Protein and antibody profiling using small molecule microarrays

• US 20070003954A1
• Filed: 05/12/2006
• Published: 01/04/2007
• Est. Priority Date: 05/12/2005
• Status: Abandoned Application

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Kodadek, Thomas
• Application Number:
  US11/433,069
• Publication Number:
  US 20070003954A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• 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 33/6854 Immunoglobulins

Abstract

Aspects of the present invention describe methodology by which arrays of synthetic molecules can be created and employed for various types of proteomics profiling experiments. The most important of these from a clinical standpoint are the visualization of antibody and T cell binding patterns, which could be employed as a tool for monitoring the state of the immune system of a patient. This may be a generally useful tool for the diagnosis of many types of disease states. Similar techniques are employed to detect the post-translational modification of specific proteins, a tool for the visualization of induction of signal transduction pathways in cells and tissues treated with drugs. Finally, aspects of the invention teache a method for the creation of simpler arrays with less than 100 features that are, nonetheless, effective for protein profiling experiments.

Claims

1. A method of profiling a plurality of distinct ligand binding moiety in a sample comprising:

(a) providing an array of ligands having a plurality of random structures;

(b) contacting said array with a biological sample comprising ligand binding moiety; and

(c) assessing binding of ligand binding moiety to said array, wherein binding of ligand binding moiety to said array detects ligand binding moieties in said sample.

2. The method of claim 1, wherein said ligand binding moiety is comprised in a body fluid or on a cell surface.

3. The method of claim 2, wherein the cell surface is on a non-adherent cell.

4. The method of claim 3, wherein the cell is a T cell.

5. The method of claim 1, wherein said ligand binding moiety is an antibody.

6. The method of claim 5, wherein assessing binding of the ligand binding moiety to the array features comprises contacting the array with labeled anti-Ig.

7. The method of claim 1, wherein said ligand binding moieties are enzymes.

8. The method of claim 7, wherein assessing comprises labeling the enzymes with fluorescently labeled or otherwise labeled mechanism-based inhibitors or other covalent inhibitors.

9. The method of claim 1, wherein said ligand binding moieties are nucleotide-binding proteins, glycosylated proteins, post-translationaly modified proteins, peptide hormones or ligands.

10. The method of claim 9, wherein assessing comprises detecting fluorescently or otherwise labeled nucleotides or nucleotide analogues, fluorescently or otherwise-labeled sugar-binding molecules, or fluorescently or otherwise-labeled antibodies.

11. The method of claim 1, wherein said random ligands comprise peptides, peptoids, oligonucleotides, oligosaccharides or small molecules.

12. The method of claim 1, wherein said random ligands are preselected based on known reactivity to said ligand binding moieties or a class of ligand binding moieties.

13. The method of claim 12, wherein the profile of binding of said ligand binding moiety is predictive of a disease state in a subject from which said sample was obtained.

14. The method of claim 13, wherein the disease state is selected from the group consisting of cancer, autoimmune disease, inflammatory disease, infectious disease, neurodegenerative disease, or cardiovascular disease.

15. The method of claim 14, wherein profile of binding differentiates between different forms of a disease state.

16. The method of claim 15, wherein the profile of binding differentiates forms of a disease state as mild or aggressive.

17. The method of claim 14, wherein the profile of binding differentiates between a disease state that is or is not responsive to a treatment or therapy.

18. The method of claim 14, wherein the disease state is breast cancer, lung cancer, prostate cancer, cervical cancer, head and neck cancer, testicular cancer, ovarian cancer, skin cancer, brain cancer, pancreatic cancer, liver cancer, stomach cancer, colon cancer, rectal cancer, esophageal cancer, lymphoma, or leukemia, such as antibodies that recognize epitopes unique to these disease states.

19. The method of claim 14, wherein autoimmune disease is lupus, myestenia gravis, multiple sclerosis, narcolepsy, rheumatoid arthritis, nephritis, Chagas disease, scleroderma, or Sjogren'"'"'s disease.

20. The method of claim 14, wherein infection is a result of infection with viruses, bacteria or fungi.

21. The method of claim 14, wherein the neurodegenerative disease is Alzheimer'"'"'s disease, dementia, or Creutzfeld-Jacob disease.

22. The method of claim 1, wherein said random ligands comprise a purely random feature and/or a non-random feature.

23. The method of claim 1, wherein said random ligands are not preselected based on known reactivity to said ligand binding proteins.

24. The method of claim 1, wherein said array comprises between about 1000 and 100,000 distinct random ligands.

25. The method of claim 1, wherein said array comprises between about 2000 and 50,000 distinct random ligands.

26. The method of claim 1, wherein said array comprises between about 4000 and 25,000 distinct random ligands.

27. The method of claim 1, wherein said array comprises between about 6000 and 15,000 distinct random ligands.

28. The method of claim 1, wherein said array comprises between about 7000 and 12,500 distinct random ligands.

29. The method of claim 1, wherein said array comprises between about 8000 and 10,000 distinct random ligands.

30. The method of claim 1, wherein said sample is urine, serum, whole blood, cerebrospinal fluid, sputum, saliva, or semen.

31. The method of claim 1, wherein said array is a microscope slide, plate, a chip, or a population of beads.

32. The method of claim 1, wherein said sample is from a cow, horse, chicken, or human subject.

33. The method of claim 1, further comprising cross-linking said ligand binding moiety to said array.

34. The method of claim 1, further comprising associating a ligand structure with binding to a ligand binding moiety.

35. The method of claim 1, further comprising assessing binding of a control ligand binding moiety to a ligand.

36. A method of profiling the binding of one or more isoforms of a ligand binding moiety in a sample comprising:

(a) providing an array of ligands having a plurality of random structures;

(b) contacting said array with a biological sample comprising one or more isoforms; and

(c) assessing binding of said one or more isoforms to said array,

wherein binding of said one or more isoforms detects said one or more isoforms in said sample.

37. The method of claim 36, wherein a plurality of isoforms are bound to the array, wherein specific isoforms are detected by specific binding patterns.

38. The method of claim 36, wherein said one or more isoforms are enzymes.

39. The method of claim 38, wherein assessing comprises photometric or non-photometric means.

40. The method of claim 36, wherein said one or more isoforms are phosphorylation isoforms, glycosylation isoforms, myristoylation isoforms, length isoforms, amino acid substitution isoforms, ubiquitylation isoforms, SUMOylation isoforms, NEDDylation isoforms, splice variants, methylation isoforms, acetylation isoforms, citrullation isoforms, nitrosylation isoforms, or formylation isoforms.

41. The method of claim 36, wherein said random ligands comprise peptides, peptoids, oligonucleotides, oligosaccharides, amino acid derivatives, or small molecules.

42. The method of claim 36, wherein said random ligands are preselected based on known reactivity to said isoforms.

43. The method of claim 42, wherein the pattern of binding of said one or more isoforms is predictive of a disease state in a subject from which said sample was obtained.

44. The method of claim 42, wherein the pattern of binding of said one or more isoforms is predictive of activation or inhibition of a cellular pathway.

45. The method of claim 36, wherein said random ligands are not preselected based on known reactivity to said one or more isoforms.

46. The method of claim 36, wherein said array comprises between about 1000 and 100,000 distinct random ligands.

47. The method of claim 36, wherein said array comprises between about 2000 and 50,000 distinct random ligands.

48. The method of claim 36, wherein said array comprises between about 4000 and 25,000 distinct random ligands.

49. The method of claim 36, wherein said array comprises between about 6000 and 15,000 distinct random ligands.

50. The method of claim 36, wherein said array comprises between about 7000 and 12,500 distinct random ligands.

51. The method of claim 36, wherein said array comprises between about 8000 and 10,000 distinct random ligands.

52. The method of claim 36, wherein said sample is urine, serum, whole blood, cerebrospinal fluid, sputum, saliva, or semen.

53. The method of claim 36, wherein said array is a plate, a chip, or a population of beads.

54. The method of claim 36, wherein said isoforms are phosphorylation isoforms, glycosylation isoforms, myristoylation isoforms, length isoforms, amino acid substitution isoforms, ubiquitylation isoforms, SUMOylation isoforms, NEDDylation isoforms, splice variants, methylation isoforms, acetylation isoforms, citrullation isoforms, nitrosylation isoforms, or formylation isoforms.

55. The method of claim 36, wherein said sample has been stimulated prior to step (a).

56. The method of claim 55, wherein said sample has been stimulated with a drug.

57. The method of claim 56, wherein said sample comprises cells stimulated in vitro.

58. The method of claim 56, wherein said sample is obtained from a subject suffering from a disease state.

59. The method of claim 55, wherein said sample has been stimulated with an environmental condition.

60. The method of claim 59, wherein said environmental condition is light, heat, cold, sleep deprivation, elevated noise, sound deprivation, light deprivation, or chemical exposure.

61. The method of claim 36, wherein said sample is from a human subject.

62. The method of claim 36, further comprising cross-linking said ligand binding proteins to said array.

63. The method of claim 36, further comprising associating a ligand structure with binding to an isoform.

64. The method of claim 36, further comprising assessing binding of more than one isoform to a single ligand.

65. The method of claim 36, wherein said random ligands comprise a purely random feature and a non-random feature.