Post-translational modifications identified by elemental analysis
AssigneeDvs Sciences Inc.
InventorOLGA ORNATSKY
Life of this patent
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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.
Claims59
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;
- and
- d) Performing solution elemental analysis of said solution.
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.
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.
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;
- and
- d) Measuring the solution by elemental analysis
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;
- and
- c) Measuring the multitude of phosphorylated substrate immobilized on element labeled supports having attached metal ion coordination complex by elemental analysis.
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.
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.
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;
- and
- c) Measuring the metal ion coordination complex attached to the multitude of phosphorylated substrate immobilized to uniquely labeled supports by elemental analysis.
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;
- and
- g) ATP.
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;
- and
- f) ATP.
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;
- and
- b) an element tag for directly tagging an affinity product;
- and optionally
- c) an affinity product.
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 and
- g) ADP.
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, phosphatase
- f) phosphatase buffer and
- g) ADP.
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.
Dependencies are inferred from the claim text.