Kit for detecting and measuring element tagged kinases and phosphatases by inductively coupled plasma mass spectrometry
View Patent ↗Kits for the assay of enzymes involved in post-translational modifications are provided. The kits facilitate the execution of methods of assay that allow for convenient and accurate analysis using atomic mass spectrometry of post-translation modifications of substrates by enzymes involved in post-translational modifications, including kinase and phosphatase enzymes.
1. A kit for detecting and measuring a mass-to-charge ratio of an element tag or a metal ion of a metal ion coordination complex by inductively coupled plasma mass spectrometry in a sample, comprising:
(a) the element tag comprising a transition element, or a transition element isotope;
(b) a non-phosphorylated substrate;
(c) a support;
(d) the metal ion coordination complex;
(e) optionally, a kinase;
(f) optionally, a kinase buffer;
(g) and optionally, ATP;
wherein the element tag is for directly tagging the support or the non-phosphorylated substrate.
2. The kit of claim 1 , wherein the element tag is for directly tagging the non-phosphorylated substrate.
3. The kit of claim 2 further comprising a non-phosphorylated substrate, wherein the non-phosphorylated substrate is attached to an element tag.
4. The kit of claim 2 further comprising a plurality of non-phosphorylated substrates, each non-phosphorylated substrate attached to a different element tag.
5. The kit of claim 2 further comprising a support with an attached metal ion coordination complex.
6. The kit of claim 2 further comprising instructions for i) labeling a non-phosphorylated substrate with an element tag; ii) incubating a kinase with the element tag labeled non-phosphorylated substrate in a kinase buffer, iii) attaching a metal ion coordination complex to a support; iv) incubating the kinase with the element tag labeled non-phosphorylated substrate in the kinase buffer with the support having attached the metal ion coordination complex to form a bound substrate; v) separating the bound substrate from an unbound substrate; vi) eluting the bound substrate, and vii) detecting and measuring the bound substrate by detecting and measuring the element tag by inductively coupled plasma mass spectrometry.
7. The kit of claim 6 wherein instructions are provided for the solution to be analyzed by inductively coupled plasma mass spectrometry after the second incubation without the separating and eluting steps.
8. The kit of claim 6 further comprising instructions for inductively coupled plasma mass spectrometry.
9. A kit of claim 1 wherein
(a) the non-phosphorylated substrate comprises a plurality of non-phosphorylated substrates; and
(b) the support comprises a plurality of supports, each type of support being attached to a different element tag or different combination of element tags.
10. The kit of claim 9 further comprising instructions for i) immobilizing a non-phosphorylated substrate on a support; ii) incubating a kinase in a kinase buffer with a plurality of non-phosphorylated substrates immobilized on a plurality of supports, each type of support being attached to a different element tag or different combination of element tags, iii) incubating a metal ion coordination complex in the kinase buffer with the kinase and the plurality of non-phosphorylated substrates immobilized on the plurality of supports to form a bound substrate, iv) washing and separating the bound substrate from an unbound substrate; v) measuring the metal ion of the metal ion coordination complex bound to the plurality of phosphorylated substrates immobilized on the plurality of supports by inductively coupled plasma mass spectrometry.
11. The kit of claim 9 further comprising a plurality of non-phosphorylated substrates immobilized on a plurality of element tag labeled beads being, each type of bead attached to a different element tag or different combination of element tags.
12. The kit of claim 1 where the support has an attached metal ion coordination complex and the support with the attached metal ion coordination complex is a titanium oxide bead.
13. The kit of claim 1 wherein the transition element is an ion.
14. The kit of claim 1 wherein the transition element includes more than one atom of an isotope.
15. The kit of claim 1 further comprising a substance selected from the group consisting of a standard, a dilution buffer, an elution buffer, a wash buffer, an assay buffer, and combinations thereof.