Microarrays of functional biomolecules, and uses therefor

• US 20060115810A1
• Filed: 09/29/2005
• Published: 06/01/2006
• Est. Priority Date: 08/03/2000
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, Regents of the University of California (University of California)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, Regents of the University of California (University of California)
• Inventors: Marks, James D., Nielsen, Ulrik, Cardone, Michael H., MacBeath, Gavin, Sinskey, Anthony, Sorger, Peter
• Application Number:
  US11/239,652
• Publication Number:
  US 20060115810A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07K 17/06 attached to the carrier via a bridging agent
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 33/5079 Mitochondria
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6851 : Methods of protein analysis involving laser desorption ionisation mass spectrometry

Abstract

Disclosed are products and methods to facilitate the identification of compounds that are capable of interacting with biological macromolecules of interest, especially when such macromolecules are attached to a support surface in microarray. Aspects of the invention concern attachment chemistry, peptide labeling, antibody preparation, applications and so on.

Claims

1-20. -20. (canceled)

21. A method for identifying a small molecule regulator of protein binding, the method comprising the steps of:

(a) attaching a capture protein on a support surface;

(b) exposing said substrate to a ligand for said capture protein and at least one small molecule; and

(c) detecting the presence or the absence of binding between said capture protein and said ligand.

22. The method of claim 21, wherein step (a) comprises attaching said capture protein on a BSA-NHS slide.

23. The method of claim 21, wherein step (a) comprises functionalizing said support surface with aldehyde groups.

24. The method of claim 21, wherein step (a) comprises attaching said capture protein in a microarray of at least 1,000 spots per cm².

25. The method of claim 21, further comprising fusing said capture protein to a GST protein.

26. The method of claim 21, further comprising detecting said binding between said capture protein and said ligand through a fluorescent dye.

27. The method of claim 26, wherein said fluorescent dye comprises a hydrophilic polymer moiety.

28. The method of claim 27, wherein said moiety is a polyethyleneglycol.

29. The method of claim 21, wherein step (c) comprises detecting said binding between said capture protein and said ligand through a labeled phage particle displaying an antibody fragment.

30. The method of claim 21, wherein said ligand comprises a family of related proteins.

31. The method of claim 30, wherein said ligand comprises the Bcl-2 family of proteins.

32. The method of claim 21, wherein said capture protein comprises a family of related proteins.

33. A method for identifying a small molecule that selectively affects a cellular pathway, the method comprising the steps of:

(a) attaching a microarray of capture proteins on a support surface, said microarray comprises proteins that act in a cellular pathway;

(b) exposing said substrate surface to at least one ligand of said capture proteins and at least one small molecule; and

(c) detecting a change in binding between said capture proteins and said ligand, said change resulting from interaction with said small molecule.

34. The method of claim 33, wherein step (c) further comprises using mass spectrometry to quantify said change.

35. The method of claim 33, further comprising detecting said binding between said capture protein and said ligand through a fluorescent dye.

36. The method of claim 35, wherein said fluorescent dye comprises a hydrophilic polymer moiety.

37. The method of claim 36, wherein said moiety is a polyethyleneglycol.

38. The method of claim 33, wherein step (c) comprises detecting said binding between said capture protein and said ligand through a labeled phage particle displaying an antibody fragment.

39. The method of claim 33, wherein step (a) comprises attaching said capture proteins on a BSA-NHS slide.

40. The method of claim 34, wherein step (a) comprises attaching said capture protein in a microarray of at least 1,000 spots per cm².

41-72. -72. (canceled)