Bimolecular optical probes

• US 20050170442A1
• Filed: 09/09/2004
• Published: 08/04/2005
• Est. Priority Date: 07/29/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Somberg, Richard, Kleman-Leyer, Karen M., Lasky, David A., Klink, Tony A., Beebe, Jane A., Werner, Elizabeth A., Kupcho, Kevin R., Vogel, Kurt
• Granted Patent: US 7,619,059 B2
• Time in Patent Office: Days
• US Class Current: 435/7.920
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2440/14 phosphorylation

Abstract

Compositions, methods, and kits for detecting and monitoring post-translational modification activities, including kinase or phosphatase activities, are described. The compositions typically include a peptide, a first detectable moiety, a first binding member, and a protease cleavage site. Modification of a composition by a post-translational modification enzyme, such as a kinase or phosphatase, alters the proteolytic sensitivity of the peptide, resulting in a change of a detectable property of the composition when it is associated noncovalently with a probe composition that includes a second binding member and a second detectable moiety. Panel assays for determining substrates or modulators of enzymatic activities are also described.

Claims

1. A composition of matter comprising:

a) a peptide having a length from five to fifty amino acids, said peptide comprising a motif selected from the group consisting of;

AIYAA (SEQ ID NO;

     1),

QDYLS (SEQ ID NO;

     4),

EIYGV (SEQ ID NO;

     7),

TX₁YVA, where X₁ can be G, A, or E (SEQ ID NO;

     110),

EEYIQ (SEQ ID NO;

     17), and

DYSQV (SEQ ID NO;

     20);

b) a first detectable moiety; and

c) a first binding member.

6. The composition of claim 1, 2, 4, or 5, wherein said first detectable moiety and said first binding member are independently covalently bound to said peptide either directly or through a Linker (L).

7. The composition of any of claims 1-5, wherein said peptide comprises a protease cleavage site.

8. The composition of claim 7, wherein said protease cleavage site is selected from the group consisting of a chymotrypsin protease cleavage site, a caspase 3 protease cleavage site, a cathepsin G protease cleavage site, a trypsin protease cleavage site, an elastase protease cleavage site, an endoproteinase Asp-N protease cleavage site, and an endoproteinase Glu-N protease cleavage site.

9. The composition of claim 1, 2, 4, or 5, wherein said first binding member is selected from the group consisting of a polypeptide, a polynucleotide, a lipid, a polysaccharide, a hormone, a vitamin, and a small organic compound.

10. The composition of any of claims 1-5, wherein said peptide has a length from 8 to 50 amino acids.

11. The composition of claim 10, wherein said peptide has a length from 8 to 25 amino acids.

12. The composition of claim 11, wherein said peptide has a length from 8 to 15 amino acids.

13. The composition of claim 1, 2, 4, or 5, wherein said composition exhibits an optical property, a magnetic property, or a radioactive property.

14. The composition of claim 13, wherein said composition exhibits an optical property selected from the group consisting of a molar extinction coefficient at an excitation wavelength, a quantum efficiency, an excitation spectrum, an emission spectrum, an excitation wavelength maximum, an emission wavelength maximum, a ratio of excitation amplitudes at two wavelengths, a ratio of emission amplitudes at two wavelengths, an excited state lifetime, an anisotropy, a polarization of emitted light, a resonance energy transfer, and a quenching of emission at a wavelength.

15. The composition of claim 14, wherein said optical property is selected from the group consisting of a fluorescence excitation spectrum, a fluorescence emission spectrum, a fluorescence excitation wavelength maximum, a fluorescence emission wavelength maximum, a ratio of fluorescence excitation amplitudes at two wavelengths, a ratio of fluorescence emission amplitudes at two wavelengths, a fluorescence excited state lifetime, a fluorescence anisotropy, and a quenching of fluorescence emission at a wavelength.

16. The composition of claim 13, wherein said composition exhibits a fluorescence excitation maximum in the range from 250 to 750 nm and/or a fluorescence emission maximum in the range from 450 to 800 nm.

17. The composition of claim 16, wherein said first detectable moiety is selected from the group consisting of 5-FAM, 6-FAM, 7-hydroxycoumarin-3-carboxamide, 6-chloro-7-hydroxycoumarin-3-carboxamide, fluorescein-5-isothiocyanate, dichlorotriazinylaminofluorescein, tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, succinimidyl ester of 5-carboxyfluorescein, succinimidyl ester of 6-carboxyfluorescein, 5-carboxytetramethylrhodamine, 6-carboxymethylrhodamine, and 7-amino-4-methylcoumarin-3-acetic acid.

18. The composition of claim 1, 2, 4, or 5, wherein said first binding member is biotin or a derivative thereof.

20. The probe composition of claim 19, wherein said second detectable moiety is capable of forming a FRET pair, a TR-RET pair, or a dark quenching RET pair with a first detectable moiety of a composition of claims 1, 2, 4, or 5 upon noncovalent association of a first binding member of said composition of claims 1, 2, 4, or 5 with said second binding member of said probe composition.

21. The composition of claim 1, 2, 4, or 5, wherein said first detectable moiety is 5-FAM.

22. The composition of claim 1, 2, 4, or 5, wherein said first detectable probe moiety is covalently linked to said peptide via a linker (L).

23. The composition of claim 22, wherein said L is selected from the group consisting of GABA, an amino acid, a small peptide, diaminopentanyl, and aminohexanoyl.

24. The composition of claim 1, wherein said motif is a recognition motif for a tyrosine kinase, said recognition motif selected from the group consisting of:

EAIYAAP (SEQ ID NO;

     2),

DQDYLSL (SEQ ID NO;

     5),

EEEYIQI (SEQ ID NO;

     18),

EEIYGVI (SEQ ID NO;

     8),

LTGYVAR (SEQ ID NO;

     11),

ITAYVAT (SEQ ID NO;

     12),

ITEYVAT (SEQ ID NO;

     13), and

GDYSQVL (SEQ ID NO;

     21).

26. The composition of claim 24, wherein said recognition motif for a tyrosine kinase is EAIYAAP (SEQ ID NO:

2), and wherein said tyrosine kinase is selected from the group consisting of Ab11, Ab12, BMX, CSF1R, Csk, EPHB4, Fes/Fps, FGFR1, FGFR4, Fgr, FLT3, Fyn, Hck, IGF1R, IRKβ

, ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B, c-MET, Src, Src N1, Src N2, SYK, TIE2, TRKa, and YES.

27. The composition of claim 24, wherein said recognition motif for a tyrosine kinase is DQDYLSL (SEQ ID NO:

5), and wherein said tyrosine kinase is selected from the group consisting of CaMKII, CDK7/CycH, CK16, IKKα

, and IKKβ

.

35. The composition of claim 24, wherein said recognition motif for a tyrosine kinase is EEIYGVI (SEQ ID NO:

8), and wherein said tyrosine kinase is selected from the group consisting of Ab11, Ab12, BMX, CSF1R, Csk, EPHB4, Fes/Fps, FGFR1, Fgr, FLT3, Fyn, Hck, IGF1R, IRKβ

, IRTK, ITK, Jak3, KDR, c-KIT, Lck, Lyn A, Lyn B, c-MET, Src, Src N1, Src N2, SYK, TIE2, TRKa, and YES.

36. The composition of claim 24, wherein said recognition motif for a tyrosine kinase is LTGYVAR (SEQ ID NO:

1), and wherein said tyrosine kinase is selected from the group consisting of CSF1R, FLT3, and c-KIT.

37. The composition of claim 24, wherein said recognition motif for a tyrosine kinase is EEEYIQI (SEQ ID NO:

18), and wherein said tyrosine kinase is selected from the group consisting of EGFR, Zap-70, PDGFR, FGFR4, Abl 1, and Lyn B.

38. The composition of claim 1, wherein said peptide is selected from the group consisting of:

EAEAIYAAPGDK (SEQ ID NO;

     3),

GDQDYLSLDK (SEQ ID NO;

     6),

EEEEYIQIVK (SEQ ID NO;

     19),

EEEIYGVIEK (SEQ ID NO;

     9),

GVLTGYVARRK (SEQ ID NO;

     14),

DDEITAYVATRK (SEQ ID NO;

     15),

TGIITEYVATRK (SEQ ID NO;

     16), and

EGDYSQVLEK (SEQ ID NO;

     22).

40. The composition of claim 7, wherein said protease cleavage site is FS, FT, or Y.

41. A method for characterizing a kinase, said method comprising:

a) contacting a composition of claims 1, 2, or 4 with a protein kinase under conditions effective for said protein kinase to phosphorylate said composition; and

b) measuring the ability of said protein kinase to phosphorylate said composition.

42. A method for identifying a substrate of a kinase, comprising:

a) contacting a composition of claims 1, 2, or 4 with a protein kinase;

b) contacting said composition and said protein kinase with a protease to form a protease mixture;

c) contacting said protease mixture with a probe composition comprising a second detectable moiety and a second binding member to form a detection mixture; and

d) comparing a measurable property in said detection mixture with the measurable property in a control detection mixture lacking said protein kinase, wherein said composition is identified as a substrate of said protein kinase if said measurable property in said detection mixture is different from said measurable property in said control detection mixture.

43. The method of claim 42, wherein ATP is present during said contacting step a).

44. A method for identifying a substrate of a kinase, comprising:

a) contacting a composition of claims 1, 2, or 4 with a protein kinase and ATP;

b) contacting said composition, said protein kinase, and said ATP with a protease to form a protease mixture;

c) contacting said protease mixture with a probe composition comprising a second detectable moiety and a second binding member to form a detection mixture; and

d) comparing a measurable property in said detection mixture with a measurable property in a control detection mixture lacking said ATP, wherein said composition is identified as a substrate of said kinase if said measurable property in said detection mixture is different from said measurable property in said control detection mixture.

45. The method of claim 44, wherein two or more different compositions of claims 1, 2, or 4 are contacted independently with said protein kinase and said ATP during said contacting step a) to form two or more kinase mixtures;

wherein each of said two or more kinase mixtures is contacted independently with a protease during said contacting step b) to form two or more protease mixtures; and

wherein each of said two or more protease mixtures is contacted independently with a probe composition during said contacting step c) to form two or more detection mixtures, and wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

46. The method of claim 44, wherein two or more different protein kinases are contacted independently with said composition and said ATP during said contacting step a) to form two or more kinase mixtures;

wherein each of said two or more kinase mixtures is contacted independently with a protease during said contacting step b) to form two or more protease mixtures;

wherein each of said two or more protease mixtures is contacted independently with a probe composition during said contacting step c) to form two or more detection mixtures; and

wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

47. The method of claim 44, wherein said comparing step d) occurs concurrently with said contacting step c).

48. The method of claim 44, wherein said comparing step d) occurs after said contacting step c).

49. The method of claim 48, wherein said contacting step b) is completed by inhibiting a proteolytic activity of said protease.

50. The method of claim 49, wherein said proteolytic activity is inhibited by adding a reagent to said protease mixtures or by heating said protease mixtures.

51. The method of claim 50, wherein said reagent is aprotinin, PMSF, TPCK, AEBSF, chymotrypsin inhibitor 1, and chymotrypsin inhibitor 2.

52. A method for identifying a modulator of activity of a kinase, comprising:

a) forming a mixture of a protein kinase, a substrate for said protein kinase, wherein said substrate is a composition according to claim 1, 2, 4, or 5, and a test compound;

b) contacting said mixture with a protease to form a protease mixture;

c) contacting said protease mixture with a probe composition comprising a second detectable moiety and a second binding member to form a detection mixture; and

d) comparing a measurable property in said detection mixture to the measurable property in a control mixture of said substrate, said protein kinase, and said protease in the absence of said test compound, wherein said test compound is identified as a modulator of activity of said kinase if said measurable property in said detection mixture is different from said measurable property in said control mixture.

53. The method of claim 52, wherein ATP is present during said contacting step a).

54. A method for identifying a modulator of activity of a kinase, comprising:

a) contacting a protein kinase, a substrate for said protein kinase, wherein said substrate is a composition according to claim 1, 2, 4, or 5, ATP, and a test compound to form a kinase mixture;

b) contacting said kinase mixture with a protease to form a protease mixture;

c) contacting said protease mixture with a probe composition comprising a second detectable moiety and a second binding member to form a detection mixture; and

d) comparing a measurable property in said detection mixture with the measurable property in a control mixture comprising said composition, said protein kinase, and said protease in the absence of said test compound, wherein said composition is identified as a modulator of activity of said kinase if said measurable property in said detection mixture is different from said measurable property in said control mixture.

55. The method of claim 54, wherein two or more different test compounds are contacted independently with said protein kinase, said ATP, and said substrate in said contacting step a) to form two or more kinase mixtures;

wherein each of said kinase mixtures is contacted independently with a protease in said contacting step b) to form two or more protease mixtures;

wherein each of said protease mixtures is contacted independently with a probe composition in said contacting step c) to form two or more detection mixtures; and

wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

56. The method of claim 54, wherein two or more different protein kinases are contacted independently with said ATP, said test compound, and said substrate in said contacting step a) to form two or more kinase mixtures;

wherein each of said kinase mixtures is contacted independently with a protease in said contacting step b) to form two or more protease mixtures;

wherein each of said protease mixtures is contacted independently with a probe composition in said contacting step c) to form two or more detection mixtures; and

wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

57. The method of claim 54, wherein said comparing step d) occurs during said contacting step c).

58. The method of claim 54, wherein said comparing step d) occurs after said contacting step c).

59. The method of claim 58, wherein said contacting step b) is completed by inhibiting a proteolytic activity of said protease.

60. The method of claim 59, wherein said proteolytic activity is inhibited by adding a reagent to said protease mixtures or by heating said protease mixtures.

61. The method of claim 60, wherein said reagent is aprotinin, PMSF, TPCK, AEBSF, chymotrypsin inhibitor 1, and chymotrypsin inhibitor 2.

62. The composition of claim 1, wherein said Y in said motif is phosphorylated.

64. The composition of claim 24, wherein said recognition motif for a tyrosine kinase is phosphorylated at the Y, and wherein said phosphorylated recognition motif is a recognition motif for a protein phosphatase.

66. The composition of claim 1, wherein said peptide comprises a protein phosphatase recognition motif, and wherein said peptide is selected from the group consisting of:

EAEAIY*AAPGDK (SEQ ID NO;

     25),

GDQDY*LSLDK (SEQ ID NO;

     28),

EEEEY*IQIVK (SEQ ID NO;

     41),

EEEIY*GVIEK (SEQ ID NO;

     31),

GVLTGY*VARRK (SEQ ID NO;

     36),

DDEITAY*VATRK (SEQ ID NO;

     37),

TGIITEY*VATRK (SEQ ID NO;

     38), and

EGDY*SQVLEK (SEQ ID NO;

     44),

wherein Y* represents a phosphorylated tyrosine.

68. The composition of claim 64, wherein said phosphatase recognition motif is EAIY*AAP, wherein Y* is a phosphorylated tyrosine (SEQ ID NO:

24), and wherein said phosphatase is selected from the group consisting of PTP1B, LAR, and LCA.

77. The composition of claim 64, wherein said phosphatase recognition motif is EEIY*GVI, wherein Y* is a phosphorylated tyrosine (SEQ ID NO:

30), and wherein said phosphatase is selected from the group consisting of PTP1B, LAR, and LCA.

78. The composition of claim 64, wherein said phosphatase recognition motif is LTGY*VAR, wherein Y* is a phosphorylated tyrosine (SEQ ID NO:

33), and wherein said phosphatase is selected from the group consisting of PTP1B, LAR, and LCA.

79. A method for characterizing a phosphatase, said method comprising:

a) contacting a composition of claim 62 with a protein phosphatase under conditions effective for said protein phosphatase to dephosphorylate said composition; and

b) measuring the ability of said protein phosphatase to dephosphorylate said composition.

80. A method for identifying a substrate of a phosphatase, comprising:

a) contacting a composition of claim 62 with a protein phosphatase;

b) contacting said composition and said protein phosphatase with a protease to form a protease mixture;

c) contacting said protease mixture with a probe composition to form a detection mixture; and

d) comparing a measurable property in said detection mixture with a measurable property in a control detection mixture lacking said phosphatase, wherein said composition is identified as a substrate of said phosphatase if said measurable property in said detection mixture is different from said measurable property in said control detection mixture.

81. The method of claim 80, wherein two or more different compositions of claim 62 or claim 63 are contacted independently with said phosphatase during said contacting step a) to form two or more phosphatase mixtures;

wherein each of said phosphatase mixtures is contacted independently with a protease during said contacting step b) to form two or more protease mixtures;

wherein each of said protease mixtures is contacted independently with a probe composition during said contacting step c) to form two or more detection mixtures; and

wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

82. The method of claim 80, wherein two or more different phosphatases are contacted independently with said composition during said contacting step a);

wherein each of said phosphatase mixtures is contacted independently with a protease during said contacting step b) to form two or more protease mixtures;

wherein each of said two or more protease mixtures is contacted independently with a probe composition during said contacting step c) to form two or more detection mixtures; and

wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

83. A method for identifying a modulator of activity of a phosphatase, comprising:

a) contacting a mixture of a protein phosphatase, a substrate for said protein phosphatase, wherein said substrate is a composition according to claim 62, and a test compound to form a phosphatase mixture;

b) contacting said phosphatase mixture with a protease to form a protease mixture;

c) contacting said protease mixture with a probe composition to form a detection mixture; and

d) comparing a measurable property in said detection mixture to the measurable property in a control detection mixture lacking said test compound, wherein said test compound is identified as a modulator of activity of said phosphatase if said measurable property in said detection mixture is different from said measurable property in said control mixture.

84. The method of claim 83, wherein two or more different test compounds are contacted independently with said phosphatase and said substrate during said contacting step a) to form two or more phosphatase mixtures;

wherein each of said phosphatase mixtures is contacted independently with a protease during said contacting step b) to form two or more protease mixtures;

wherein each of said two or more protease mixtures is contacted independently with a probe composition during said contacting step c) to form two or more detection mixtures; and

wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

85. The method of claim 83, wherein two or more different phosphatases are contacted independently with said test compound and said substrate during said contacting step a) to form two or more phosphatase mixtures;

wherein each of said phosphatase mixtures is contacted independently with a protease during said contacting step b) to form two or more protease mixtures wherein each of said protease mixtures is contacted independently with a probe composition during said contacting step c) to form two or more detection mixtures; and

wherein a measurable property in each of said detection mixtures is compared with the measurable property in a corresponding control mixture.

86. An article of manufacture comprising:

a) packaging matter; and

b) a composition of matter according to claim 1, 2, 4, 5 or 62 associated with said packaging material.

87. The article of claim 86, further comprising a protein kinase or a protein phosphatase.

88. The article of claim 86, further comprising a protease.

89. The article of claim 87, further comprising ATP.

90. The article of claim 86, further comprising a probe composition.

2. A composition of matter comprising:

a) a peptide having a length from five to fifty amino acids, said peptide comprising a motif selected from the group consisting of;

RR(S/T)L (SEQ ID NO;

     145),

L(S/T)TT (SEQ ID NO;

     146),

L(S/T)LD (SEQ ID NO;

     147),

RX₂(S/T)X₂, where X₁ can be V, A, or Q and X₂ can be V or L (SEQ ID NO;

     148),

TS(S/T)L (SEQ ID NO;

     149),

X₁(S/T)PX₂ where X₁ can be P or I and X₂ can be Q K, or D (SEQ ID NO;

     150),

(S/T)X₁X₂VA, where X₁ can be A, E, or Q and X₂ can be Y or H (SEQ ID NO;

     151),

I(S/T)IAN (SEQ ID NO;

     152),

SIA(S/T)I (SEQ ID NO;

     153),

(S/T)VPPS*P, where S* is a phosphorylated serine (SEQ ID NO;

     154),

DX₁(S/T)X₂, where X₁ can be A or E and X₂ can be I or Q (SEQ ID NO;

     155), and

D(S/T)QV (SEQ ID NO;

     156);

b) a first detectable moiety; and

c) a first binding member.

63. The composition of claim 2, 3, 4, or 5, wherein said S/T in said motif is phosphorylated.

3. A composition of matter comprising:

a) a peptide having a length from five to fifty amino acids, said peptide comprising a motif selected from the group consisting of;

RRX₂(S/T)L, where X₁ can be F, W, or Y (SEQ ID NO;

     45),

LX₁(S/T)TT, where X₁ can be F, W, or Y (SEQ ID NO;

     48),

X₁L(S/T)LD, where X₁ can be F, W, or Y (SEQ ID NO;

     51),

RX₁X₂(S/T)X₃, where X₁ can be V, A, or Q, X₂ can be F, W, or Y, and X₃ can be V or L (SEQ ID NO;

     54),

TX₁S(S/T)L, where X₁ can be F, W, or Y (SEQ ID NO;

     61),

X₁X₂(S/T)PX₃ where X₁ can be P or I, X₂ can be F, W, or Y, and X₃ can be G, K, or D (SEQ ID NO;

     64),

X₁(S/T)X₂X₃VA, where X₁ can be F, W, or Y, X₂ can be A, E, or Q, and X₃ can be Y or H (SEQ ID NO;

     71),

IX₁(S/T)IAN, where X₁ can be F, W, or Y (SEQ ID NO;

     78),

SIAX₁(S/T)I, where X₁ can be F, W, or Y (SEQ ID NO;

     81),

(S/T)VPPS*P, where S* is a phosphorylated serine (SEQ ID NO;

     84),

DX₁X₂(S/T)X₃, where X₁ can be A or E, X₂ can be F, W, or Y, and X₃ can be I or Q (SEQ ID NO;

     87);

DX₂(S/T)QV, where X₁ can be F, W, or Y (SEQ ID NO;

     92); and

PF(S/T)VP (SEQ ID NO;

     145).

4. The composition of claim 3, further comprising b) a first detectable moiety;

and

c) a first binding member.

5. The composition of claim 3, wherein said peptide comprises a motif selected from the group consisting of:

RRF(S/T)L (SEQ ID NO;

     157),

LF(S/T)TT (SEQ ID NO;

     158),

YL(S/T)LD (SEQ ID NO;

     159),

RX₁F(S/T)X₂, where X₁ can be V, A, or Q and X₂ can be V or L (SEQ ID NO;

     160),

TFS(S/T)L (SEQ ID NO;

     161),

X₁F(S/T)PX₂ where X₁ can be P or I and X₂ can be G, K, or D (SEQ ID NO;

     162),

F(S/T)X₁X₂VA, where X₁ can be A, E, or Q and X₂ can be Y or H (SEQ ID NO;

     163),

IF(S/T)IAN (SEQ ID NO;

     164),

SIAF(S/T)I (SEQ ID NO;

     165),

DX₁F(S/T)X₂, where X₁ can be A or E and X₂ can be I or Q (SEQ ID NO;

     166); and

DY(S/T)QV (SEQ ID NO;

     167).

25. The composition of claim 3 or 5, wherein said motif is a recognition motif for a serine/threonine kinase, said recognition motif selected from the group consisting of:

LRRFSLG (SEQ ID NO;

     46),

GLFSTTP (SEQ ID NO;

     49),

DYLSLDK (SEQ ID NO;

     52),

NRVFSVA (SEQ ID NO;

     55),

PRAFSVG (SEQ ID NO;

     56),

RRQFSLR (SEQ ID NO;

     57),

RTFSSLA (SEQ ID NO;

     62),

APFSPGG (SEQ ID NO;

     65),

HPFSPKK (SEQ ID NO;

     66),

KIFSPDV (SEQ ID NO;

     67),

EFTAYVA (SEQ ID NO;

     72),

IFTEYVA (SEQ ID NO;

     73),

VFTQHVA (SEQ ID NO;

     74),

RIFSIANS (SEQ ID NO;

     79),

DSIAFSIV (SEQ ID NO;

     82),

FSVPPS*PD, where S* is a phosphorylated serine (SEQ ID NO;

     85),

EDAFSII (SEQ ID NO;

     88),

EDEFSQN (SEQ ID NO;

     89),

EGDYSQV (SEQ ID NO;

     93), and

RPFSVPP (SEQ ID NO;

146).

28. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is LRRFSLG (SEQ ID NO:

46), and wherein said serine/threonine kinase is selected from the group consisting of Akt1, Akt2, Akt3, Aurora A, CaMKII, CDK2/CycA, CDK3/CycE, CDK7/CycH, MAPKAP-K1α

, MAPKAP-K1β

, MAPKAP-K1γ

, MSK1, PAK2, PKA, PKG, and ROCK.

29. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is GLFSTTP (SEQ ID NO:

49), and wherein said serine/threonine kinase is selected from the group consisting of p38γ

, p38δ

, and REDK.

30. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is NRVFSVA (SEQ ID NO:

55), and wherein said serine/threonine kinase is selected from the group consisting of Akt3, AMPK, CaMKII, CDK7/CycH, CHK2, IKKα

, MAPKAP-K1α

, MAPKAP-K2, MAPKAP-K3, MAPKAP-K5, PAK2, PKA, PKCβ

II, REDK, ROCK, ROCK2, and SGK1.

31. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is PRAFSVG (SEQ ID NO:

56), and wherein said serine/threonine kinase is selected from the group consisting of Akt1, Akt2, Akt3, CaMKII, CDK7/CycH, IKKβ

, MAPKAP-K1α

/RSK1, MAPKAP-K1β

/RSK2, MAPKAP-K1γ

/RSK3, MSK1, PAK2, PIM1, PKA, PKG, REDK, and SGK1.

32. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is RRQFSLR (SEQ ID NO:

57), and wherein said serine/threonine kinase is selected from the group consisting of Akt1, Akt2, Akt3, CaMKII, CHK1, CHK2, MAPKAP-K1α

, MAPKAP-K1β

, MAPKAP-K1γ

, MSK1, p70 S6 Kinase, PAK2, PIM1, PKA, PKCα

, PKCβ

1, PKCβ

II, PKCγ

, PKCδ

, PKCε

, PKCξ

, PKCη

, PKCθ

, PKCt, PKG, ROCK, ROCK2, and SGK1.

33. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is RTFSSLA (SEQ ID NO:

62), and wherein said serine/threonine kinase is selected from the group consisting of Akt1, CDK2/CycA, CDK6, IKKβ

, MAPKAP-K1α

, MAPKAP-K1β

, MAPKAP-K1γ

, p70 S6 Kinase, PAK2, and PKA.

34. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is APFSPGG (SEQ ID NO:

65), and wherein said serine/threonine kinase is selected from the group consisting of CDK2/CycA, CDK3/CycE, ERK1, ERK2, IKKα

, p38β

, p38γ

, and p38δ

.

39. The composition of claim 3, wherein said peptide is selected from the group consisting of:

ALRRFSLGEK (SEQ ID NO;

     47),

VAPFSPGGRAK (SEQ ID NO;

     68),

RGGLFSTTPGGTK (SEQ ID NO;

     50),

KLNRVFSVAC (SEQ ID NO;

     58),

GDQDYLSLDK (SEQ ID NO;

     53),

ARPRAFSVGK (SEQ ID NO;

     59),

RRRQFSLRRKAK (SEQ ID NO;

     60),

RPRTFSSLAEGK (SEQ ID NO;

     63),

AKHPFSPKKAK (SEQ ID NO;

     69),

DDEFTAYVATRK (SEQ ID NO;

     75),

TGIFTEYVATRK (SEQ ID NO;

     76),

TGVFTQHVATRK (SEQ ID NO;

     77),

QRIFSIANSIVK (SEQ ID NO;

     80),

RIDSIAFSIVGK (SEQ ID NO;

     83),

PRPFSVPPS*PDK, where S* is a phosphorylated serine (SEQ ID NO;

     86),

EEDAFSIIGK (SEQ ID NO;

     90),

REDEFSQNEEK (SEQ ID NO;

     91),

IIKIFSPDVEK (SEQ ID NO;

     70),

EGDYSQVLEK (SEQ ID NO;

     22), and

CPRPFSVPPS*PDK (SEQ ID NO;

     147), where S* is a phosphorylated serine.

65. The composition of claim 5, wherein said peptide comprises a protein phosphatase recognition motif selected from the group consisting of:

LRRFS*LG (SEQ ID NO;

     96),

GLFS*TTP (SEQ ID NO;

     99),

DYLS*LDK (SEQ ID NO;

     102),

NRVFS*VA (SEQ ID NO;

     105),

PRAFS*VG (SEQ ID NO;

     106),

RRQFS*LR (SEQ ID NO;

     107)

RTFSS*LA (SEQ ID NO;

     112),

APFS*PGG (SEQ ID NO;

     115),

HPFS*PKK (SEQ ID NO;

     116),

KIFS*PDV (SEQ ID NO;

     117),

EFT*AYVA (SEQ ID NO;

     122),

IFT*EYVA (SEQ ID NO;

     123),

VFT*QHVA (SEQ ID NO;

     124),

RIFS*IANS (SEQ ID NO;

     129),

DSIAFS*IV (SEQ ID NO;

     132),

FS*VPPS*PD (SEQ ID NO;

     135),

EDAFS*II (SEQ ID NO;

     138),

EDEFS*QN (SEQ ID NO;

     139),

EGDYS*QV (SEQ ID NO;

     143), and

PF(S/T)*VP (SEQ ID NO;

148).

wherein S* represents a phosphorylated serine and T* represents a phosphorylated threonine.

67. The composition of claim 3, wherein said peptide has a sequence selected from the group consisting of:

ALRRFS*LGEK (SEQ ID NO;

     97),

VAPFS*PGGRAK (SEQ ID NO;

     118),

RGGLFS*TTPGGTK (SEQ ID NO;

     100),

KLNRVFS*VAC (SEQ ID NO;

     108),

GDQDYLS*LDK (SEQ ID NO;

     103),

ARPRAFS*VGK (SEQ ID NO;

     109),

RRRQFS*LRRKAK (SEQ ID NO;

     110),

RPRTFSS*LAEGK (SEQ ID NO;

     113),

AKHPFS*PKKAK (SEQ ID NO;

     119),

DDEFT*AYVATRK (SEQ ID NO;

     125),

TGIFT*EYVATRK (SEQ ID NO;

     126),

TGVFT*QHVATRK (SEQ ID NO;

     127),

QRIFS*IANSIVK (SEQ ID NO;

     130),

RIDSIAFS*IVGK (SEQ ID NO;

     133),

PRPFS*VPPS*PDK (SEQ ID NO;

     136)

EEDAFS*IIGK (SEQ ID NO;

     140),

REDEFS*QNEEK (SEQ ID NO;

     141),

IIKIFS*PDVEK (SEQ ID NO;

     120),

EGDYS*QVLEK (SEQ ID NO;

     144), and

CPRPFS*VPPS*PDK (SEQ ID NO;

150),

wherein S* represents a phosphorylated serine and T* represents a phosphorylated threonine.

69. The composition of claim 65, wherein said phosphatase recognition motif is DQDYLS*L, wherein S* is a phosphorylated serine (SEQ ID NO:

27), and wherein said phosphatase is selected from the group consisting of PP1α

, PP2A, PP2B, and PP2C.

70. The composition of claim 65, wherein said phosphatase recognition motif is LRRFS*L, wherein S* is a phosphorylated serine (SEQ ID NO:

96), and wherein said phosphatase is selected from the group consisting of PP1α

, PP2A, and PP2C.

71. The composition of claim 65, wherein said phosphatase recognition motif is GLFS*TTP, wherein S* is a phosphorylated serine (SEQ ID NO:

99), and wherein said phosphatase is selected from the group consisting of PP1α

, PP2A, PP2B, and PP2C.

72. The composition of claim 65, wherein said phosphatase recognition motif is NRVFS*VA, wherein S* is a phosphorylated serine (SEQ ID NO:

105), and wherein said phosphatase is selected from the group consisting of PP1α

, PP2A, PP2B, and PP2C.

73. The composition of claim 65, wherein said phosphatase recognition motif is PRAFS*VG, wherein S* is a phosphorylated serine (SEQ ID NO:

106), and wherein said phosphatase is selected from the group consisting of PP1α

, PP2A, and PP2B.

74. The composition of claim 65, wherein said phosphatase recognition motif is RRQFS*LR, wherein S* is a phosphorylated serine (SEQ ID NO:

107), and wherein said phosphatase is selected from the group consisting of PP1α

, PP2A, and PP2B.

75. The composition of claim 65, wherein said phosphatase recognition motif is RTFSS*LA, wherein S* is a phosphorylated serine (SEQ ID NO:

112), and wherein said phosphatase is selected from the group consisting of PP1α

, PP2A, and PP2B.

76. The composition of claim 65, wherein said phosphatase recognition motif is APFS*PGG, wherein S* is a phosphorylated serine (SEQ ID NO:

115), and wherein said phosphatase is selected from the group consisting of PP1α and

PP2A.

91. The composition of claim 25, wherein said recognition motif for a serine/threonine kinase is RPFSVPP (SEQ ID NO:

146), and wherein said serine/threonine kinase is GSK-3α

or GSK-3β

.

19. A probe composition comprising a second binding member and a second detectable moiety.

92. A probe composition comprising a streptavidin tetramer having 1 to 12 CS-124-DTPA-Phe-Tb chelates linked thereto.