Claims
- 1. A method of analyzing the activity or level of one or more protein or enzyme, said method comprising:
(a) providing a pool of substrates (peptides, antibodies, binding domains, other molecules acting as substrates or control substrates) each with a specific tag and representing a substrate of one or more of said proteins or enzymes, or substrates derived therefrom using said tagged substrates as substrates; (b) hybridizing said pool of tagged substrates to an ordered array of specific and complementary tags immobilized on a surface, said array comprising more different tags, at least some of which comprise control tags, wherein each tag is localized in a predetermined region of said surface, the density of said different tags is greater than about 100 different tags per 1 cm.sup.2, and all tags in the substrates derived therefrom using said proteins or enzymes are complementary to at least some of the immobilized tags; (c) quantifying the hybridization of said substrates tagged with nucleic acids or PNAs to said array, wherein said quantification is proportional to the activity of proteins or enzymes modifying or attaching to the substrates tagged with nucleic acids or PNAs.
- 2. The method of claim 1, wherein said pool of substrates each tagged with a single tag comprises substrates tagged with nucleic acids or PNAs and wherein said ordered array of specific and complementary tags immobilized on a surface comprises ordered array of specific and complementary nucleic acids or PNAs immobilized on a surface .
- 3. The method of claim 2, wherein said quantifying comprises calculating the difference in hybridization signal intensity between each of said substrates tagged with a single nucleic acid or PNA and its corresponding related elements.
- 4. The method of claim 3, wherein said quantifying comprises calculating the average difference in hybridization signal intensity between each of said substrates tagged with a single nucleic acid or PNA and its corresponding control substrate for each protein or enzyme, where the control substrate has either an identical tag or a different tag.
- 5. The method of claim 1, wherein said multiplicity of substrates tagged with a nucleic acids or PNAs is 100 or more.
- 6. The method of claim 1, wherein for each said protein or enzyme, said array comprises at least 8 different substrates tagged with a nucleic acids or PNAs acting as substrates.
- 7. The method of claim 1, wherein said hybridization is performed with a fluid volume of about 200 .mu.l or less.
- 8. The method of claim 1, wherein said quantifying comprises detecting a hybridization signal that is proportional to the concentration of modified substrates tagged with a nucleic acids or PNAs in said tagged substrates pool.
- 9. The method of claim 1, wherein said substrates nucleic acid or PNA tags are at least 21 nucleotides in length.
- 10. The method of claim 1, wherein said control substrates comprise either premodified substrates or substrates which are substrates of constitutively expressed control proteins or enzymes.
- 11. The method of claim 10, wherein said tagged substrates include GST-Pin1, GST-14-3-3, GSTFyn SH2, GST-p85, GST-Shc PTB, GST-Shc SH2 and GST-Grb2, and said control substrates are selected from the group consisting of substrates for protein kinase C alpha., protein kinase C .beta.1 , protein kinase C .beta.2, protein kinase C .gamma. phosphatidylinositol 3-kinase alpha., phosphatidylinositol 3-kinase beta., phosphatidylinositol 3-kinase C2 .beta., phosphatidylinositol 3-kinase C2 gamma, src, abl, PDGF receptor.
- 12. The method of claim 1, wherein said hybridization comprises a hybridization at low stringency of 42.degree. C. to 54.degree. C. and 3× TBST and a wash at higher stringency.
- 13. The method of claim 1, wherein said pool of substrates each tagged with a single nucleic acid or PNA comprises fluorescently labeled substrates.
- 14. The method of claim 1, wherein said quantifying comprises quantifying fluorescence of a label on said hybridized tagged substrate at a spatial resolution of about 100 .mu.m or higher.
- 15. The method of claim 1, wherein said providing comprises:
(i) treating said pool of tagged substrates with protein or enzyme samples, thereby modifying the tagged substrates and leaving intact the tag single stranded component of each tagged substrate; (ii) isolating the tagged substrates pool thereby leaving a pool of substrates modified by those protein or enzymes present and active in the protein or enzyme sample.
- 16. A method of analyzing the activity of one or more protein or enzyme, said method comprising:
(a) providing a pool of molecules (peptides, antibodies, binding domains, other molecules acting as substrates or control substrates) and representing a substrate of one or more of said proteins or enzymes, or substrates derived therefrom; (b) reacting said pool of molecules to an array of proteins, peptides, or other non DNA molecules, immobilized on a surface, wherein each different protein, peptide, or other non DNA molecule is localized in a predetermined region of said surface, the density of said different proteins, peptides, or other molecules, is greater than about 60 different oligonucleotides per 1 cm.sup.2,; (c) quantifying the reactivity of said array, wherein said quantification is proportional to the activity of proteins or enzymes modifying or attaching to the substrates tagged with nucleic acids or PNAs.
- 17. The method of claim 16, wherein said pool of molecules further comprises the same substrate for more than one different element in the said array.
- 18. The method of claim 17, wherein said quantifying comprises calculating the difference in signal intensity between each of said array elements.
- 19. The method of claim 18, wherein said quantifying comprises calculating the average difference in signal intensity between each of said array element and its corresponding control substrate for each protein or enzyme.
- 20. The method of claim 16, wherein said multiplicity of array elements is 100 or more.
- 21. The method of claim 16, wherein for each said protein or enzyme, said array comprises at least different reactive elements.
- 22. The method of claim 16, wherein said hybridization is performed with a fluid volume of about 200 .mu.l or less.
- 23. The method of claim 16, wherein said quantifying comprises detecting a hybridization signal that is proportional to reacted array element.
- 24. The method of claim 16, wherein said control substrates comprise either premodified substrates or substrates which are substrates of constitutively expressed control proteins or enzymes.
- 25. The method of claim 24, wherein said tagged substrates include GST-Pin1, GST-14-3-3, GST-Fyn SH2, GST-p85, GST-Shc PTB, GST-Shc SH2 and GST-Grb2, and said control substrates are selected from the group consisting of substrates for protein kinase C alpha., protein kinase C beta. 1 , protein kinase C .beta.2, protein kinase C gamma. phosphatidylinositol 3-kinase .alpha., phosphatidylinositol 3-kinase .beta., phosphatidylinositol 3-kinase C2 beta., phosphatidylinositol 3-kinase C2 gamma, src, abl, PDGF receptor.
- 26. The method of claim 16, wherein said reacting comprises a reaction at 4.degree. C. to 37.degree. C. and 1.5× TBST and a wash at 2xTBST.
- 27. The method of claim 16, wherein said pool of molecules comprises fluorescently labeled molecules.
- 28. The method of claim 16, wherein said quantifying comprises quantifying fluorescence of a label on said reacted substrate at a spatial resolution of about 100 .mu.m or higher.
Parent Case Info
[0001] This application claims the priority of U.S. Provisional Application No. 60/174,171, Methods for the Detection of Modified Peptides, Proteins, and other Biological Molecules, filed Jan. 3, 2000, which is incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60174171 |
Jan 2000 |
US |