# US 2016/0252501 A1 · Enzyme coupled assay for quantification of protein and peptide binding by samdi mass spectrometry > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2016/0252501 A1 - Document ID: US20160252501A1 - Status: Published application - Kind: A1 - Application number: 15/051,338 - Filed: 2016-02-23 - Published: 2016-09-01 - Earliest claimed: 2015-02-23 - Assignees: Northwestern University - Applicants: Northwestern University - Inventors: Milan Mrksich; Patrick O'Kane - Web page: https://patentgrep.com/patent/US20160252501A1 ## CPC - G01N 33/54306: {Solid-phase reaction mechanisms} - G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)} - G01N 2333/47: Assays involving proteins of known structure or function as defined in the subgroups - G01N 2333/98: acting on amide bonds in linear amides (3.5.1) - G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 1 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%. ### Claim 2 (depends on 1) The method of claim 1, wherein the total density of the ligand and the substrate on the surface is from about 3% to about 7% or from about 5% to about 6%. ### Claim at position 3 3-4. (canceled) ### Claim 5 (depends on 1) The method of claim 1, wherein extent of product formation in the presence of the fusion protein is greater than extent of product formation in the presence of the enzyme not fused to the receptor. ### Claim 6 (canceled) ### Claim 7 (depends on 1) The method of claim 1, wherein at least one of the substrate and ligand is bound to the surface via a linker. ### Claim 8 (depends on 7) The method of claim 7, wherein the linker has a structure of formula I: wherein L is the substrate or ligand. ### Claim 9 (depends on 8) The method of claim 8, wherein L is attached to the pyrrolindinone of formula (I) via a thiol bond. ### Claim 10 (depends on 7) The method of claim 7, wherein the linker is attached to the SAM via an ethylene glycol moiety. ### Claim 11 (depends on 1) The method of claim 1, wherein the SAM is attached to the surface via a thiol bond. ### Claim 12 (depends on 1) The method of claim 1, wherein the surface comprises gold, silver, or copper. ### Claim at position 12 13-14. (canceled) ### Claim 15 (depends on 1) The method of claim 1, wherein at least one of the substrate and the ligand comprises a peptide. ### Claim 16 (depends on 15) The method of claim 15, wherein the peptide is bound to the SAM via a cysteine residue. ### Claim 17 (depends on 1) The method of claim 1, wherein the enzyme is a deacetylase, acetyltransferase, esterase, phosphorylase/kinase, phosphatase, protease, methylase, demethylase, or a DNA or RNA modifying enzyme. ### Claim at position 16 18-20. (canceled) ### Claim 21 (depends on 17) The method of claim 17, wherein the substrate comprises an acylated peptide and the product comprises a deacylated peptide, the substrate comprises a phosphorylated peptide and the product comprises a dephosphorylated peptide, or the substrate comprises a methylated peptide and the product comprises a demethylated peptide. ### Claim at position 18 22-26. (canceled) ### Claim 27 (depends on 1) The method of claim 1, wherein the receptor is a chromodomain, a bromodomain, a PDZ domain, a Src homology 2 (SH2) domain SH2 domain, or a PDZ domain. ### Claim 28 (canceled) ### Claim 29 (depends on 25) The method of claim 25, wherein the ligand is a peptide and comprises a methylated lysine residue. ### Claim 30 (depends on 1) The method of claim 1, wherein the substrate density on the surface is greater than the ligand density on the surface. ### Claim 31 (depends on 1) The method of claim 1, wherein the ligand has a dissociation constant (KD) of about 10 pM to about 100 μM, about 100 nM to about 10 μM, or less than 10 μm. ### Claim at position 24 32-33. (canceled) ### Claim 34 (depends on 1) The method of claim 1, further comprising contacting the immobilized substrate and the immobilized ligand with the fusion protein in the presence of one or more potential modulators of the receptor-ligand binding; subjecting the substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal; and 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 in the presence of the one or more potential modulators. ### Claim 35 (depends on 34) The method of claim 34, wherein the modulator is an inhibitor of the receptor-ligand binding. ### Claim 36 (canceled) ### Claim 37 (depends on 1) The method of claim 1, further comprising admixing a matrix to the substrate and product prior to subjecting the substrate and product to mass spectrometry.