Enzyme coupled assay for quantification of protein and peptide binding by samdi mass spectrometry
AssigneeNorthwestern University
InventorsMilan Mrksich, Patrick O'Kane
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Abstract
Disclosed herein are methods of using an immobilized substrate, immobilized ligand, and a fusion protein of an enzyme for the substrate and a receptor for the ligand, where the immobilized substrate can react to form an immobilized product that has a different mass than the immobilized substrate, and using this transformation to indirectly determine the binding of the receptor and the ligand. These methods can be used for high-throughput screening for possible modulators (e.g., inhibitors or activators) of the ligand-receptor interaction.
Claims28
A method comprising
- (a) contacting an immobilized substrate and an immobilized ligand with a fusion protein under conditions to transform the immobilized substrate to an immobilized product, wherein
- the immobilized product has a mass different from the immobilized substrate;
- the fusion protein comprises (1) an enzyme capable of transforming the substrate to the product and (2) a receptor, and
- the immobilized substrate for the enzyme and the immobilized ligand for the receptor are each bound to a self-assembled monolayer (SAM) on a surface;
- (b) subjecting the substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal;
- and
- (c) detecting binding of the receptor and the ligand by correlating a signal intensity of the product to a signal intensity of the substrate to determine the extent of product formation and thereby detecting the binding of the receptor and the ligand,
- wherein the total density of the ligand and the substrate on the surface is less than or equal to about 10%.
3-4. (canceled)
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13-14. (canceled)
18-20. (canceled)
22-26. (canceled)
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The method of claim 25, wherein the ligand is a peptide and comprises a methylated lysine residue.
32-33. (canceled)
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Dependencies are inferred from the claim text.