POST-TRANSLATIONAL MODIFICATIONS IDENTIFIED BY ELEMENTAL ANALYSIS

Abstract

Methods and kits for enzymes involved in post-translational modifications are provided. The methods employ elemental analysis, including ICP-MS. The methods allow for the convenient and accurate analysis of post-translation modifications of substrates by enzymes involved in post-translational modifications, including kinase and phosphatase enzymes.

Description

BRIEF DESCRIPTION OF THE FIGURES

The invention is illustrated in the figures, which are meant to be exemplary and not limiting.

FIG. 1. Dose-dependence curve of EGFR kinase activity. Increasing amounts of EGFR-GST were incubated with 2 ug biotinylated substrate peptide (PTP1B) per reaction per well of 96-well streptavidin coated plate. All reactions were set up in triplicate. Detected signal is presented as normalized response of Ti ions to Ir internal standard.

FIG. 2. Peptide concentration dependence of EGFR kinase activity. Increasing amounts of biotinylated substrate peptide (PTP1B) were incubated with 50 ng of EGFR-GST per reaction per well of 96-well Streptavidin coated plate. All reactions were set up in triplicate. Detected signal is presented as normalized response of Ti ions to Ir internal standard.

FIG. 3. Intracellular EGFR kinase activity in human cells. Lysates prepared from A431 and KG1-a cells were mixed with PTP1B(Tyr66) substrate linked to agarose beads in the presence of ATP and cations. Washed agarose beads with phosphorylated substrate were incubated with Ti02 particle suspension. Analysis of titanium content in solution was done by ICP-MS. Triplicate samples were set up for analysis.

FIG. 4. Schematic representation of solution ICP-MS analysis of kinase activity in cellular lysates, in accordance with the invention.

FIG. 5. Flow chart showing method of specific kinase(s) assay, in accordance with the invention.

FIG. 6. Flow chart showing method of specific phosphatase(s) assay, in accordance with the invention.

FIG. 7. Flow chart showing method of specific kinase(s) assay using uniquely labeled beads, in accordance with the invention.

FIG. 8. Flow chart showing method of specific phosphatase(s) assay using uniquely labeled beads, in accordance with the invention.

Claims

1. A method for a kinase assay, comprising: a) Incubating ATP, at least one kinase, and a free non-phosphorylated substrate labeled with an element tag, with a support having attached thereto metal ion coordination complexes under conditions to enable the kinase to phosphorylate the substrate;b) Separating free non-phosphorylated substrate from bound phosphorylated substrate labeled with an element tag to the support;c) Eluting the element tag associated with the resultant phosphorylated substrate into a solution; andd) Performing solution elemental analysis of said solution.

2. The method of claim 1 where in step (a) a multitude of free non-phosphorylated substrates each labeled with a unique element tag are incubated.

3. The method of claim 1 wherein the metal ion coordination complex attached to the support is a titanium oxide bead.

4. The method of claim 1 where in step (a) the conditions to enable the kinase to phosphorylate the substrate include a kinase reaction buffer.

5. The method of claim 1 where in step (a) antagonists or agonists of kinase are incubated.

6. The method of claim 1 wherein the kinase is delivered in the form of a cell lysate.

7. A method for a phosphatase assay, comprising: a) Incubating free phosphorylated substrate labeled with an element tag with a support having attached thereto metal ion coordination complexes;b) Separating free phosphorylated substrate from bound phosphorylated substrate labeled with an element tag attached to the metal ion coordination complexes attached to the support;c) Incubating ADP and at least one phosphatase with the bound phosphorylated substrate labeled with an element tag attached to the metal ion coordination complexes attached to the support under conditions to enable the phosphatase to dephosphorylate the substrate;d) Separating free non-phosphorylated substrate labeled with an element tag from bound phosphorylated substrate attached to the metal ion coordination complexes attached to the support;e) Measuring the tag element in a solution of the free non-phosphorylated substrate.

8. The method of claim 7 wherein a multitude of free phosphorylated substrates each labeled with an element tag that is characteristic of the substrate are incubated in step (a) and the tag elements characteristic of the substrate are measured in step (e).

9. The method of claim 7 where in step (c) the conditions to enable the phosphatase to dephosphorylate the substrate include incubation with a phosphatase reaction buffer.

10. The method of claim 7 where in step (c) antagonists or agonists of phosphatase are incubated.

11. The method of claim 7 wherein the metal ion coordination complex is attached to element labeled beads.

12. The method of claim 7 wherein the metal ion coordination complex attached to the support is a titanium oxide bead.

13. The method of claim 7 wherein the phosphatase is in the form of a cell lysate.

14. A method for a phosphatase assay, comprising: a) Incubating, in a multitude of solutions, each solution comprising a different free phosphorylated substrate labeled with an element tag, which can optionally be the same element tag for all substrates, a plurality of element labeled supports having attached thereto a metal ion coordination complex, in such manner that each type of phosphorylated substrate labeled with an element tag, which can optionally be the same element tag for all substrates, is attached to a single type of element labeled support;b) Separating free phosphorylated substrate from the bound substrate attached to the metal ion coordination complex attached to the multitude of element labeled supports in the multitude of separate solutions;c) Incubating the multitude of element labeled supports having attached thereto the multitude of phosphorylated substrates labeled with an element tag, which can optionally be the same element tag for all substrates, through attachment to the metal ion coordination complex that is attached to the supports in a single solution with ADP and at least one phosphatase in conditions that enable the phosphatase to dephosphorylate the phosphorylated substrates;d) Separating free non-phosphorylated substrate from bound phosphorylated substrate labeled with an element tag, which can optionally be the same element tag for all substrates, attached to the metal ion coordination complex attached to said multitude of element labeled supports;e) Performing particle elemental analysis of bound phosphorylated substrate labeled with an element tag, which can optionally be the same element tag for all substrates, attached to the metal ion coordination complex attached to the multitude of element labeled supports.

15. A method for a kinase assay, comprising: a) Incubating ATP, at least one kinase, and a free metal ion coordination complex, with an immobilized non-phosphorylated substrate under conditions which enable the kinase to phosphorylate the substrate;b) Separating immobilized phosphorylated substrate attached to the metal ion coordination complex from the free ion coordination complex and the immobilized non-phosphorylated substrate;c) Eluting the metal ion coordination complex attached to the immobilized phosphorylated substrate into a solution; andd) Measuring the solution by elemental analysis

16. A method for a kinase assay, comprising: a) Incubating ATP, at least one kinase, a free metal ion coordination complex, and a multitude of non-phosphorylated substrates immobilized on element labeled supports in such manner that a single type of non-phosphorylated substrate is attached to a single type of element labeled support, in conditions to enable the kinase to phosphorylate the substrates;b) Separating the multitude of phosphorylated substrates immobilized on element labeled supports having attached metal ion coordination complex from the free metal ion coordination complexes and the multitude of immobilized non-phosphorylated substrates; andc) Measuring the multitude of phosphorylated substrate immobilized on element labeled supports having attached metal ion coordination complex by elemental analysis.

17. A method for a kinase assay, comprising: a) Introducing a multitude of non-phosphorylated substrates with element tags into live cells;b) Incubating the cells having the introduced non-phosphorylated substrates with an agonist or an antagonist of kinase activity;c) Fixing and permeabilizing the cells;d) Incubating the cells with element-labeled antibodies directed against phosphospecific kinase substrates;e) Separating the cells from unbound antibodies;f) Measuring the phosphorylated substrates with element tags and attached element-labeled antibodies by elemental analysis.

18. The method of claim 17 wherein the step of introducing a multitude of non-phosphorylated substrates is selected from the group consisting of microinjection, transfection of peptide expressing plasmid, liposome delivery, and incubation with PTD-conjugated substrate.

19. The method of claim 17 wherein the step of separating the cells from the unbound antibodies is selected from the group consisting of low speed centrifugation and filtration.

20. The method of claim 17 wherein the non-phosphorylated substrates are labeled with different element tags, specific to each substrate.

21. The method of claim 17 wherein the non-phosphorylated substrates are labeled with the same element tag.

22. A. method for a phosphatase assay, comprising: Incubating ADP and at least one phosphatase, with an immobilized phosphorylated substrate with attached metal ion coordination complexes in conditions that enable the phosphatase to dephosphorylate the substrate;b) Separating the free metal ion coordination complex from the immobilized non-phosphorylated substrate and the immobilized phosphorylated substrate with attached metal ion coordination complex;c) Eluting the metal ion coordination complex into a solution;d) Measuring the solution by elemental analysis.

23. A method for a phosphatase assay, comprising: a) Incubating ADP, at least one phosphatase, and a multitude of phosphorylated substrates with attached metal ion coordination complex immobilized to element labeled supports in such manner that a single type of phosphorylated substrate is attached to a single type of element labeled support in conditions that enable the phosphatase to dephosphorylate the phosphorylated substrates;b) Separating the free metal ion coordination complex from the multitude of non-phosphorylated substrates immobilized to element labeled supports and the multitude of immobilized phosphorylated substrate; andc) Measuring the metal ion coordination complex attached to the multitude of phosphorylated substrate immobilized to uniquely labeled supports by elemental analysis.

24. A kit for the detection and measurement of elements in a sample, where the measured elements include an element tag attached to a non-phosphorylated substrate and a metal ion coordination complex, comprising: a) an element tag for directly tagging non-phosphorylated substrate;b) non-phosphorylated substrate;c) a solid support;d) metal ion coordination complex;e) and optionally, kinase;f) kinase buffer; andg) ATP.

25. The kit of claim 24 further comprising instructions for i) directly tagging the non-phosphorylated substrate with an element tag; ii) incubating kinase with element labeled non-phosphorylated substrate in kinase buffer, iii) attaching metal ion coordination complex to the support; iv) incubating the kinase with element labeled non-phosphorylated substrate in kinase buffer with the support having attached metal ion coordination complex; v) separating bound substrate from unbound substrate; vi) eluting the bound substrate, and vii) detecting and measuring the bound substrate by elemental analysis.

26. A kit for the detection and measurement of elements in a sample, where the measured elements include element labels of uniquely labeled supports and an element of a metal ion coordination complex, comprising: a) a multitude of non-phosphorylated substrates;b) uniquely labeled supports;c) metal ion coordination complex;d) and optionally, kinase;e) kinase buffer; andf) ATP.

27. The kit of claim 26 further comprising instructions for i) immobilizing the non-phosphorylated substrates on element labeled supports in separate solutions; ii) incubating kinase in kinase buffer with the multitude of non-phosphorylated substrates immobilized on uniquely labeled supports, iii) incubating the metal ion coordination complex in the kinase buffer with the kinase and the multitude of non-phosphorylated substrates immobilized on uniquely labeled supports, iv) washing and separating bound substrate from unbound substrate; v) measuring the metal ion coordination complex bound to the multitude of phosphorylated substrate immobilized on uniquely labeled supports by elemental analysis.

28. A kit for the detection and measurement of elements in a sample, where the measured elements include element tags attached to affinity products that recognize phosphorylated substrates, comprising: a) non-phosphorylated substrate ready to be introduced into a cell; andb) an element tag for directly tagging an affinity product; and optionallyc) an affinity product.

29. The kit of claim 28 further comprising instructions for i) introducing the non-phosphorylated substrate into a cell; ii) directly tagging an affinity product that recognizes phosphorylated substrates; iii) fixing and permeabilizing the cells; iv) combining the labeled affinity product with the cells; v) separating bound affinity product from unbound affinity product, and vi) detecting and measuring the amount of the bound affinity product labeled with an element tag by particle elemental analysis.

30. A kit for the detection and measurement of elements in a sample, where the measured elements include an element tag attached to a phosphorylated substrate and a metal ion coordination complex, comprising: a) an element tag for directly tagging phosphorylated substrate;b) phosphorylated substrate;c) a solid support;d) metal ion coordination complex;e) and optionally, phosphatase;f) phosphatase buffer andg) ADP.

31. The kit of claim 30 further comprising instructions for i) direct tagging of the phosphorylated substrate with an element tag; ii) attaching the metal ion coordination complex to the support; iii) incubating the element labeled phosphorylated substrate with the support with attached metal ion coordination complex; iv) separating bound substrate rom unbound substrate; v) incubating the phosphatase in phosphatase buffer with the support with the attached metal ion coordination complex; vi) separating bound substrate from unbound substrate; vii) eluting the bound substrate, and viii) measuring the bound substrate by solution elemental analysis.

32. A kit for the detection and measurement of elements in a sample, where the measured elements include an element tag attached to a phosphorylated substrate, an element of a metal ion coordination complex, and elements of uniquely labeled supports, comprising: a) an element tag for directly tagging phosphorylated substrate;b) a multitude of phosphorylated substrates;c) uniquely labeled supports;d) metal ion coordination complex;e) and optionally, phosphatasef) phosphatase buffer andg) ADP.

33. The kit of claim 32 further comprising instructions for i) direct tagging the phosphorylated substrates with an element tag; ii) attaching a metal ion coordination complex to the uniquely labeled support; iii) adding element labeled phosphorylated substrates to the uniquely labeled support with attached metal ion coordination complex in separate volumes, iv) incubating the substrates; v) washing the supports; vi) combining the multitude of uniquely labeled supports having attached thereto the multitude of resultant phosphorylated substrate labeled with an element tag through coordination to the metal ion coordination complex that is attached to the supports; vii) incubating the phosphatase, the phosphatase buffer and the supports; viii) separating bound substrate from unbound substrate; and ix) measuring the phosphorylated substrate labeled with an element tag coordinated to the metal ion coordination complex attached to said multitude of uniquely labeled supports by particle elemental analysis.

34. The kit of claim 32 further comprising a multitude of phosphorylated substrates directly labeled with the same element tag.

35. The kit of claim 32 further comprising a multitude of phosphorylated substrates directly labeled with unique element tags.

36. The kit of claim 24 further comprising a non-phosphorylated substrate, wherein the non-phosphorylated substrate is directly labeled with an element tag.

37. The kit of claim 24 further comprising a multitude of non-phosphorylated substrates directly labeled with unique element tags.

38. The kit of claims 24 or 30 where the support with attached metal ion coordination complex is a titanium oxide bead.

39. The kit of claims 24 or 30 further comprising a support with an attached metal ion coordination complex.

40. The kit of claim 26 further comprising a multitude of non-phosphorylated substrates immobilized on uniquely labeled beads.

41. The kit of claim 28 further comprising a multitude of non-phosphorylated substrates to be introduced into a cell.

42. The kit of claim 28 wherein the non-phosphorylated substrate with or without an element tag is in a sterile solution at a concentration compatible with microinjection into the cell.

43. The kit of claim 28 further comprising an affinity product that recognizes phosphorylated substrates, wherein the affinity product is directly labeled with an element tag.

44. The kit of claim, 28 further comprising a multitude of affinity products that recognize phosphorylated substrates, wherein the affinity products are directly labeled with unique element tags.

45. The kit of claim 28 further comprising an expression plasmid and wherein the non-phosphorylated substrate is produced by an expression plasmid transfected or electroporated into the cell.

46. The kit of claim 28 wherein the non-phosphorylated substrate with or without an element tag is in a liposome solution.

47. The kit of claim 32 further comprising a multitude of uniquely labeled supports with attached metal ion coordination complex.

48. The kit of claim 30 further comprising a phosphorylated substrate, wherein the phosphorylated substrate is directly labeled with an element tag.

49. The kit of claim 30 further comprising a multitude of phosphorylated substrates directly labeled with unique element tags.

50. The kit of claim 32 wherein the non-phosphorylated substrate with or without an element tag is attached to a protein transfer domain (PTD) in a sterile solution.

51. A kit of any one of claims 25, 29 or 31 wherein instructions are provided for the solution to be analyzed by solution elemental analysis after the second incubation without the separating and eluting steps.

52. A kit of claim 33 wherein instructions are provided for a cell lysate to be incubated wherein the cell lysate comprises a phosphatase.

53. The kit of claim 24 wherein the element is measured using a mass spectrometer.

54. The kit of claims 24 wherein the element is an isotope or ion.

55. The kit of claim 24 wherein the element is selected from a group consisting of the noble metals, transition elements, lanthanides, rare earth elements, gold, silver, platinum, rhodium, iridium and palladium.

56. The kit of claim 28 wherein the affinity product that recognizes the phosphorylated substrates is selected from a group consisting of antibody, Fab′, aptamer, antigen, hormone, growth factor, receptor, protein, peptide, SH2 peptide, and nucleic acid.

57. The kit of claims 24 wherein the element includes more than one atom of an isotope.

58. The kit of claims 24 further comprising any combination of the substances wherein the substances are selected from the group consisting of standards, a dilution buffer, an elution buffer, a wash buffer and an assay buffer.

59. The kit of claim 25 further comprising instructions for particle elemental analysis.