eruptions such as blisters and abscesses or can be extracts of tissues from biopsies of normal, malignant, or suspect tissues.
  • a sample can be further fractionated, if desired, to a fraction containing particular components or cell types.
  • a blood sample can be fractionated into serum or into fractions containing particular types of blood cells such as red blood cells or white blood cells (leukocytes).
  • a sample can be a combination (pool) of samples from an individual such as a combination of a tissue and fluid sample, and the like.
  • a sample can be processed to facilitate detection of enzymes.
  • the sample can be treated with freeze/thaw treatment, drying and rehydrating, a dounce, detergent or other methods.
  • Releasing or solubilizing enzymes can also be used provided they do not interfere with the assay, e.g., by interfering with the activity of a phospho-transfer enzyme of interest.
  • the sample can be obtained from body fluids and tissues in which particular enzymes being tested are typically expressed.
  • Samples can be treated with customary care to preserve enzymatic activity. Suitable methods for obtaining samples that preserve the activity or integrity of enzymes in the sample are well known to those skilled in the art. Such methods include the use of appropriate buffers and/or inhibitors, including nuclease, protease and phosphatase inhibitors that preserve or minimize changes in enzymes in the sample.
  • Such inhibitors include, for example, chelators such as ethylenediamne tetraacetic acid (EDTA), ethylene glycol bis(P-aminoethyl ether) N,N,Nl,Nl-tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain and the like, and phosphatase inhibitors such as phosphate, sodium fluoride, vanadate and the like.
  • chelators such as ethylenediamne tetraacetic acid (EDTA), ethylene glycol bis(P-aminoethyl ether) N,N,Nl,Nl-tetraacetic acid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, antipain and the like, and phosphatase
  • the enzymatic activity to be detected is a protease
  • methods for obtaining samples that preserve the activity or integrity of the enzyme would not include protease inhibitors that adversely affect the particular protease activity.
  • Appropriate buffers and conditions for enzyme-containing samples are well known (see, for example, Ausubel et al. Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999); Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press (1988); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1999); Tietz Textbook of Clinical Chemistry, 3rd ed. Burtis and Ashwood, eds. W.B. Saunders, Philadelphia, (1999)).
  • a sample can be processed to eliminate or minimize the presence of interfering substances, as appropriate.
  • a sample can be fractionated by a variety of methods well known to those skilled in the art, including subcellular fractionation, and chromatographic techniques such as ion exchange, hydrophobic and reverse phase, size exclusion, affinity, hydrophobic charge-induction chromatography, and the like (Ausubel et al. supra, 1999; Scopes, Protein Purification: Principles and Practice, third edition, Springer- Verlag, New York (1993); Burton and Harding, J. Chromatogr. A 814:71-81 (1998)).
  • a sample can be in a variety of physical states.
  • a sample can be a liquid or solid, can be dissolved or suspended in a liquid, can be in an emulsion or gel, and can be absorbed onto a material.
  • a sample can be a liquid blood sample, liquid serum sample, liquid white blood cell sample, dried blood, serum, or white cell sample, or such a sample absorbed onto a paper or polymer substrate.
  • Some of the methods described herein can be carried out under conditions that allow an enzyme to act on a substrate.
  • Conditions under which proteins retain activity are well known to those skilled in the art and generally include roughly physiologically salt levels, a buffering agent, and a temperature in the range of 4-37°C.
  • a sample can be adjusted or placed into a solution or environment to have a specified characteristic such as a specified pH (e.g., a pH of about 7.0), salt concentration, surfactant property, viscosity and the like.
  • a specified characteristic such as a specified pH (e.g., a pH of about 7.0), salt concentration, surfactant property, viscosity and the like.
  • the ability of an enzyme to act on a substrate can be improved, enhanced and/or stabilized in the presence of sample ingredients such as inorganic salts, alcohols, detergents and surfactants, if desired.
  • the methods generally include the steps of contacting a sample with a phospho-transfer substrate attached to a solid-phase (e.g., an encoded particle), wherein the phospho-transfer substrate comprises a recognition site specific to the phospho-transfer activity (e.g., the activity possessed by a phospho-transfer enzyme).
  • a solid-phase e.g., an encoded particle
  • the phospho-transfer substrate comprises a recognition site specific to the phospho-transfer activity (e.g., the activity possessed by a phospho-transfer enzyme).
  • the methods can be used to, e.g., determine the substrate specificity of one or more enzymatic activities; (ii) to identify compounds capable of modifying one or more phospho-transfer activities; (iii) optimize reaction conditions for one or more phospho- transfer activities, or (iv) identify one or more phospho-transfer activities (e.g., identify an unknown kinase activity in a sample).
  • a method described herein can be used to detect the presence, or absence, of a phospho-transfer activity associated with disease. For example, the presence of a kinase associated with cancer (e.g., Aurora kinase, Akt, BCR-AbI, or RET/PTC) in a biological sample can be detected using any of the methods described herein.
  • suitable conditions under which a phospho-transfer reaction can occur include pH ranges of greater than 5, or greater than 6.
  • the reactions can be performed at a pH range of about 6.0 to 8.0 (e.g., about between pH 6.0 and pH 6.5, about between pH 6.0 and pH 7.0, about between pH 6.5 and pH 7.0, about between pH 6.5 and pH 7.5, about between pH 6.8 and pH 7.5; about between pH 6.9 and pH 7.2; about between pH 7.0 and pH 7.5; about between pH 7.0 and pH 8.0; or about between pH 7.5 and pH 8.0).
  • the disclosure provides a method for detecting the presence of a kinase in a sample, the activity of that kinase, and/or the incubation conditions appropriate for kinase activity.
  • a solid phase (such the bottom of a well or tube, the surface of a slide or a Petri dish, or a bead or a particle such as an encoded particle) is contacted with a substrate, wherein the substrate is subsequently immobilized on the solid phase.
  • a substrate such as shown in Fig. 37, numerous identical peptide substrates having a phosphoacceptor site recognized by a specific kinase are immobilized on an encoded particle.
  • the particle (one or more particles, which may be in approximately known numbers) can then be exposed to a sample (e.g., a sample containing a kinase activity that recognizes the recognition site (e.g., phosphoacceptor site) contained in the substrate) under phosphorylation conditions.
  • a sample e.g., a sample containing a kinase activity that recognizes the recognition site (e.g., phosphoacceptor site) contained in the substrate
  • phosphorylation reaction mixture is meant that a reaction mixture contains components in which a kinase activity, if present in the reaction mixture, is able to phosphorylate a substrate (i.e., containing a recognition site, in this case, a phosphoacceptor site for the kinase activity) present in the reaction mixture.
  • a phosphorylation reaction mixture contains components including, without limitations, ATP, divalent cation such as magnesium or manganese and suitable aqueous buffer. Exemplary phosphorylation conditions are also
  • Fig. 38 shows, if the sample contains a kinase activity that recognizes the phosphoacceptor site in the substrate, then some or all of the substrates bound to the beads will become phosphorylated. For example, under appropriate incubation conditions, the number of phosphorylated peptides will be approximately proportional to the concentration of the specific kinase.
  • the particles are then washed to remove the free kinase and any other components of the phosphorylation reaction mixture (e.g., free ATP).
  • the particles can then be exposed to a counter-anion- detectable phosphomonoester-selective binding agent complex (see Fig. 39) under conditions whereby the detectable phosphomonoester-selective binding agent will bind to the particles if peptides immobilized on the particles are phosphorylated.
  • the phosphomonoester-selective binding agent can be any of those described herein.
  • the excess ubound complexes are washed away, and a signal indicative of the number of phosphorylated peptides is read, for example on a flow cytometric instrument such as a Luminex xMAP 200 instrument (commercially available from the Luminex Corp., Austin, TX).
  • a flow cytometric instrument such as a Luminex xMAP 200 instrument (commercially available from the Luminex Corp., Austin, TX).
  • the phosphomonoester-selective binding agent is labeled with phycoerythrin, which is easily read by the Luminex xMAP 200 instrument.
  • the method includes contacting a sample containing the kinase (or suspected of containing a kinase) with a substrate (e.g., a peptide substrate) in a phosphorylation reaction mixture, wherein the substrate comprises a phosphoacceptor site specific to the kinase.
  • a substrate e.g., a peptide substrate
  • the substrate is immobilized on a solid phase surface prior to contact with the sample in a phosphorylation reaction mixture.
  • the substrate can immobilized on a solid phase surface after contacting the substrate with the sample in a phosphorylation reaction mixture.
  • the substrate immobilized on the solid phase is separated from the sample, and then contacted with a detectable phosphomonoester-selective binding agent.
  • the substrate is contacted with the detectable phosphomonoester-selective binding agent prior to immobilizing the peptide on a solid phase. The presence of the detectable phosphomonoester-selective binding agent bound to the peptide indicates the presence of the kinase in the sample.
  • the substrate can be the kinase itself.
  • the methods described herein also relate to the detection and/or identification of a phosphatase.
  • the methods can include contacting a sample with a phosphorylated substrate under dephosphorylation conditions, wherein the phosphorylated substrate comprises a recognition site specific to the phosphatase.
  • substrate can be immobilized on a solid phase prior to contact with the sample or immobilized on a solid phase surface after contact with the sample.
  • the methods further includes separating the solid phase from the phosphatase reaction mixture and contacting the solid phase with a counter-anion-detectable phosphomonoester-selective binding agent complex under conditions whereby the detectable phosphomonoester-binding agent will bind to the solid phase if the immobilized peptide is phosphorylated. After separating the immobilized peptide from the unbound complex, the detectable phosphomonoester-selective binding agent bound to the immobilized peptide is detected. A decrease in the detectable phosphomonoester- selective binding agent bound to the immobilized peptide indicates the presence of the phosphatase in the sample.
  • Phosphodiesterase Activity Also provided are methods for detecting the presence of a phosphodiesterase in a sample.
  • phosphodiesterase is meant an enzyme that catalyzes the hydrolysis of phosphodiester bonds.
  • a phosphodiesterase can cleave a cAMP into AMP.
  • the methods for detecting a phosphodiesterase include, e.g., contacting the sample suspected of containing the phosphodiesterase with a cyclic nucleotide, such as a cAMP or a cGMP molecule immobilized on a solid phase in a phosphodiesterase reaction mixture under conditions that allow phosphodiesterase activity.
  • the methods further include separating the solid phase from the phosphorylation reaction mixture and contacting the solid phase with a counter-anion-detectable phosphomonoester-selective binding agent complex under conditions whereby the detectable phosphomonoester- binding agent will bind to the solid phase if the cyclic nucleotide is cleaved by the phosphodiesterase.
  • the methods can further include separating the solid phase from the unbound complex and detecting the detectable phosphomonoester-selective binding agent bound to the solid phase wherein the presence of the detectable phosphomonoester- selective binding agent bound to the solid phase indicates the presence of the phosphodiesterase in the sample.
  • phosphodiesterases Numerous purified phosphodiesterases are commercially available. For example, recombinant human Phosphodiesterase 3B, recombinant human Phosphodiesterase 1 IAl, recombinant human Phosphodiesterase 4A4, recombinant human Phosphodiesterase 4D3, and calf spleen Phosphodiesterase (Type II) are commercially available from the Calbiochem branch of EMD Biosciences, San Diego, CA. Such enzymes can be used in the methods described herein as, e.g., positive controls.
  • Nucleotide Cyclase Activity Nucleotide cyclases are enzymes that catalyze the formation of a cyclic nucleotide from a non-cyclic nucleotide. For example, adenylate cyclase catalyzes the formation of cyclic AMP (cAMP) from ATP. The methods described herein can be employed to detect the presence or the activity of a nucleotide cyclase.
  • cAMP cyclic AMP
  • the invention provides methods for detecting the presence of a nucleotide cyclase in a sample. These methods include contacting the sample suspected of containing the nucleotide cyclase with nucleotide triphosphate, such as an ATP or GTP molecule, immobilized on a solid phase in a cyclase reaction mixture under cyclase reaction conditions.
  • Cyclase reaction mixtures can contain components including, without limitations, free phosphate ⁇ e.g., ATP, GTP, or ADP).
  • Non-limiting cyclase reaction conditions include a pH which allows the activity of the nucleotide cyclase and a temperature which allows the activity of the nucleotide cyclase. In some embodiments, cyclase reaction conditions include having a pH of about 7.0 and a temperature of between about 25°C and about 40 0 C.
  • the solid phase can be separated from the cyclase reaction mixture, and then contacted with a counter-anion-detectable phosphomonoester-selective binding agent complex under conditions whereby the detectable phosphomonoester-selective binding agent will bind to the immobilized ATP if the immobilized nucleotide triphosphate is not cyclized by a nucleotide cyclase.
  • the detectable phosphomonoester-selective binding agent bound to the solid phase is detected.
  • a decrease in the detectable phosphomonoester-selective binding agent bound to the solid phase indicates the presence of the nucleotide cyclase in the sample.
  • screening methods useful for identifying a compound that modulates the activity of a phospho-transfer enzyme such as any of the phospho-transfer enzymes described herein.
  • the method can include the steps of contacting a phospho- transfer substrate with a phospho-transfer enzyme in the presence of a candidate compound; contacting the phospho-transfer substrate with any of the counter-anion- phosphomonoester-selective binding agent complexes described herein; and determining whether the candidate compound modulates the activity of the phospho-transfer enzyme.
  • a difference in the amount of binding of the phosphomonester-selective binding agent (dinuclear metal chelating moiety/divalent cation complex) to the substrate in the presence of the compound as compared to the amount of binding in the absence of the candidate compound indicates that the candidate compound is a compound capable of modulating the activity of the phospho-transfer enzyme.
  • a known substrate of a phospho-transfer enzyme can be attached to a solid support such as a multi-well assay plate and contacted with the phospho-transfer enzyme in the presence or absence of a candidate compound. Suitable conditions for phospho-transfer enzyme reactions are described above. In some embodiments, a series of reactions can be performed under varying concentrations of a candidate compound, which can be used to determine a 50% inhibitory concentration (IC50). For example, a phospho-transfer enzyme reaction can be done in the absence of a candidate compound, or in the presence of from about 0.01 nM to about 10 mM of a candidate compound.
  • the compound can be one that inhibits or stimulates the activity of a phospho- transfer enzyme.
  • a disease state e.g., cancer, an inflammatory condition, or a metabolic disorder
  • the methods described herein can be used to identify a compound that is capable of specifically inhibiting the activity of the phospho-transfer enzyme (e.g., a kinase such as Akt, Aurora kinase, Plkl, HER2, VEGF receptor, or PDGF receptor).
  • a kinase such as Akt, Aurora kinase, Plkl, HER2, VEGF receptor, or PDGF receptor
  • the methods described herein can be used to identify a compound that stimulates the activity of the phospho-transfer enzyme.
  • Candidate compounds that can be used in the methods described herein include various chemical classes and include small organic molecules having a molecular weight in the range of, e.g., 50 to 2,500 daltons.
  • Candidate compounds can optionally contain functional groups that promote interaction with proteins (e.g., hydrogen bonding) and can include at least an amine, carbonyl, hydroxyl, or carboxyl group (or at least two of the functional chemical groups).
  • Candidate compounds can optionally contain cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures (e.g., purine core) substituted with one or more of the above functional groups.
  • Candidate compounds can also include biomolecules including, but not limited to, peptides, polypeptides, proteins, antibodies, peptidomimetics, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives or structural analogues thereof.
  • Candidate compounds can also include nucleic acids, for example, nucleic acids that inhibit the mRNA or protein expression of a phospho-transfer enzyme, for example, an antisense oligonucleotide that hybridizes to a phospho-transfer enzyme mRNA transcript, or a small interference RNA (siRNA) specific for a particular phospho-transfer enzyme.
  • nucleic acids for example, nucleic acids that inhibit the mRNA or protein expression of a phospho-transfer enzyme, for example, an antisense oligonucleotide that hybridizes to a phospho-transfer enzyme mRNA transcript, or a small interference RNA (siRNA) specific for a particular phospho-transfer enzyme.
  • siRNA small interference RNA
  • Candidate compounds can be identified from a number of potential sources, including chemical libraries, natural product libraries, and combinatorial libraries comprised of random peptides, oligonucleotides (e.g., small inhibitory RNAs (siRNAs)), or organic molecules.
  • Chemical libraries can consist of random chemical structures, some of which are analogs of known compounds or analogs or compounds that have been identified as hits or leads in other drug discovery screens, while others are derived from natural products, and still others arise from non-directed synthetic organic chemistry.
  • Natural product libraries can include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science (1998) 282:63-68.
  • Combinatorial libraries can be composed or large numbers of peptides, oligonucleotides, or organic compounds as a mixture.
  • Peptide libraries can be prepared by traditional automated synthesis methods or by use of recombinant nucleic acids.
  • Libraries of interest include peptide combinatorial, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
  • Identification of candidate compounds through the use of the various libraries permits subsequent modification of the candidate compound hit or lead to optimize the capacity of the hit or lead to modulate the activity of a phospho-transfer enzyme.
  • Candidate compounds identified herein can be synthesized by any chemical or biological method.
  • the candidate compounds can also be pure, or may be in a heterologous composition, and can be prepared in an assay-, physiologic-, or pharmaceutically- acceptable diluent or carrier.
  • This composition can also contain additional compounds or constituents that do not bind to or modulate the activity of a phospho-transfer enzyme.
  • multiplex assay methods to detect more than one phospho- transfer activities (e.g., more than one kinase, phosphatase, nucleotide cyclase, or phosphodiesterase activity) simultaneously or, e.g., to determine the substrate specificity of a phospho-transfer activity.
  • a set of encoded particles is used.
  • Subsets of particles, where each subset member is encoded with the same particle identification (ID) code (such as a bar code) each have different kinase substrates (in this case peptide substrate) immobilized on them.
  • ID particle identification
  • the plurality of assays can be carried out simultaneously.
  • a sample containing two kinase activities A and B
  • the mixture of encoded particles comprises one or more pluralities of encoded particles, each plurality of particles having a different attached kinase substrate, the kinase substrate being identifiable by a particle code, and each different kinase substrate containing a phosphoacceptor site for kinase A or kinase B.
  • Fig. 41 shows, as described above where just one type of bead is employed, the multiplex bead set is washed after incubation with the sample and then labeled with a counter-anion-detectable phosphomonoester-selective binding agent complex. After a final wash, the particles are read in a detection instrument that both detects the encoded identification of the particle and also quantifies the signal from the detectable binding agent.
  • An exemplary instrument is the Luminex xMAP 200TM (Luminex Corporation, Austin, TX).
  • the substrates are immobilized on a plurality of solid phases prior to contact with the sample in a phosphorylation reaction mixture, wherein substrates having different sequences are immobilized on different solid phases and wherein the substrates immobilized on the same solid phase have the same sequence. Further, in some embodiments, the substrates are immobilized on solid phases after contacting the substrates with the sample in a phosphorylation reaction mixture. The substrates immobilized on the solid phases are separated from the sample, and then contacted with a detectable phosphomonoester-selective binding agent.
  • the substrates are contacted with the counter-anion-detectable phosphomonoester-selective binding agent complex prior to immobilizing the on a solid phase.
  • the presence of the detectable phosphomonoester-selective binding agent bound to a particular solid phase indicates the presence in the sample of a kinase that recognizes a phosphoacceptor site present in the substrates immobilized on that particular solid phase.
  • the substrates can be contacted with a phospho-transfer activity prior to being immobilized on a solid phase.
  • Fig. 42 each have a sequence-specific tag on one end, such as a single-stranded oligonucleotide, such that substrates having the same amino acid sequence have the same tag attached to them.
  • each molecule of each peptide substrate with the same sequence has the same tag sequence coupled to it, and that tag is different for each different peptide substrate (i.e., peptide substrates having different amino acid sequences also have different tags).
  • the substrates for example, in approximately known numbers, are then added to a biological sample in which the presence of some quantity of the phospho-transfer activity (e.g., a kinase activity) specific to the phosphoacceptor site in the substrates, in a phosphorylation reaction mixture.
  • some quantity of the phospho-transfer activity e.g., a kinase activity
  • a phospho-transfer activity e.g., kinase activity
  • some or all of the sequence-tagged peptides are phosphorylated.
  • the number of phosphorylated substrates will be approximately proportional to the specific activity of the phospho-transfer activities (e.g., kinase activities) in the sample.
  • the tagged substrates After incubation with the kinase-containing sample, the tagged substrates exhibit phosphorylation states in approximate proportion to the specific activity of the respective kinases in the sample (Fig. 43).
  • the tagged peptides are then incubated with a multiplex particle set comprising encoded particles.
  • Each particle encoded with a particular ID has a particular complementary tag sequence immobilized on its surface.
  • Fig. 44 shows, the tag sequences on the peptides thus specifically hybridize to their complementary tags on the encoded particles.
  • the encoded particles with the specifically hybridized peptides captured are then incubated with the counter-anion-detectable phosphomonoester-selective binding agent complex, resulting in the binding of the detectable binding agent to the encoded particles if the substrates bound to the particles contained a phosphoacceptor site recognized by a kinase in the sample (Fig. 45). Then the bead set is read on an appropriate instrument that can read the bead ID and quantify the reporter signal for each bead.
  • the invention provides a method for detecting the presence of one or more kinase activities in a sample.
  • the method includes contacting the sample suspected of containing one or more kinases with a plurality of peptides in a phosphorylation reaction mixture under phosphorylation conditions, wherein at least one peptide in the plurality comprises a phosphoacceptor site specific to one or more kinase.
  • the plurality of peptides is immobilized on a plurality of solid phases, wherein each peptide having a different amino acid sequence is immobilized on a separate and distinguishable solid phase.
  • each solid phase can be separated and can be distinguished from one another, for example, by using encoded solid phases (e.g., solid phases encoded with different bar codes, different colored solid phases, or solid phases having different sizes).
  • the immobilized peptides are contacted with a counter-anion-detectable phosphomonoester- selective binding agent complex under conditions whereby the detectable phosphomonoester-selective binding agent will bind to the immobilized peptide if the immobilized peptide is phosphorylated.
  • the solid phase to which the detectable phosphomonoester-selective binding agent is bound is detected and distinguished.
  • the binding of the detectable phosphomonoester-selective binding agent to a particular solid phase indicates the presence in the sample of a kinase that recognizes the phosphoacceptor site in the peptide immobilized on that solid phase.
  • Example 13 also featured are methods useful in determining the substrate specificity of a phospho-transfer activity (e.g., a kinase activity or a phosphatase activity).
  • a mixture of encoded particles can be contacted with a sample containing a kinase activity of interest, wherein the mixture of encoded particles includes one or more pluralities of encoded particles, each plurality of particles having a different attached phospho-transfer substrate.
  • Each different substrate is identifiable by a particle code.
  • At least two pluralities of encoded particles include different phospho-transfer substrates, which substrates contain a variant recognition site (phosphoacceptor site) specific to one kinase activity.
  • substrates immobilized on the solid phases are separated from the sample, and then contacted with a counter-anion-detectable phosphomonoester- selective binding agent complex.
  • the substrates are contacted with a counter-anion-detectable phosphomonoester-selective binding agent complex prior to immobilizing the on a solid phase.
  • the substrates are contacted with the counter-anion-detectable phosphomonoester-selective binding agent complex at the same time as the sample.
  • the presence of the detectable phosphomonoester-selective binding agent bound to a particular solid phase indicates the presence in the sample of a kinase that recognizes a phosphoacceptor site present in the substrates immobilized on that particular solid phase. Such results would indicate that the preferentially phosphorylated substrate contains a preferred recognition site for that particular kinase activity.
  • the detectable phosphomonoester-selective binding agent bound to the solid phase is detected using a flow cytometer.
  • the flow cytometer is a Luminex system, such as a Luminex 200TM system.
  • any of the proceeding methods can utilize phosphomonoester-selective binding agents that are not detectably labeled.
  • the counter-anion of a counter-anion-phosphomonoester-selective binding agent complex can be a dye such as a catechol-based dye (e.g., Pyrocatechol Violet or any other catechol-based dye described herein).
  • the biniding of the phosphomonoester-selective binding agent to a phosphomonoester containing molecule can be detected as a measure of the free counter-anion dye liberated from the complex.
  • any of the methods for detection can be performed in any format that allows for rapid preparation, processing, and analysis of multiple reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 386 wells).
  • Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the detectable phosphomonoester-selective binding agent.
  • detectors include, but are not limited to, spectrophotometers, luminometers, fluorometers, and devices that measure radioisotope decay.
  • kits for detecting the presence of one or more phospho- transfer activities, which kits can include a mixture of encoded particles and any of the counter-anion-phosphomonoester-selective binding agent complexes described herein.
  • the kits can include counter-anion- phosphomonoester-selective binding agent complex, wherein the phosphomonoester-selective binding agent is detectably labeled.
  • the kits can include counter-anion- phosphomonoester-selective binding agent complex, wherein the counter-anion is a dye such as a catechol-based dye.
  • the mixture includes one or more pluralities of encoded particles, each plurality of particles having a different attached phospho-transfer substrate.
  • the phospho-transfer substrate is identifiable by a particle code.
  • At least some of the encoded particles can be magnetic.
  • the kit can optionally contain more than one plurality of encoded particles.
  • the kit can at least two pluralities of encoded particles, which particles contain different nucleic acid particle codes.
  • a TRITC labeled phosphomonoester-binding molecule was synthesized as depicted in Fig. 7.
  • 34 mg (0.063 mmol) of N,N,N'-Tri(2- pyridylmethyl)-N ' - [5 -N"-(2-aminoethyl)carbamoyl-2-pyridylmethyl]- 1,3- diaminopropane-2-ol 34 mg, 0.063 mmol
  • the resulting solution containing the reaction mixture was sonicated at room temperature for 20 minutes.
  • reaction mixture was purified on a C 18(2) preparative column using the following conditions:
  • a FITC labeled phosphomonoester-binding molecule was synthesized as depicted in Fig. 8. For this synthesis, 10 mg (0.018 mmol) of N,N,N'-Tri(2-pyridylmethyl)-N'-[5- N"-(2-aminoethyl)carbamoyl-2-pyridylmethyl]-l,3-diaminopropane-2-ol was dissolved in 1 mL of methanol.
  • reaction mixture was purified on a Cl 8(2) preparative column using the following conditions:
  • Solvent A 1 % TEAAc pH 4.2
  • a coumarin labeled phosphomonoester-binding molecule was synthesized as depicted in Fig. 9.
  • 10 mg (0.018 mmol) of N,N,N'-Tri(2- pyridylmethyl)-N ' - [5 -N"-(2-aminoethyl)carbamoyl-2-pyridylmethyl]- 1,3- diaminopropane-2-ol and 16 mg (0.084 mmol) Coumarin-3-carboxylic acid (CAS# 531- 81-7) were dissolved in 5 mL of methanol.
  • reaction mixture was purified on a C 18(2) preparative column using the following conditions:
  • Solvent A 1 % TEAAc pH 4.2
  • Solvent B Acetonitrile Gradient: 100% A -» 5 min 100% A -> 100% B over 50 min Flow Rate: 10 mL/min UV: 300 nm
  • Triethylamine (MW 101.19; Dens. 0.726) - Aldrich 471283
  • the reaction mixture was purified by ion exchange chromatography (CM- Sephadex C-50). Briefly, 0.8 g of resin was allowed to swell overnight in 40 mL of 0.6 mM phosphate buffer, pH 7.2. The resin was added to a 60 mL column and was allowed to settle for 1 hour. The column was then rinsed with approximately 200 mL of phosphate buffer. 3 mL of phosphate buffer was added to the product and methanol was added until the residue dissolved. The reaction mixture was added to the column and eluted with a 0.6 mM phosphate buffer -> 10OmM NaCl gradient. A yellowish band eluted at approximately 5-10 mM NaCl.
  • the Ruthenium Dye labeled labeled phosphomonoester-binding molecule (main red band) eluted with 40 mM NaCl.
  • the product was collected and the solvents were reduced to about 20 mL under reduced pressure.
  • To this red solution was added 12 mg (0.074 mmol) of Ammonium Hexafluorophosphate.
  • the mixture was stirred for 10 minutes, and then extracted with methylene chloride (2 x 10 mL).
  • the combined organic layers were washed with a 10 mM NH 4 PF 6 solution (2 x 20 mL).
  • the organic layer was dried over magnesium sulfate, filtered and concentrated to produce an orange-red solid.
  • the first lane of the gel was stained with the total protein stain, SYPRO® Ruby dye (commercially available from Molecular Probes Inc.).
  • the second lane of the gel was stained with the TRITC labeled phosphomonoester-selective binding agent.
  • the third lane of the gel was stained with the Ru-Dye labeled phosphomonoester-selective binding agent.
  • the staining protocol using the TRITC labeled phosphomonoester-selective binding agent was as follows. After electrophoresis, gels are fixed for one hour in 10% ethanol / 7% acetic acid with gentle agitation. Typically the gels are placed in a volume of fixative that is equivalent to about ten times the volume of the gel. This step is repeated using fresh fixative for an additional 30 minutes to ensure complete removal of SDS. The gel is then washed three times for 10 minutes each in deionized water to remove the ethanol and acetic acid.
  • the lyophilized TRITC labeled phosphomonoester-selective binding agent from example 1 (0.1 ⁇ mol per vial) is diluted in 100 ⁇ l dimethyl formamide.
  • the gel is destained three times for 30 minutes each in 25 ml 10 mM Bis-Tris, 100 mM Na 2 SO 4 , 15 % propylene glycol, pH 6.5. Afterwards, the gel is washed twice for 5 minutes each with deionized water. The gel is imaged using a xenon-arc lamp-based CCD camera system (ProXPRESS 2D imager, PerkinElmer) with band pass filters optimized to allow excitation illumination at 541 nm and emission readings at 572 nm.
  • a xenon-arc lamp-based CCD camera system ProXPRESS 2D imager, PerkinElmer
  • the staining protocol using the Ru-Dye labeled phosphomonoester-selective binding agent was as follows. After electrophoresis, gels are fixed for one hour in 10% ethanol / 7% acetic acid with gentle agitation. Typically the gels are placed in a volume of fixative that is equivalent to about ten times the volume of the gel. This step is repeated using fresh fixative for an additional 30 minutes to ensure complete removal of SDS. The gel is then washed three times for 10 minutes each in deionized water to remove the ethanol and acetic acid.
  • the lyophilized Ru-Dye labeled phosphomonoester-selective binding agent from example 4 (0.15 ⁇ mol per vial) is diluted in 150 ⁇ l dimethyl formamide. Then 15 ⁇ l of 20 mM ZnCl 2 is added, followed by 1.334 mL 10 mM Tris- HCl/ 0.1 M Na 2 SO 4 , pH 8.0 to generate 1.5 ml of dye concentrate. Typically this dye concentrate is incubated for 30 minutes at room temperature to ensure binding of the zinc ions to the chelate.
  • the staining solution is prepared by adding 750 ⁇ l concentrate to 25 mL of 10 mM Tris-HCl, 100 mM Na 2 SO 4 , 15 % propylene glycol, pH 8.0 (0.075 ⁇ mol dye per gel).
  • the gel is incubated in the staining solution with gentle agitation for 90 minutes without direct exposure to light. Then, the gel is destained three times for 30 minutes each in 25 ml 10 mM Bis-Tris, 100 mM Na 2 SO 4 , 15 % propylene glycol, pH 6.5. Afterwards, the gel is washed twice for 5 minutes each with deionized water.
  • the gel is imaged using a xenon-arc lamp-based CCD camera system (ProXPRESS 2D imager, PerkinElmer) with band pass filters optimized to allow excitation illumination at 456 nm and emission readings at 654 nm.
  • Fig. 11 As shown in Fig. 11 (left column), SYPRO ® Ruby dye clearly stained all of the proteins in the gel. In contrast, the TRITC labeled phosphomonoester-selective binding agent and the Ru-Dye labeled phosphomonoester-selective binding agent stained only ovalbumin (Fig. 11, middle and right lanes , respectively). Not surprisingly, the only protein to be stained by the TRITC labeled phosphomonoester-selective binding agent and the RU-Dye labeled phosphomonoester-selective binding agent was the phosphorylated ovalbumin protein.
  • Fig. 11 shows detection of phosphoproteins using counter anion-dinuclear transition metal cation-pair complexes.
  • Fig. 11, left lane shows SYPRO ® Ruby dye staining of broad-range molecular weight marker proteins in an SDS-polyacrylamide gel.
  • Fig. 11, middle lane shows the selective staining of the phosphoprotein ovalbumin using TRITC conjugated to a phosphomonoester-selective binding agent, as synthesized in example 1.
  • Fig. 11, right lane shows the selective staining of the phosphoprotein ovalbumin using a ruthenium complex conjugated to a phosphomonoester-selective binding agent as synthesized in example 4.
  • the counter-anion employed was sulfate, as explained above.
  • the labeled phosphomonoester-selective binding agents described above are able to specifically bind gel-immobilized phosphorylated proteins.
  • the labeled phosphomonoester-selective binding agent can bind phosphomonoester residues on phosphoproteins, in this case ovalbumin, immobilized either in gels (see lower left quadrant of Fig. 12, lower panel) or immobilized on a PVDF membrane (see lower left quadrant of Fig. 12, upper panel).
  • the detectably labeled phosphomonoester-selective binding agent allowed the visualization of phosphomonoester residue-containing proteins in rat liver cytosol.
  • rat liver cytosol 100 ⁇ g of rat liver cytosol was subjected to isoelectric focusing (IEF), and the IEF then placed on top of an SDS-PAGE gel (10%) and sealed into place, and then elctrophoretically resolved for the second dimension.
  • IEF isoelectric focusing
  • Fig. 13 both the TRITC-labeled Phos-tagTM (left) and the ruthenium labeled Phos-tagTM (right) are capable of selectively binding to phosphorylated proteins.
  • TRITC-labeled Phos-tagTM From the gel labeled with TRITC-labeled Phos-tagTM, five spots were excised, the gel mashed up and treated with trysin, and the peptides extracted and dried down for MALDI mass spectrometry analysis (Fig. 14, upper panel).
  • the lower panel of Fig. 14 shows that at least three of the five spots are known phosphoproteins.
  • the same proteins in Example 5 i.e., myosin, ⁇ -galactosidase, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, lysozyme, and aprotinin
  • the proteins are next transferred to a polyvinylidene difluoride (PVDF) membrane. Following transfer, the PVDF membrane is cut up between the lanes, and phosphoprotein blotting analysis is performed using, as reporter, TRITC labeled phosphomonoester-selective binding agent.
  • PVDF polyvinylidene difluoride
  • the fifth lane is subjected to Western blotting analysis with an anti-ovalbumin antibody.
  • the sixth and final lane is subjected to Western blotting analysis with an anti-bovine serum albumin antibody.
  • Secondary, TRITC-labeled antibodies are used in the Western blots of the control lanes.
  • the staining protocol using the TRITC labeled phosphomonoester-selective binding agent was as follows. After Western blotting, the blots are allowed to air dry completely, and are then pre-wetted with 100% ethanol. The membranes are washed once in deionized water and then are incubated for one hour in 10% ethanol / 7% acetic acid with gentle agitation.
  • the gels are placed in a volume of fixative that is equivalent to about five to ten times the volume of the original gel used for Western blotting.
  • the membrane is agitated in 25 mL of blocking solution (0.5% Tween-20 / 0.25% poly(vinylpyrrolidone)-40 (PVP-40) in 10 mM Bis-Tris, pH 6.5) for 30 minutes or over night.
  • the membrane is then washed once with 25 mL 10 mM Bis-Tris, 100 mM Na 2 SO 4 , pH 6.5.
  • the TRITC Blot staining buffer is made as follows. Sample lyophilized at 0.1 umol per vial per example 1 is diluted in 100 ⁇ l dimethyl formamide.
  • the TRITC Blot Staining Solution is then prepared by adding 250 ⁇ l dye concentrate to 25 ml of 10 mM Bis-Tris, 100 mM Na 2 SO 4 , pH 6.5 (0.025 umol dye per gel).
  • the membrane is placed face down in 25 mL TRITC Blot Staining Solution (prepared in the last step) and incubated with gentle agitation for 30 minutes.
  • the membrane is washed three times for five minutes each in a destain buffer of 10 mM Bis-Tris, 100 mM Na 2 SO 4 , 15% propylene glycol, pH 6.5.
  • the blots are imaged using a xenon-arc lamp-based CCD camera system (ProXPRESS 2D imager, PerkinElmer) with band pass filters optimized to allow excitation illumination at 541 nm and emission readings at 572 nm.
  • a xenon-arc lamp-based CCD camera system ProXPRESS 2D imager, PerkinElmer
  • the labeled phosphomonoester-selective binding agent described above is able to specifically bind PVDF-immobilized phosphorylated proteins.
  • a standard set of proteins as in Example 5 (i.e., myosin, ⁇ - galactosidase, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, lysozyme, and aprotinin) was separated by electrophoresis on a 12% SDS polyacrylamide gel.
  • One of the proteins in the set of molecular weight markers was ovalbumin, a known phosphorylated protein. Each lane was loaded with approximately 1 ⁇ g of each standard protein. After separation, the gel was fixed in a solution of 10% ethanol and 7% acetic acid. The gel was then washed in distilled water to remove the fixing solution. The phosphoprotein was detected using the TRITC labeled phosphomonoester-selective binding agent as described in example 5.
  • the ovalbumin bands were cut out of the gel.
  • Two gel slices were placed in each tube in order to digest 2 ⁇ g ovalbumin per tube.
  • the gel slices were incubated in 200 ⁇ L of 25 mM ammonium bicarbonate with 5% acetonitrile ambient temperature for 30 minutes. After aspirating the supernatant, the gel slices were incubated with 200 ⁇ L of 25 mM ammonium bicarbonate with 50% acetonitrile at ambient temperature for 30 minutes. This step is repeated and then the gel slices were washed twice with 100% acetonitrile. The supernatant was removed and the gel slices were dried at 60 0 C for 10 minutes.
  • Trypsin was solubilized in 1 mM HCl and diluted in 25 mM ammonium bicarbonate. Trypsin solution (0.5 ⁇ g trypsin per tube) was added to each sample and incubated at 37°C overnight.
  • the phosphorylated peptides in the digest were isolated and enriched using TiO 2 thin-film coated magnetic beads (Per: Patton, W., Mikulskis, A., Golenko, E. (July 6, 2005) Methods and compositions for detecting and isolating phosphorylated molecules using hydrated metal oxides. US Application Serial No. PCT/US 05/23810.).
  • a suspension of TiO 2 coated magnetic beads in 70% ethanol was diluted 1 : 10 in water and dispensed into a low binding polypropylene 96-well plate. The beads are washed twice with 200 ⁇ L of 1% formic acid and 50% acetonitrile.
  • Each trypsin digested sample was mixed with 50 ⁇ L of 1% formic acid and 50% acetonitrile.
  • the supernatant containing the digested protein is added to the TiO 2 coated magnetic beads and incubated at ambient temperature for 30 minutes with agitation.
  • the supernatant is aspirated from each tube and the beads are washed four times with 1% formic acid and 50% acetonitrile. This is followed by a wash of 50% acetonitrile.
  • the beads are then incubated for 5 minutes in 20 ⁇ L of 5 mM ammonium phosphate (pH 9.5) with 75% acetonitrile.
  • the supernatant containing the phosphorylated peptides is aspirated and placed in a clean well. The plate is incubated at 50 0 C for 30 minutes to completely dry the sample.
  • the dried sample is then prepared for mass spectroscopy analysis.
  • the matrix solution is prepared by dissolving 5 mgs of ⁇ -cyano-4-hydroxycinnamic acid (CHCA) in 1.25 mL of 1% formic acid and 50% acetonitrile.
  • CHCA ⁇ -cyano-4-hydroxycinnamic acid
  • the dried peptides are completely dissolved in 8 ⁇ L of matrix solution.
  • This mixture is then spotted on a sample plate suitable for matrix-assisted laser desorption ionization-time-of- flight mass spectrometry (MALDI-TOF MS) analysis.
  • MALDI-TOF MS matrix-assisted laser desorption ionization-time-of- flight mass spectrometry
  • protein of interest is cleaved into smaller fragments; the resultant peptide fragments are measured and used for database search (with database searching engine such as ProFound) to identify the protein.
  • Fig. 15 shows a MS spectrum of the phosphorylated peptide isolated from a tryptic digest of ovalbumin using Ti ⁇ 2-coated magnetic beads. In addition to the phosphorylated peptide, there is a peak indicating the neutral loss of 98 Da owing to H 3 PO 4 loss. The samples were analyzed on a PerkinElmer prOTOFTM 2000 MALDI O- TOF Mass Spectrometer. Phosphorylated protein and peptide identification with mass spectrometry-based techniques have been developed using MS an MS/MS.
  • the MASCOT database search result identified the protein as ovalbumin [validated]-chicken (OACH) with high score 100.
  • Fig. 17 shows MS/MS spectra of the fragment ion from parent mass ion 2088.9 Da.
  • the peptide MW 1088.9 is EVVGSAEAGVD AASVSEEFR (SEQ ID NO:22); phosphorylated at the first S (G-pS-A).
  • the experiment confirms compatibility of phosphoprotein detection with the phosphomonoester-selective binding agent with enrichment of phosphorylated peptides, their mass spectrometry-based characterization and identification of phosphorylated residues within the phosphopeptides.
  • a non-limiting phosphomonoester- selective binding agent of the invention (Phos-tagTM molecule) complexed (non- covalently) to Zn2+ cations was incorporated into a colorimetric sensor capable of detecting phosphorylated species in aqueous media at neutral pH.
  • the assay is based upon the selective binding of pyrocatechol violet to the Phos-tagTM complex. As shown in Fig.
  • pyrocatechol violet (upper left) interacts with a Zn Phos-tagTM molecule to form a complex which can be disrupted upon the addition of a phosphomonoester (i.e., H3PO4), where the phosphomonoester displaces the pyrocatechol violent from the phosphomonoester (see also Fig. 19).
  • a phosphomonoester i.e., H3PO4
  • the phosphomonoester displaces the pyrocatechol violent from the phosphomonoester
  • a number of other compounds each of which having a vicinal diol moiety that could potentially interact with the dinuclear zinc complex of Phos-tagTM as Pyrocatechol Violet, were screened and the changes in UV-VIS absorbance and fluorescence properties of these reagents in the presence and absence of dinuclear zinc Phos-tagTM complex were examined. Fluorescence scans showed that three of the screened compounds are fluorescent when excited at the proper wavelength.
  • Fig. 29 shows the fluorescence spectra of Piceatannol (uppers left), Tiron (upper right), and Quercetin (bottom), each prepared at a concentration of 100 ⁇ M in 10 mM HEPES buffer at pH 7.
  • model phosphopeptides were purchased from AnaSpec, Inc (San Jose, CA): pT, sequence RRREEE[pT]EEEAA (catalog #24514) (the amino acid sequence of SEQ ID NO:2 containing a phosphorylated threonine residue); pS, sequence RRREEE[pS]EEEAA (catalog # 24512) (the amino acid sequence of SEQ ID NO:3 containing a phosphorylated serine residue); and pY, sequence EPQ[p Y]EEIPIYL (catalog #25062) (the amino acid sequence of SEQ ID NO:4 containing a phosphorylated tyrosine residue).
  • a non-phosphorylated peptide, P2 (amino acids 521-533), sequence TSTEPQYQPGENL (SEQ ID NO:23; catalog # 04-10-22-02-PEK), was purchased from SynPep Corporation (Dublin, CA).
  • the "p” refers to the presence of a phosphate group attached to the following amino acid residue (e.g., "pS” mean phosphorylated serine).
  • Bovine serum albumin (BSA), ⁇ -casein, and ovalbumin were used as model proteins.
  • Fig. 30 shows the responses of two fluorescent sensing systems of Quercetin and Tiron to three different phosphorylated peptides and one non-phosphorylated peptide.
  • Phenylfluorone (PF) solution was made by dissolving 32.0 mg of PF in ethanol with addition of 1 mL concentrated HCl and dilution up to 20 ml with ethanol. 10 mM of Triton X-100 and Cetylpyridinium chloride was each prepared in distilled water.
  • a colorimetric sensing ensemble was prepared by mixing the Phos-tagTM amine (50 ⁇ M), MnC12 (100 ⁇ M), and PF (50 ⁇ M) at a molar ratio of 1 :2: 1 in a reaction system containing 10 mM borate buffer at pH 9.7 with addition of 0.6 ⁇ M cetylpyridinium chloride and 2 mM Triton X-100.
  • 50 ⁇ l of 1 mM sodium phosphate dibasic solution was added to the above sensing mixture to have a final concentration of 50 ⁇ M.
  • UV- Vis spectra were obtained for the PF solution (50 ⁇ M diluted in the reaction buffer), the PF sensing ensemble, and the sensing mixture with the presence of phosphate anions, and the absorbance intensities and wavelength shift were compared.
  • Association of PF with the Phos-tagTM Mn complex decreased the absorbance of PF, and the absorbance was brought back upon addition of phosphate anions into the sensing mixture due to the replacement of PF with the phosphate anions (Fig. 33).
  • BPR Bromopyrogallol red
  • UV -Vis spectra were obtained for the diluted BPR solution (20 ⁇ M), the BPR sensing ensemble, and the sensing mixture with the presence of phosphate anions, and the absorbance intensities were compared at 570 nm, where BPR has the maximum absorbance.
  • Association of BPR with the Phos-tagTM Zn complex decreased the absorbance of BPR, and the absorbance was increased upon addition of phosphate anions into the sensing mixture due to the replacement of BPR with the phosphate anions that freed the bound BPR into the solution (Fig. 34).
  • Several different polypeptides were subjected to SDS polyacrylamide gel electrophoresis on two identical sets of gels (Fig. 35).
  • the polypeptides were a mix of phosphorylated and non-phosphorylated proteins.
  • Lane 1 is Peppermintstick molecular weight markers (Invitrogen) which contains two phosphoproteins, ovalbumin and ⁇ - casein, as well as several nonphosphorylated proteins.
  • Lane 2 contained Candycane markers (Invitrogen), a mixture of glycosylated and nonglycosylated proteins, but no phosphoproteins.
  • Lane 3 contained Broad-range molecular weight markers (Bio-Rad) which includes the phosphoprotein ovalbumin, as well as various nonphosphorylated proteins.
  • Lane 4 contained the phosphoprotein ⁇ -casein enzymatically treated (alkaline phosphatase) to remove most phosphorylated residues, but still retains 1-2 phosphate residues per protein.
  • Lane 5 contains the fully phosphorylated protein ⁇ -casein, not treated with alkaline phosphatase. It contains 8-9 phosphate residues per protein.
  • Lane 6 contained the monophosphorylated protein pepsin.
  • Lane 7 contained the phosphoprotein phosvitin, which contains roughly 100 phosphate residues per protein.
  • Lane 8 contains the glycophosphoprotein fetuin.
  • Lane 9 contains fibrinogen, a phosphoprotein with three subunits of 63.4, 56 and 47 kDa mass.
  • Lane 10 contained riboflavin-binding protein, which contains 5 phosphate residues per protein.
  • One gel was subjected to staining protocol A and the other gel to staining protocol B.
  • Protocol B comprised immersion of the the gel in 10 mM Tris-acetate buffer (pH 7.4) containing the TRITC-Phos-tagTM conjugate shown in Fig. 7 and 100 ⁇ M zinc acetate for 30 minutes. The gel was then removed from the solution and twice washed with 100 mL of 10 mM Tris-acetate buffer (pH 7.4) for 10 minutes. A fluorescent image of the gel was photographed at an excitation wavelength of 473 nm with use of a fluorescence detecting filter of 510 nm.
  • a gel was subjected to Protocol A, which is described in Example 5 for the counter-anion-TRITC-Phos-tagTM conjugate.
  • Protocol A which is described in Example 5 for the counter-anion-TRITC-Phos-tagTM conjugate.
  • phosphorylated proteins stained with the counter-anion-TRITC-Phos-tagTM conjugate left were clearly distinguished from the non-phosphorylated proteins resolved in the gel.
  • the gel stained by protocol B exhibited non-specific binding to the gel and proteins.
  • Figs. 36A-36C depict fluorescence intensity traces of ovalbumin and alpha-casein (Fig. 36A), pepsin (Fig. 36B), and riboflavin (Fig. 36C) generated by the two staining methods.
  • Group I has the sequence Nterm-ARRSEDEGPPN-Cterm (SEQ ID NO:24).
  • Group II has the sequence Nterm-FRASEDEQARHL-Cterm (SEQ ID NO:25).
  • Group III has the sequence Nterm-LARSEDECDGTH-Cterm (SEQ ID NO:26).
  • Group IV has the sequence Nterm-EDSESEDEEDED-Cterm (SEQ ID NO:27).
  • a Control Group of peptides is generated, where the control group peptides have the sequence Nterm- SSSSSSSSSS-Cterm (SEQ ID NO:28).
  • the peptides are generated, such that there are 100 peptides in each of Group I, Group II, Group III, Group IV, and the Control Group where all the peptides in each group have the identical sequence.
  • Luminex beads are internally labeled with different amounts of dye, and thus can be distinguished from one another following analysis with a Luminex system (e.g., a Luminex 100 System).
  • a Luminex system e.g., a Luminex 100 System
  • the beads of all Groups, each coated with a different peptide, are now mixed together (there are equal numbers of beads in each group) and exposed to the casein kinase II (Casein Kinase II commercially available from New England Biolabs, Ipswich, MA) in a phosphorylation reaction mixture.
  • the phosphorylation reaction mixture provided by New England Biolabs comprises 20 mM Tris-HCl, 50 mM KCl. 10 mM MgCl 2 , 200 ⁇ M ATP, pH 7.5, and recommends incubation for about two hours at 30 0 C.
  • the beads are separated from the kinase and from the phosphorylation reaction mixture by centrifugation, and resuspended in saline.
  • the beads are then contacted with a phosphomonoester-selective binding agent covalently linked to a cyanine 5 dye. After the unbound phosphomonoester-selective binding agent is removed by centrifuging the beads away from them, the beads are analyzed using a Luminex 100 System.
  • the outlined approach is superior to the "one-bead one-peptide" methods known in the art for defining protein kinase substrate specificity, which involve generating a peptide library on polymeric beads, where each bead has only one peptide sequence associated with it, incorporating [ ⁇ - 32 P]ATP into the immobilized peptides using a protein kinase of interest, suspending the beads in 1% agarose, immobilizing the agarose on glass plates and exposing the plates to X-ray film for 20-30 hours for identification of beads containing labeled peptides. The labeled beads are then typically excised from the agarose gel and subjected to automated peptide sequencing for identification of the optimal sequence.
  • Example 13 is essentially repeated, except that the Luminex beads are magnetic, and following incubation of the peptides with casein Kinase II (plus free ATP), the beads are separated from the phosphorylation reaction mixture by simply exposing the mixture to a magnet, allowing the beads to adhere, and rinsing away the components in the reaction mixture that are not bound to the magnet.
  • the magnetic beads are then exposed to a detectable phosphomonoester-selective binding agent, where the unbound binding agent is removed by allowing the beads to adhere to a magnet and rinsing.
  • the beads are then analyzed using a Luminex 100 System.
  • Peptides containing the phosphoacceptor site of the Ca 2+ /Calmodulin-Dependent Protein Kinase II are generated. Groups of these peptides are created, where each group contains an identical number of peptides, each having the same sequence, where different groups have peptides having different sequences.
  • the groups are: Group I: Nterm-ATRRRLSWRN-Cterm (SEQ ID NO:29)
  • Group II Nterm-PTWRFLSEAG-Cterm (SEQ ID NO:30)
  • Group III Nterm-VDYRNLSPKL-Cterm (SEQ ID NO:31)
  • Control Group Nterm-SSSSSSSSSS-Cterm (SEQ ID NO:28)
  • biotinylation of a peptide can be accomplished using standard methods.
  • the biotin-PEG-NHS reagent is commercially available from Nektar Pharmaceuticals (San Carlos, CA). Mixing a peptide with the biotin-PEG-NHS reagent will allow a reaction to spontaneously occur whereby the NHS (the N-Hydroxysuccinimide ester group) on the biotin-PEG-NHS reagent reacts with the NH 2 of the Nterm (i.e., N-terminus) of the peptide. As a result, the biotin label is added to the N-terminus of the peptide.
  • the biotinylated peptides are next contacted with avidin-coated magnetic beads and allowed to bind to the beads (through the biotin on the peptide binding to the avidin on the bead), such that one group of peptide is bound with one group of beads.
  • the beads used are similar to the LumAvidin beads available from Luminex Corp. (Austin, TX). LumAvidin beads are internally labeled with different amounts of dye, and thus can be distinguished from one another (and, the groups of peptides can thus be distinguished) on a Luminex system.
  • the bound peptides attached to the beads are exposed in a phosphorylation reaction mixture to Ca 2+ /Calmodulin-Dependent Protein Kinase II (CaMKII), the active portion of which is commercially available from New England Biolabs (Ipswich, MA).
  • the phosphorylation reaction mixture comprises 50 mM Tris-HCl, 10 mM MgCl 2 , 2 mM dithiothreitol, 0.1 mM EGTA, pH 7.5, and 200 ⁇ M ATP, and incubated at 25 0 C for two hours. Following the incubation, the kinase is inactivated by heating the phosphorylation reaction mixture (including the beads and kinase) for twenty minutes at 60 0 C.
  • the beads (and the peptides bound to them) are then separated from the rest of the components in the phosphorylation mixture by placing the mixture against a magnet, allowing the beads to adhere to the magnet, and removing the remaining components of the phosphorylation mixture.
  • the beads are then contacted with a phosphomonoester- selective binding agent covalently linked to a FITC (fluorescein isothiocyanate) dye.
  • FITC fluorescein isothiocyanate
  • Example 13 is essentially repeated; however, each peptide in each group is synthesized covalently linked to a specific nucleic acid tag covalently linked that is designed to be complementary to one of the tags present on the FlexMap beads commercially available from Luminex Corp., such that each group has a different nucleic acid tag.
  • the groups are thus designed to have the following sequences:
  • Control Group Nucleic acid tag 5-Nterm-SSSSSSSSSSSS-Cterm (SEQ ID NO:
  • peptide/nucleic acid molecules per group There are 100 peptide/nucleic acid molecules per group.
  • the groups are pooled in incubated with casein kinase II (CKII) in a phosphorylation reaction mixture as described in Example 13.
  • CKII casein kinase II
  • the mixture is added to five different types of FlexMap beads, where each of the five types of beads is complementary to the nucleic acid tags on the peptides in one of the Groups.
  • the FlexMap beads are next separated by centrifugation from the other components of the phosphorylation reaction mixture.
  • the beads are next contacted with a detectable phosphomonoester-selective binding agent.
  • the beads are analyzed using a Luminex 100 System.
  • Nterm-YLRRRLSDSN-Cterm (SEQ ID NO:32), is contained in synapsin site 1, the naturally occurring substrate for CaMKII (see, e.g., Pearson, R. B., and Kemp, B. E. (1991). In T. Hunter and B. M. Sefton (Eds.), Methods in Enzymology Vol. 200, (pp. 62-81). San Diego: Academic Press). An experiment is performed to determine the best conditions for phosphorylating the serine residue in this sequence.
  • Peptides having the sequence Nterm-YLRRRLSDSN-Cterm are generated and attached to different groups of magnetic Luminex beads.
  • the groups differ based on the number of peptides attached to the beads.
  • the groups are as follows:
  • Group III 100 peptides per bead
  • Group IV 200 peptides per bead.
  • Group V 500 peptides per bead.
  • Group VI 1000 peptides per bead.
  • the beads are pooled (i.e., 600 beads are pooled) and all groups are incubated at the same time with CaMKII, and, following staining with a FITC-labeled phosphomonoester-selective binding agent as described in Example 12, the beads are analyzed with a Luminex 100 system.
  • the group of peptides where the level of the fluorescence to peptide ratio is the highest indicates the best conditions for phosphorylation of the peptide by CaMKII.
  • Example 18 A biological sample is tested to determine if it contains Ca 2+ /Calmodulin- Dependent Protein Kinase II (CaMKII) and/or Casein Kinase II (CKII, CK-2).
  • CaMKII Ca 2+ /Calmodulin- Dependent Protein Kinase II
  • CKII Casein Kinase II
  • Peptides having the sequence Nterm-YLRRRLSDSN-Cterm (SEQ ID NO:32; which is present in the naturally occurring substrate for CaMKII) are generated and attached to one group of magnetic Carboxylated Luminex beads.
  • Peptides having the sequence Nterm- ADSESEDEED -Cterm (SEQ ID NO:27; which is present in the naturally occurring substrate for CKII) are generated and attached to a second group of magnetic Carboxylated Luminex beads.
  • Control peptides with the sequence Nterm- SSSSSSSSSS-Cterm (SEQ ID NO:28) are generated and attached to a third group of magnetic Carboxylated Luminex beads.
  • the beads are pooled (i.e., 300 beads are pooled) and all groups are incubated at the same time with the sample in a phosphorylation reaction mixture that allows the activity of both CaMKII and CKII.
  • the beads are removed by adherence to a magnet, and contacted with a phycoerythrin-labeled phosphomonoester-selective binding agent. Free (i.e., non- binding) binding agent is removed, again by adhering the beads to a magnetic), and then the beads are analyzed with a Luminex 100 system.
  • the results of this experiment will allow the simultaneous determination of which, if any, of CaMKII or CKII is present in the sample. If the binding agent binds to the group of beads attached to the Nterm-YLRRRLSDSN-Cterm (SEQ ID NO:32) peptide, then CaMKII is present in the sample. If the binding agent binds to the group of beads attached to the Nterm- ADSESEDEED-Cterm peptide, then CKII is present in the sample.
  • Protein phosphatase type 2A is a protein serine/threonine phosphatase that controls a number of cellular processes, including transcription, translation, metabolism, cell growth, and apoptosis (Janssens and Goris (2001) Biochem. J. 353: 417-439; Zabrocki et al. (2002) MoI. Microbiol. 43: 835-842; Milward et al. (1999) Trends Biochem. Sci. 24: 186-191).
  • Nterm- RRA(pT)VA-Cterm (where the indicated threonine residues is phosphorylated), is the consensus phosphatase recognition site for several serine/threonine phosphatases, including protein phosphatases 2A.
  • An experiment is performed to determine the best conditions required for protein phosphatase type 2A to remove the phosphate group from the threonine residue in the Nterm- RRA(pT)VA-Cterm consensus phosphatase recognition sequence (the amino acid sequence "RRATV A"(SEQ ID NO:33) containing a phosphorylated threonine residue).
  • the groups have the following sequences (where the pT symbol denotes a phosphorylated threonine residue):
  • TRRRATV AHS (SEQ ID NO:35) containing a phosphorylated threonine residue
  • EKRRATV ATN (SEQ ID NO:37) containing a phosphorylated threonine residue
  • Nterm-SSSSSpTSSSS-Cterm the amino acid sequence
  • the peptides are generated, such that there are 100 peptides in each of Group I, Group II, Group III, Group IV, and the Control Group where all the peptides in each group have the identical sequence.
  • the peptides in each group are attached to the same type of Carboxylated Luminex bead, according to manufacturer's instructions (Luminex Corp., Austin, TX). Luminex beads are internally labeled with different amounts of dye, and thus can be distinguished from one another following analysis with a Luminex system (e.g., a Luminex 100 System).
  • Phosphatase 2 A Purified Protein Phosphatase 2 A (PP2A) is purchased from Promega Corp. (Madison WI). The beads of all Groups (there are equal numbers of beads in each of the groups), each coated with a different peptide, are now mixed together and exposed to the PP2A in a phosphatase reaction mixture containing 50 mM Tris-HCl (pH 8.5), 20 mM MgC12, and ImM DTT.
  • the beads are separated from the PP2A and from the phosphatase reaction mixture by centrifugation, and resuspended in saline.
  • the beads are then contacted with a phosphomonoester-selective binding agent covalently linked to a cyanine 5 dye. After the unbound phosphomonoester-selective binding agent is removed by centrifuging the beads away from them, the beads are analyzed using a Luminex 100 System.
  • the results will show that one group of beads (which is coated with one Group of peptides) is less phosphorylated than the other groups.
  • the peptides in this Group are found to contain the amino acid residues outside of the consensus phosphatase recognition site which are most conducive in allowing dephosphorylation of the threonine residue in the consensus phosphatase recognition site by casein kinase II. This peptide sequence is likely to be similar to that found in PP2A's actual biological target.
  • the phosphomonoester-selective binding agent can also be used to detect the presence of a phosphodiesterase.
  • a phosphodiesterase can cleave a cAMP into AMP.
  • AMP unlike cAMP, has a free phosphomonoester, which can be specifically bound by a phosphomonoester-selective binding agent of the invention.
  • cAMP 3 '5' cyclic AMP
  • Group I 10 molecules of cAMP per bead
  • Group II 50 molecules of cAMP per bead
  • Group III 100 molecules of cAMP per bead
  • Group IV 200 molecules of cAMP per bead.
  • Group V 500 molecules of cAMP per bead.
  • Group VI 1000 molecules of cAMP per bead.
  • the beads are separated from the phosphodiesterase I and from the phosphatase reaction mixture by centrifugation, and resuspended in saline.
  • the beads are then contacted with a phosphomonoester-selective binding agent covalently linked to a cyanine 5 dye. After the unbound phosphomonoester-selective binding agent is removed by centrifuging the beads away from them, the beads are analyzed using a Luminex 100 System.

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Abstract

La présente invention concerne, en partie, l'utilisation de compositions qui incluent un groupement chélateur susceptible de se lier sélectivement avec un groupe phosphomonoester, et une concentration suffisante de contre-anions pour empêcher une liaison non spécifique du groupement chélateur. Les compositions peuvent être utilisées pour détecter sélectivement un groupe phosphomonoester, notamment sur une molécule cible.
PCT/US2007/076343 2006-08-18 2007-08-20 Procédés et réactifs de détection de groupes phosphomonoester WO2008022355A2 (fr)

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CN102702154A (zh) * 2012-05-31 2012-10-03 西北师范大学 用于比色检测铜离子的受体化合物及其制备和应用
CN103969325A (zh) * 2014-05-23 2014-08-06 复旦大学 一种聚多巴胺修饰的maldi靶板的合成方法及其应用
CN109765289A (zh) * 2019-02-01 2019-05-17 上海市计量测试技术研究院(中国上海测试中心、华东国家计量测试中心、上海市计量器具强制检定中心) 一种定量检测脱氧核糖核酸中有机磷和无机磷的方法
CN116157679A (zh) * 2020-09-16 2023-05-23 沃特世科技公司 用于惰性液相色谱系统和柱的系统适用性测试的化合物

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CN102266758A (zh) * 2011-03-11 2011-12-07 中国科学院过程工程研究所 一种以聚蔗糖分子为延伸物的分离纯化介质及其制备方法
CN102702154A (zh) * 2012-05-31 2012-10-03 西北师范大学 用于比色检测铜离子的受体化合物及其制备和应用
CN103969325A (zh) * 2014-05-23 2014-08-06 复旦大学 一种聚多巴胺修饰的maldi靶板的合成方法及其应用
CN109765289A (zh) * 2019-02-01 2019-05-17 上海市计量测试技术研究院(中国上海测试中心、华东国家计量测试中心、上海市计量器具强制检定中心) 一种定量检测脱氧核糖核酸中有机磷和无机磷的方法
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