Kinase and phosphatase assays
Compositions, methods, and kits for detecting and monitoring kinase, phosphatase and protein post-translational modification activity are described. The compositions typically include a peptide, a detectable moiety, and a protease cleavage site. Modification of a peptide by a kinase, phosphatase or other protein post-translational modification alters the proteolytic sensitivity of the peptide, resulting in a change of a detectable property of the composition. Panel assays for determining substrates or modulators of kinase, phosphatase or other protein post-translational modification activity are also described.
1. A composition of matter comprising:
(a) a peptide having a length up to fifty amino acids, the peptide comprising
EEEYIQI (SEQ ID NO: 18) or EEEEYIQIVK (SEQ ID NO: 19); and
(b) a first detectable moiety associated with the peptide.
2. The composition of claim 1 , wherein the first detectable moiety is covalently linked to the peptide.
3. The composition of claim 1 , wherein the peptide comprises a protease cleavage site.
4. The composition of claim 3 , wherein the 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.
5. The composition of claim 1 , wherein the peptide has a length selected from the group consisting of from 8 to 50 amino acids, from 8 to 25 amino acids and from 8 to 15 amino acids.
6. The composition of claim 1 , further comprising a second detectable moiety associated with the peptide.
7. The composition of claim 1 , wherein the composition exhibits an optical property, a magnetic property, or a radioactive property.
8. The composition of claim 6 , wherein the first or second detectable moieties are 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, fluorescein maleimide, and 7-amino-4-methylcoumarin-3-acetic acid.
9. The composition of claim 6 , wherein the first detectable moiety, the second detectable moiety or the first and second detectable moiety is a member of a specific binding pair.
10. The composition of claim 6 , wherein the first detectable moiety and the second detectable probe moiety form a dark quenching RET pair or a FRET pair.
11. The composition of claim 6 , wherein the first detectable moiety is 7-hydroxycoumarin-3-carboxamide and the second detectable moiety is 5-FAM.
12. The composition of claim 6 , wherein the first or second detectable probe moiety is covalently linked to the peptide via a linker (L).
13. A method for identifying a modulator of activity of a tyrosine kinase, comprising:
(a) forming a mixture of a protein kinase, a composition of claim 1 , and a test compound;
(b) contacting the mixture with a protease to form a protease mixture; and
(c) comparing a measurable property in the protease mixture to the measurable property in a control mixture, wherein the test compound is identified as a modulator of activity of the kinase if the measurable property in the protease mixture is different from the measurable property in the control mixture.
14. An article of manufacture comprising:
(a) packaging matter; and
(b) a composition of matter according to claim 1 associated with the packaging material; and
(c) at least one member selected from the group consisting of a tyrosine protein kinase, a tyrosine protein phosphatase, a protease and ATP.
15. A composition of matter comprising:
(a) a peptide having a length from five to fifty amino acids, the peptide comprising EEYIQ (SEQ ID NO: 17); and
(b) a first detectable moiety covalently linked to the peptide, wherein the 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, fluorescein maleimide, and 7-amino-4-methylcoumarin-3-acetic acid.
16. The composition of claim 15 , wherein the peptide comprises a protease cleavage site.
17. The composition of claim 16 , wherein the 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.
18. The composition of claim 15 , wherein the peptide has a length selected from the group consisting of from 8 to 50 amino acids, from 8 to 25 amino acids and from 8 to 15 amino acids.
19. The composition of claim 15 , wherein the composition further comprises a second detectable moiety.
20. The composition of claim 19 , wherein the first detectable moiety and the second detectable probe moiety form a dark quenching RET pair or a FRET pair.
21. The composition of claim 19 , wherein the first or second detectable probe moiety is covalently linked to the peptide via a linker (L).
22. A method for identifying a modulator of activity of a tyrosine kinase, comprising:
(a) forming a mixture of a protein kinase, a composition of claim 15 , and a test compound;
(b) contacting the mixture with a protease to form a protease mixture; and
(c) comparing a measurable property in the protease mixture to the measurable property in a control mixture, wherein the test compound is identified as a modulator of activity of the kinase if the measurable property in the protease mixture is different from the measurable property in the control mixture.
23. An article of manufacture comprising:
(a) packaging matter; and
(b) a composition of matter according to claim 15 associated with the packaging material; and
(c) at least one member selected from the group consisting of a protein kinase, a protein phosphatase, a protease and ATP.