Materials and methods for controlling isotope effects during fractionation of analytes

• US 20030186326A1
• Filed: 09/27/2002
• Published: 10/02/2003
• Est. Priority Date: 09/27/2001
• Status: Active Grant

• Current Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Original Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Inventors: Zhang, Roujian, Regnier, Fred E.
• Granted Patent: US 7,449,170 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• 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
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Compositions and methods for controlling or eliminating isotope effects during fractionation of chemically equivalent but isotopically distinct compounds. Isotope coding agents contain heavy isotopes other than deuterium. The invention facilitates intelligent data acquisiton. After sample fractionation, isotope abundance ratios are calculated using mass spectrometry, and analytes of interest are identified in real time.

Claims

1. An isotope coding agent comprising:

a reactive functional group that reacts with an amine, a thiol, a hydroxyl or a carboxyl; and

an isotopic linker comprising at least three heavy non-deuterium isotopes.

2. The isotope coding agent of claim 1 further comprising an affinity functional group for affinity selection.

3. The isotope coding agent of claim 1 wherein each heavy non-deuterium isotope is independently selected from the group consisting of ¹³C, ¹⁸O and ¹⁵N.

4. The isotope coding agent of claim 1 wherein the heavy non-deuterium isotopes are identical.

5. The isotope coding agent of claim 1 comprising at least three ¹³C isotopes.

6. The isotope coding agent of claim 1 comprising at least three ¹⁸O isotopes.

7. The isotope coding agent of claim 1 wherein at least two of the heavy non-deuterium isotopes differ.

8. The isotope coding agent of claim 1 wherein the reactive functional group iodoacetic acid or iodoacetamide and wherein the affinity functional group biotin.

9. An isotope coding agent comprising:

a reactive functional group selected from the group consisting of iodoacetic acid and iodoacetamide;

an isotopic linker having the structural formula C₁₀H₁₇N₃O₃ and comprising about nine ¹³C isotopes; and

a functional group for affinity selection comprising biotin.

10. An isotope coding agent comprising a compound selected from the group consisting of succinic anhydride, N-acetoxysuccinimide and propionate-N-hydroxysuccinimide, wherein the compound comprises at least three heavy non-deuterium isotopes.

11. An isotope coding agent comprising at least three ¹³C isotopes, wherein the isotope coding agent reacts with an amine, a carboxyl, a hydroxyl or a thiol.

12. An isotope coding agent comprising at least three ¹⁸O isotopes, wherein the isotope coding agent reacts with an amine, a carboxyl, a hydroxyl or a thiol.

13. An isotope coding agent comprising at least three ¹⁵N isotopes, wherein the isotope coding agent reacts with an amine, a carboxyl, a hydroxyl or a thiol.

14. A method for making an isotope coding agent comprising incorporating at least three heavy isotopes independently selected from the group consisting of ¹³C, ¹⁸O and ¹⁵N into a compound to yield the ¹³C-, ¹⁸O- and/or ¹⁵N-containing isotope coding agent, wherein a deuterated isoform of the compound is in use as a isotope coding agent in mass spectrometry.

15. The method of claim 14 wherein the isotope effect observed for the ¹³C-, ¹⁸O- and/or ¹⁵N-containing isotope coding agent is smaller than the isotope effect observed for the deuterated isoform.

16. A method for making an isotope coding agent comprising:

identifying a deuterated isotope coding agent; and

synthesizing an isoform of the denterated isotope coding agent, wherein the isoform comprises at least three heavy isotopes independently selected from the group consisting of ¹³C, ¹⁸O and ¹⁵N.

17. The method of claim 16 wherein the isotope effect observed for the ¹³C-, ¹⁸O- or ¹⁵N-containing isotope coding agent is smaller than the isotope effect observed for the deuterated isotope coding agent.

18. A peptide covalently linked to an isotope coding agent, wherein the isotope coding agent comprises at least three ¹³C isotopes.

19. The peptide of claim 18 wherein the isotope coding agent is selected from the group consisting of an ICAT reagent, succinic anhydride, N-acetoxysuccinimide and propionate-N-hydroxysuccinimide.

20. A peptide covalently linked to an isotope coding agent, wherein the isotope coding agent comprises at least three ¹⁸O isotopes.

21. The peptide of claim 20 wherein the isotope coding agent is selected from the group consisting of an ICAT reagent, succinic anhydride, N-acetoxysuccinimide and propionate-N-hydroxysuccinimide.

22. A peptide covalently linked to at least two different isotope coding agents, wherein each isotope coding agent comprises at least one heavy isotope independently selected from the group consisting of ¹³C, ¹⁸O and ¹⁵N.

23. A method for isotopically coding an analyte comprising covalently linking the analyte to an isotope coding agent, wherein the isotope coding agent comprises at least three heavy isotopes independently selected from the group consisting of ¹³C, ¹⁸O and ¹⁵N.

24. A device for detecting a difference in the concentration of an analyte present in a first sample and in a second sample, the device comprising:

a sample fractionator having an outlet;

a mass spectrometer coupled to the outlet of the sample fractionator; and

software for determining the abundance ratio for the analyte using the mass spectrum of a combined sample immediately following elution of isotopically labeled analytes from the sample fractionator.

25. The device of claim 24 wherein the software determines a normalized abundance ratio characterizing analytes whose concentration is the same in the first and second samples and an abundance ratio of the first and second isotopically labeled analytes, and wherein a difference in the abundance ratio of the first and second isotopically labeled analytes and the normalized abundance ratio is indicative of a difference in concentration of the analyte in the first and second samples.

26. The device of claim 24 wherein the abundance ratios are determined in real time such that an eluted fraction comprising analytes that have changed in concentration can be immediately subjected to further analysis using MS-MS.

27. The device of claim 24 wherein the software is designed for proteomics applications.

28. The device of claim 24 wherein the software is designed for blood sample analysis.

29. The device of claim 24 wherein the analytes further analyzed on-line, in real time, using MS-MS.

30. The device of claim 24 wherein the decision of whether to perform MS-MS on a peak is made by the software without input from a human operator.

31. The device of claim 24 wherein the sample fractionator is a chromatography column.

32. The device of claim 24 wherein the sample fractionator is a reverse phase column.

33. A method for using the device of claim 24 comprising:

fractionating a combined sample using the fractionator, wherein each constituent sample comprises an isoform of each of a plurality of analytes, at least one isoform of each analyte comprising an isotope coding reagent comprising at least one non-deuterium heavy atom and having a mass of at least 3 amu greater than the mass of an analyte isoform comprising no heavy isotopes, said fractionation yielding a plurality of elution peaks;

performing MS on each elution peak;

determining the relative abundance ratio in real time of different isoforms of the analyte to detect isoforms exhibiting a change in concentration; and

performing MS-MS on the elution peak comprising the isoforms exhibiting a change in concentration to further analyze said isoforms.

34. The method of claim 33 further comprising identifying the analyte exhibiting the change in concentration.

35. The method of claim 33 wherein the analytes are peptides produced by proteolysis of samples containing proteins, and wherein MS-MS analysis of the elution peak comprising isoforms of peptides exhibiting a change in concentration is used to identify the proteins from which the peptides were derived.

36. The method of claim 33 wherein the isotope coding reagent contains no deuterium atoms.

37. The method of claim 33 wherein the isotope coding reagent comprises at least one heavy isotope selected from the group consisting ¹³C, ¹⁸O and ¹⁵N.

38. The method of claim 33 wherein the different isoforms of an analyte do not resolve during fractionation.

45. The method of claim 38 wherein mass spectrometric analysis is performed using matrix assisted laser desorption ionization (MALDI), electrospray ionization (ESI), fast atom bombardment (FAB), electron impact ionization, atmospheric pressure chemical ionization (APCI), time-of-flight (TOF), quadrapole, ion trap, magnetic sector, ion cyclotron resonance mass, or combinations thereof.

39. A method for detecting a difference in the concentration of an analyte present in a first sample and in a second sample, each sample comprising a plurality of analytes, the method comprising:

covalently attaching a first isoform of a labeling agent to the analyte in the first sample to yield at least one first isotopically labeled analyte;

covalently attaching a second isoform of the labeling agent to the analyte in the second sample to yield at least one second isotopically labeled analyte, wherein the first isoform comprises at least one non-deuterium heavy isotope, and wherein the first and second isoforms differ in mass by at least 3 amu;

mixing at least portions of the first and second samples to yield a combined sample; and

subjecting the combined sample to mass spectrometric analysis to determine a normalized abundance ratio characterizing analytes whose concentration is the same in the first and second samples and an abundance ratio of the first and second isotopically labeled analytes, wherein a difference in the abundance ratio of the first and second isotopically labeled analytes and the normalized abundance ratio is indicative of a difference in concentration of the analyte in the first and second samples.

40. The method of claim 39 wherein the analyte is a protein.

41. The method of claim 39 further comprising fractionating the combined sample using a sample fractionator to yield at least one fraction comprising the first and second isotopically labeled analytes prior to determining the abundance ratios.

42. The method of claim 41 wherein the isotope effect observed during fractionation of a mixture of the first and second isotopically labeled analytes is smaller than the isotope effect observed during fractionation of a mixture comprising the second isotopically labeled analyte and an analyte labeled with a deuterated isoform of the labeling agent.

43. The method of claim 41 wherein fractionating the combined sample comprises subjecting the proteins to single or multi-dimensional reversed phase chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, capillary gel electrophoresis, capillary zone electrophoresis, and capillary electrochromatography, capillary isoelectric focusing, immobilized metal affinity chromatography, affinity electrophoresis or any combination thereof.

44. The method of claim 41 wherein mass spectrometric analysis is performed on the fraction immediately following elution of the fraction from the sample fractionator, and wherein abundance ratios are determined in real time such that an eluted fraction comprising analytes that have changed in concentration can be immediately subjected to further analysis using MS-MS.

46. A method for detecting a difference in the concentration of a protein present in a first sample and in a second sample, each sample comprising a plurality of proteins, the method comprising:

covalently attaching a first isoform of a labeling agent to the protein in the first sample to yield at least one first isotopically labeled protein;

covalently attaching a second isoform of the labeling agent to the protein in the second sample to yield at least one second isotopically labeled protein, wherein the first isoform comprises at least one non-deuterium heavy isotope, and wherein the first and second isoforms differ in mass by at least 3 amu;

cleaving proteins in the first and second samples to yield first and second isotopically labeled peptides in the first and second samples, respectively;

mixing at least portions of the first and second samples to yield a combined sample, wherein mixing is performed before or after fragmentation; and

subjecting the combined sample to mass spectrometric analysis to determine a normalized abundance ratio characterizing peptides derived from proteins whose concentration is the same in the first and second samples and an abundance ratio of the first and second isotopically labeled peptides, wherein a difference in the abundance ratio of the first and second isotopically labeled peptides and the normalized abundance ratio is indicative of a difference in concentration in the first and second samples of a protein from which the peptide is derived.

47. A method for detecting a difference in the concentration of a protein present in a first sample and in a second sample, each sample comprising a plurality of proteins, the method comprising:

cleaving proteins in the first and second samples to yield at least one peptide in each sample;

covalently attaching a first isoform of a labeling agent to a peptide in the first sample to yield at least one first isotopically labeled peptide;

covalently attaching a second isoform of the labeling agent to a peptide in the second sample to yield at least one second isotopically labeled peptide, wherein the first isoform comprises at least one non-deuterium heavy isotope, and wherein the first and second isoforms differ in mass by at least 3 amu;

mixing at least portions of the first and second samples to yield a combined sample; and

subjecting the combined sample to mass spectrometric analysis to determine a normalized abundance ratio characterizing peptides derived from proteins whose concentration is the same in the first and second samples and an abundance ratio of the first and second isotopically labeled peptides, wherein a difference in the abundance ratio of the first and second isotopically labeled peptides and the normalized abundance ratio is indicative of a difference in concentration in the first and second samples of a protein from which the peptide is derived.

48. The method of claim 47 wherein the first and second isoforms of the labeling agent are attached to at least one amino group on peptides in the first and second samples.

49. The method of claim 47 wherein the first and second isotopically labeled peptides comprise at least one affinity ligand, the method further comprising, prior to determining the abundance ratios, contacting the peptides with a capture moiety to select peptides comprising the at least one affinity ligand.

50. The method of claim 47 further comprising identifying the protein from which the detected peptide was derived.

51. The method of claim 47 further comprising fractionating the combined sample using a sample fractionator to yield at least one fraction comprising the first and second isotopically labeled analytes prior to determining the abundance ratios.

52. The method of claim 51 wherein the isotope effect observed during fractionation of a mixture of the first and second isotopically labeled peptides is smaller than the isotope effect observed during fractionation of a mixture comprising the second isotopically labeled peptide and a peptide labeled with a deuterated isoform of the labeling agent.

53. The method of claim 51 wherein fractionating the combined sample comprises subjecting the proteins to single or multi-dimensional reversed phase chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, capillary gel electrophoresis, capillary zone electrophoresis, and capillary electrochromatography, capillary isoelectric focusing, immobilized metal affinity chromatography, affinity electrophoresis or any combination thereof.

54. The method of claim 51 wherein mass spectrometric analysis is performed on the fraction immediately following elution of the fraction from the sample fractionator, and wherein abundance ratios are determined in real time such that an eluted fraction comprising analytes that have changed in concentration can be immediately subjected to further analysis using MS-MS.

55. The method of claim 47 wherein the sample comprises at least about 100 proteins.

56. The method of claim 47 wherein the first sample is obtained from an environment before application of a stimulus and the second sample is obtained from an environment after application of the stimulus.

57. The method of claim 47 wherein the first sample is obtained from an environment in the presence of, or after application of, a stimulus and the second sample is obtained from an environment in the absence of the stimulus.

58. The method of claim 47 wherein the first and second samples are obtained from different organisms, cells, organs, tissues or bodily fluids, the method further comprising determining differences in concentration of at least one protein in the organisms, cells, organs, tissues or bodily fluids from which the samples were obtained.

59. A method for detecting a difference in the concentration of an analyte of interest present in a multiplicity of samples, each sample comprising a plurality of analytes, the method comprising:

for each sample, covalently attaching an isoform of a labeling agent to the analyte of interest to yield an isotopically labeled analyte of interest, wherein different isoforms of the labeling agent are used for each sample, the isoforms differ from one another in mass by at least 3 amu, and all but at most one isoform comprise at least one non-deuterium heavy isotope;

mixing at least portions of the samples to yield a combined sample; and

subjecting the combined sample to mass spectrometric analysis to determine a normalized abundance ratio characterizing analytes whose concentration is the same in the samples and an abundance ratio of at least one pair of the isotopically labeled analytes of interest, wherein a difference in the abundance ratio of the isotopically labeled analytes of interest and the normalized abundance ratio is indicative of a difference in concentration of the analyte of interest in the samples.

60. The method of claim 59 wherein the analyte of interest is present in about 3 to about 5 samples.

61. A method for detecting a difference in the concentration of a protein of interest present in a multiplicity of samples, each sample comprising a plurality of proteins, the method comprising:

cleaving the proteins in the samples to yield at least one peptide of interest derived from the protein of interest;

for each sample, covalently attaching an isoform of a labeling agent to the peptide of interest to yield an isotopically labeled peptide of interest, wherein different isoforms of the labeling agent are used for each sample, the isoforms differ from one another in mass by at least 3 amu, and all but at most one isoform comprise at least one non-deuterium heavy isotope;

mixing at least portions of the samples to yield a combined sample; and

subjecting the combined sample to mass spectrometric analysis to determine a normalized abundance ratio characterizing peptides derived from proteins whose concentration is the same in the first and second samples and an abundance ratio of at least one pair of the isotopically labeled peptides of interest, wherein a difference in the abundance ratio of the isotopically labeled peptides of interest and the normalized abundance ratio is indicative of a difference in concentration in the samples of a protein from which the peptide is derived.

62. The method of claim 61 wherein the protein of interest is present in about 3 to about 5 samples.

63. A method for identifying isoforms of an analyte, the method comprising:

fractionating a combined sample, wherein each constituent sample comprises an isoform of each of a plurality of analytes, at least one isoform of each analyte comprising an isotope coding reagent comprising at least one non-deuterium heavy atom and having a mass of at least 3 amu greater than the mass of an analyte isoform comprising no heavy isotopes, and wherein the isoforms of the analyte do not resolve during fractionation, said fractionation yielding a plurality of elution peaks;

performing MS on each elution peak to detect isoforms of the analyte.