Kinase and/or phosphatase sensing via hydroxyquinoline-sensitized chelates
The present invention generally relates to assays of enzymes responsible for phosphoregulation including kinases (such as protein kinases, which mediate protein and peptide phosphorylation) and phosphatases (such as protein phosphatases, which mediate protein and peptide dephosphorylation). Certain aspects of the invention use lanthanide ions such as europium ions that exhibit chelation-enhanced luminescence. Phosphorylation of a peptide by a kinase may cause a complex to form between the lanthanide ion, the phosphate group, and a reporter group such as a hydroxyquinoline, which results in luminescence when in the complexed state. Thus, in certain embodiments, determination of luminescence may be indicative of kinase activity. Certain embodiments also include the use of substrates for detection of phosphatase activity, where dephosphorylation results in a loss of signal assay. Other aspects are generally related to techniques for making and using such peptides or complexes, kits involving such peptides or complexes, and the like.
1. A composition, comprising:
a peptide having a first portion comprising a structure:
wherein each of R 1 , R 2 , R 3 , R 4 , and R 5 independently is hydrogen or —SO 2 X such that at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is —SO 2 X, wherein X is —OR′ or —NR′R″, R′ and R″ each independently being hydrogen or an alkyl group, and n is 0 or a positive integer, and
a second portion comprising a phosphate group,
wherein the N and/or the O of the first portion, and the phosphate group of the second portion, are coordinated via an europium ion in the 3 + state (Eu(III)).
2. The composition of claim 1 , wherein the peptide comprises a structure:
wherein:
Eu is the europium ion; and
the wavy lines indicate covalent binding of the structure to the peptide.
3. The composition of claim 2 , wherein Eu is further coordinated to a plurality of H 2 O molecules.
4. The composition of claim 1 , wherein R 1 is —H and/or R 2 is —H and/or R 4 is —H and/or R 5 is —H.
5. The composition of claim 1 , wherein the structure has only one —SO 2 X.
6. The composition of claim 1 , wherein R 3 is —SO 2 X.
7. The composition of claim 6 , wherein X is —NR′R″.
8. The composition of claim 7 , wherein R′ and R″ are each an alkyl group.
9. The composition of claim 1 , wherein the phosphate group is a portion of a phosphorylated amino acid residue within the peptide.
10. The composition of claim 9 , wherein the phosphorylated amino acid is serine or threonine or tyrosine.
11. The composition of claim 1 , wherein n is 0, 1, or 2.
12. The composition of claim 1 , wherein the —(CH 2 ) n — of the structure is covalently bound to a second amino acid residue within the peptide.
13. The composition of claim 12 , wherein the second amino acid residue is cysteine.
14. The composition of claim 13 , wherein the —(CH 2 ) n — of the structure is covalently bound to the sulfur atom of the cysteine residue.
15. The composition of claim 12 , wherein the —(CH 2 ) n — of the structure is covalently bound to an amino acid backbone of the peptide.
16. The composition of claim 15 , wherein the phosphorylated amino acid and the second amino acid residue are separated by at least one amino acid residue.
17. The composition of claim 15 , wherein the phosphorylated amino acid and the second amino acid residue are separated by 1-5 amino acid residues.
18. A method, comprising:
noncovalently binding an Eu(III) ion to a peptide comprising a first portion, and a second portion comprising a phosphate group,
the first portion having a structure:
wherein:
each of R 1 , R 2 , R 3 , R 4 , and R 5 independently is hydrogen or —SO 2 X such that at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is —SO 2 X, wherein X is —OR′ or —NR′R″, R′ and R″ each independently being hydrogen or an alkyl group,
n is 0 or a positive integer, and
the wavy line indicates covalent attachment to the peptide; and
determining luminescence of the structure to determine binding of the Eu(III) ion to the first and second portions of the peptide.
19. The method of claim 18 , wherein the peptide is prepared by exposing a precursor peptide having a phosphorylatable amino acid residue to a kinase able to phosphorylate the phosphorylatable amino acid residue to produce the phosphopeptide.
20. A composition, comprising:
a solution comprising dissolved Eu(III) ions and a peptide comprising a portion having a structure:
wherein:
each of R 1 , R 2 , R 3 , R 4 , and R 5 independently is hydrogen or —SO 2 X such that at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is —SO 2 X, wherein X is —OR′ or —NR′R″, R′ and R″ each independently being hydrogen or an alkyl group,
n is 0 or a positive integer, and
the wavy line indicates covalent attachment to the peptide.
21. The method of claim 18 , wherein the phosphopeptide is exposed to a phosphopeptide phosphatase to produce the peptide.