Claims
- 1. A method for comparing the relative abundance of a protein of interest in multiple samples of biological matter comprising:
culturing a first sample of biological matter in a first medium containing a natural abundance of isotopes; culturing a second sample of biological matter in a second medium, wherein at least one isotope in the second medium has a different abundance than the abundance of the same isotope in the first medium; modulating one of the samples; combining at least a portion of each of the samples; removing at least one protein from the combined samples; subjecting the at least one removed protein to mass spectroscopy to develop a mass spectrum; computing a ratio between the peak intensities of at least one pair of closely spaced peaks; determining the relative abundance of the protein in each sample based on the at least one computed ratio; and identifying the protein.
- 2. The method of claim 1, further comprising identifying the protein by electrophoresis, antibodies, bioassay or from the mass spectrum.
- 3. The method of claim 1, wherein the removing step comprises extracting proteins from the combined cell pool, the method further comprising separating the extracted proteins by a process chosen from the group consisting of one-dimensional electrophoresis, two-dimensional electrophoresis, ultra-centrifugation, chromatography and affinity binding.
- 4. The method of claim 3, wherein the separating step comprises:
subjecting the extracted proteins to electrophoresis in a gel; staining the gel; removing gel spots of interest; and destaining the gel spots of interest; the method further comprising digesting the proteins in the destained gel spots.
- 5. The method of claim 1, further comprising digesting the at least one protein of interest.
- 6. The method of claim 1, further comprising computing ratios of the peak intensities of a plurality of pairs of closely spaced peaks of the mass spectrum.
- 7. The method of claim 1, further comprising:
removing a plurality of proteins of interest from the combined cell pool; digesting the plurality of proteins of interest into a plurality of peptides; subjecting the digested proteins to mass spectroscopy to develop the mass spectrum; selecting a plurality of pairs of closely spaced peaks on the mass spectrum; computing the ratio of the intensities of the peaks in each pair; determining the protein from which the pair of peaks in the mass spectrum are derived based on the mass spectrum; and determining the relative abundance of the protein in each cell pool.
- 8. The method of claim 1, further comprising determining the relative quantity of a modified protein of interest in each cell pool.
- 9. The method of claim 8, further comprising determining the site of the modification on the protein.
- 10. The method of claim 1, wherein the modulating step comprises subjecting the biological matter in the one sample to an environmental or chemical stimulus.
- 11. The method of claim 1, wherein the modulating step comprises genetically manipulating the biological matter in the one sample.
- 12. The method of claim 11, wherein the genetic manipulation comprises performing gene therapy.
- 13. The method of claim 1, wherein the modulating step comprises subjecting one of the cell pools to a drug or hormone.
- 14. The method of claim 1, wherein the modulating step comprises subjecting the cell pool to a treatment chosen from the group consisting of a virus, a bacteria and a carcinogen.
- 15. The method of claim 1, wherein the protein is a marker for the effect of the modulating step on a biological process.
- 16. The method of claim 1, comprising culturing the second sample in a second medium in which the at least one isotope is non-radioactive.
- 17. The method of claim 1, comprising culturing the second sample in a second medium enriched in the at least one isotope.
- 18. The method of claim 1, comprising culturing the second sample in a second medium depleted in the at least one isotope.
- 19. The method of claim 1, wherein the biological matter is chosen from the group consisting of biological cells, biological tissue, an organ, an organism, a collection of organisms, a portion of an organism, and a cell-free biological mimetic system.
- 20. The method of claim 1, comprising culturing the second sample in a second medium wherein at least one isotope chosen from the group consisting of nitrogen-15, cabon-13, oxygen-17, oxygen-18, sulfur 34 and hydrogen-2, has a different abundance than the abundance of the same isotope in the first medium.
- 21. The method of claim 20, comprising culturing the second sample in a second medium enriched to at least about 90% in nitrogen-15.
- 22. A method for comparing the relative abundance of a peptide of interest derived from the same protein from multiple samples of biological matter comprising:
culturing a first cell pool in a first medium containing a natural abundance of isotopes; culturing a second cell pool in a second medium isotopically enriched in at least one non-radioactive isotope; modulating one of the cell pools; combining at least a portion of each of the cell pools; extracting the proteins from the combined cell pools; separating the extracted proteins into a plurality of proteins; digesting at least one protein of interest from the separated proteins into a plurality of peptides; subjecting the digested peptides to mass spectroscopy to develop a mass spectrum; and determining the relative abundance of the peptide of interest from each sample based on the mass spectrum.
- 23. The method of claim 22, further comprising identifying the protein from which the peptide is derived and determining the relative quantity of the protein in each cell pool.
- 24. The method of claim 22, further comprising determining the relative occurrence of a modification of the peptide of interest in each cell pool.
- 25. The method of claim 24, wherein the modification of the peptide is chosen from the group consisting of the phosphorylation, glycosylation, and acylation of the peptide, the determining step comprising determining the difference in the relative abundance of the modified peptide in each cell pool.
- 26. The method of claim 24, further comprising determining the level of modification of the peptide.
- 27. The method of claim 24, wherein the type of peptide modification is chosen from the group consisting of the phosphorylation, glycosylation, and acylation of the peptide, the determining step comprising determining the difference in the relative abundance of the modified peptide in each cell pool.
- 28. The method of claim 22, wherein the separating step comprises subjecting the extracted proteins to a process chosen from the group consisting of one-dimensional electrophoresis, two-dimensional electrophoresis, ultra-centrifugation, chromatography and affinity bonding.
- 29. The method of claim 28, wherein the separating step comprises:
subjecting the extracted proteins to electrophoresis in a gel; staining the gel; removing gel spots of interest; and destaining the gel spots of interest; and the digesting step comprises digesting the destained gel spots of interest.
- 30. The method of claim 29, wherein the digesting step comprises mixing the destained gel spots of interest with a proteolytic enzyme.
- 31. The method of claim 30, wherein the proteolytic enzyme is trypsin.
- 32. The method of claim 29, comprising culturing the second cell pool in a medium enriched in an isotope chosen from the group consisting of nitrogen-15, carbon-13, oxygen-17, oxygen-18, sulfur-34 and hydrogen-2.
- 33. A method for comparing the relative abundance of an ionizable component of biological matter from multiple samples, comprising:
culturing a first sample of the biological matter in a first medium; culturing a second sample of the biological matter in a second medium, wherein at least one isotope in the second medium has a different abundance than the abundance of the same isotope in the first medium; modulating the biological matter in one of the samples; combining at least a portion of each of the samples; subjecting at least a portion of the combined sample to mass spectroscopy to develop a mass spectrum; and determining the relative abundance of at least one component of interest in each sample based on the mass spectrum.
- 34. The method of claim 33, wherein the at least one component of interest is chosen from the group consisting of a protein, a peptide, a nucleic acid, a carbohydrate, a lipid, a co-factor and post-synthetic derivatives thereof.
- 35. The method of claim 33, wherein the biological matter is chosen from the group consisting of biological cells, biological tissue, an organ, an organism, a collection of organisms, a portion of an organism, and a cell-free biological mimetic system.
- 36. The method of claim 33, wherein the determining step comprises computing ratios of the peak intensities of at least one pair of closely spaced peaks corresponding to the at least one component of interest of the mass spectrum.
- 37. The method of claim 33, further comprising culturing a third sample of the biological matter in a third medium, wherein at least one isotope has a different abundance than the abundance of the same isotope in the first and second media, and modulating the third sample by a different modulation than the modulation in the first modulating step.
- 38. The method of claim 33, wherein the determining step comprises computing ratios of the peak intensities of at least one pair of closely spaced peaks corresponding to the at least one component of interest of the mass spectrum.
- 39. The method of claim 33, comprising culturing the first sample in a first medium containing a natural abundance of isotopes.
- 40. A method for comparing the relative abundance of a cellular component of interest in multiple cell pools, wherein the cellular component is ionizable, the method comprising:
culturing a first cell pool in a first medium; culturing a second cell pool in a second medium, wherein at least one isotope in the second medium has a different abundance than the abundance of the same isotope in the first medium; modulating one of the cell pools; combining at least a portion of each of the cell pools; removing at least one component of interest from the combined cell pool; subjecting the at least one removed component of interest to mass spectroscopy to develop a mass spectrum; computing ratios of the peak intensities of at least one pair of closely spaced peaks corresponding to the at least one component of interest of the mass spectrum; and identifying the component of interest.
- 41. The method of claim 40, wherein the cellular component of interest is chosen from the group consisting of a protein, a peptide, a nucleic acid, a carbohydrate, a lipid, a cofactor, and post-synthetic derivatives thereof.
- 42. The method of claim 41, wherein the component of interest is an excreted by-product of the biological matter, the method further comprising removing the biological matter from the combined cell pool and subjecting at least a portion of the medium of the combined sample to mass spectroscopy.
- 43. A method for comparing the relative abundance of biological matter from two animal subjects, wherein the biological matter can be analyzed by mass spectroscopy, comprising:
feeding a first animal food; feeding a second animal food including at least one isotope having a different abundance than the same isotope in the food fed the first animal; withdrawing a sample of biological matter from the first animal; withdrawing a sample of biological matter from the second animal; combining at least a portion of the withdrawn biological matter from the first and second animals; subjecting the combined biological matter to mass spectroscopy to develop a mass spectrum; and determining the relative abundance of at least one component of interest of the combined biological matter based on the mass spectrum.
- 44. The method of claim 43, comprising feeding a second animal from a species different than the species of the first animal.
- 45. The method of claim 43, comprising providing first and second human subjects.
- 46. A method for comparing the relative abundance of biological matter from a single animal subject at different times, wherein the biological matter can be analyzed by mass spectroscopy, comprising:
withdrawing a first sample of biological matter from the subject; feeding the subject food including at least one isotope having a different abundance than the same isotope in the food fed the first animal, after withdrawing the first sample; withdrawing a sample of biological matter from the subject after feeding; mixing at least portions of the first and second samples; subjecting the combined samples to mass spectroscopy to develop a mass spectrum; and determining the relative abundance of at least one component of interest of the combined biological matter based on the mass spectrum.
- 47. The method of claim 46, comprising providing a human subject.
- 48. The method of claim 46, further comprising withdrawing at least one subsequent sample of biological matter from the subject a period of time after withdrawing the second sample, mixing at least a portion of the at least one subsequent sample with a portion of the first sample and then performing the subjecting and determining steps to monitor the metabolism of the food with time.
Government Interests
[0001] STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
[0002] The U.S. Government has certain rights to the invention, by virtue of its partial support of research under National Institute of Health Grant Nos. RR00862 (BTC), GM47021 and GM49716 (FRC).
Divisions (1)
|
Number |
Date |
Country |
Parent |
09304799 |
May 1999 |
US |
Child |
09949510 |
Sep 2001 |
US |