Mass labels

• US 20050048489A1
• Filed: 09/16/2002
• Published: 03/03/2005
• Est. Priority Date: 09/14/2001
• Status: Active Grant

• Current Assignee: Electrophoretics Limited (Proteome Sciences, PLC)
• Original Assignee: Electrophoretics Limited (Proteome Sciences, PLC)
• Inventors: Schwarz, Joseph, Kuhn, Karsten, Thompson, Andrew, Neumann, Thomas, Hamon, Christian, Schafer, Jurgen
• Granted Patent: US 7,732,378 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• 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: H01J 49/04 Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components

Abstract

Provided is a set of two or more mass labels, each label in the set comprising a mass marker moiety attached via a cleavable linker having at least one amide bond to a mass normalisation moiety, wherein the aggregate mass of each label in the set may be the same or different and the mass of the mass marker moiety of each label in the set may be the same or different, and wherein in any group of labels within the set having a mass marker moiety of a common mass each label has an aggregate mass different from all other labels in that group, and wherein in any group of labels within the set having a common aggregate mass each label has a mass marker moiety having a mass different from that of all other mass marker moieties in that group, such that all of the mass labels in the set are distinguishable from each other by mass spectromety, and wherein the mass marker moiety comprises an amino acid and the mass normalisation moiety comprises an amino acid.

Claims

1. A set of two or more mass labels, each label in the set comprising a mass marker moiety attached via a cleavable linker having at least one amide bond to a mass normalisation moiety, wherein the aggregate mass of each label in the set may be the same or different and the mass of the mass marker moiety of each label in the set may be the same or different, and wherein the set comprises a group of labels having a mass marker moiety of a common mass or the set comprises a group of labels having a common aggregate mass, and wherein in any group of labels within the set having a mass marker moiety of a common mass each label has an aggregate mass different from all other labels in that group, and wherein in any group of labels within the set having a common aggregate mass each label has a mass marker moiety having a mass different from that of all other mass marker moieties in that group, such that all of the mass labels in the set are distinguishable from each other by mass spectrometry, and wherein the mass marker moiety comprises an amino acid and the mass normalisation moiety comprises an amino acid.

2. A set of mass labels according to claim 1, in which each label in the set comprises a mass marker moiety having a common mass and each label in the set has a unique aggregate mass.

3. A set of mass labels according to claim 1, in which each label in the set comprises a mass marker moiety having a unique mass and each label in the set has a common aggregate mass.

4. A set of mass labels according to claim 3, in which each mass marker moiety in the set has a common basic structure, and each mass normalisation moiety in the set has a common basic structure that may be the same or different from the common basic structure of the mass marker moieties, and wherein each mass label in the set comprises one or more mass adjuster moieties, the mass adjuster moieties being attached to or situated within the basic structure of the mass marker moiety and/or the basic structure of the mass normalisation moiety, such that every mass marker moiety in the set comprises a different number of mass adjuster moieties and every mass label in the set has the same number of mass adjuster moieties.

5. A set of mass labels according to claim 4, each mass label in the set having the following structure:

M(A)_y-L-X(A)_z

wherein M is a mass normalisation moiety comprising an amino acid, X is a mass marker moiety comprising an amino acid, A is a mass adjuster moiety, L is a cleavable linker comprising the amide bond, y and z are integers of 0 or greater, and y+z is an integer of 1 or greater.

6. A set of mass labels according to claim 4, wherein the mass adjuster moiety is selected from:

(a) an isotopic substituent situated within the basic structure of the mass marker moiety and/or within the basic structure of the mass normalisation moiety, and

(b) substituent atoms or groups attached to the basic structure of the mass marker moiety and/or attached to the basic structure of the mass normalisation moiety.

7. A set of mass labels according to claim 6, wherein the mass adjuster moiety is selected from a halogen atom substituent, a methyl group substituent, and 2H or 13C isotopic substituents.

8. A set of mass labels according to claim 7, wherein the mass adjuster moiety is a fluorine atom substituent.

9. A set of mass labels according to claim 1, wherein the cleavable linker attaching the mass marker moiety to the mass normalisation moiety is a linker cleavable by collision induced dissociation.

10. A set of mass labels according to claim 1, wherein the cleavable linker comprises proline and/or aspartic acid.

11. A set of mass labels according to claim 1, wherein the mass marker moiety and/or the mass normalisation moiety comprises a fragmentation resistant group.

12. A set of mass labels according to claim 1, wherein the mass marker moiety comprises a sensitivity enhancing group, such as a pre-ionised group.

13. A set of mass labels according to claim 1, wherein the mass marker moiety or the mass normalisation moiety comprises a reactive functionality.

14. A set of mass labels according to claim 1, wherein each mass label in the set comprises an affinity capture ligand, such as biotin.

15. A set of two or more analytes, each analyte in the set being different and being attached to a unique mass label or a unique combination of mass labels, from a set of mass labels as defined in claim 1.

16. A set of analytes according to claim 15, wherein one or more analytes in the set is a standard analyte having a known mass, or known chromatographic properties.

17. A set of two or more probes, each probe in the set being different and being attached to a unique mass label or a unique combination of mass labels, from a set of mass labels as defined in claim 1.

18. A set of analytes or probes according to claim 15, wherein each analyte or probe is attached to a unique combination of mass labels, each combination being distinguished by the presence and absence of each mass label in the set of mass labels and/or the quantity of each mass label attached to the probe.

19. A set of analytes or probes according to claim 15, wherein each analyte or probe comprises a biomolecule.

20. A set of analytes or probes according to claim 19, wherein the biomolecule is selected from a DNA, an RNA, an oligonucleotide, a nucleic acid base, a protein and/or an amino acid.

21. A method of analysis, which method comprises detecting an analyte by identifying by mass spectrometry a mass label or a combination of mass labels relatable to the analyte, wherein the mass label is a mass label from a set of mass labels as defined in claim 1.

22. A method according to claim 21, wherein the mass labels employed are labels comprising an affinity capture ligand, and labelled analytes are separated from unlabelled analytes by capturing the affinity capture ligand with a counter ligand.

23. A method according to claim 21, in which two or more analytes are detected by simultaneously identifying their mass labels or combinations of mass labels by mass spectrometry.

24. A method according to claim 21, wherein each analyte is identified by a unique combination of mass labels from a set or array of mass labels, each combination being distinguished by the presence and absence of each mass label in the set or array and/or the quantity of each mass label.

25. A method according to claim 21, for identifying two or more analytes, wherein the analytes are separated according to their mass, prior to detecting their mass labels by mass spectrometry.

26. A method according to claim 25, wherein the analytes to be identified are mixed with one or more standard analytes, having known mass or known properties in the separation method used, to facilitate the characterisation of the analytes.

27. A method according to claim 26, wherein the standard analytes are as defined in claim 16.

28. A method according to claim 25, wherein separation is carried out by a chromatographic or electrophoretic method.

29. A method according to claim 21, wherein the mass spectrometer employed to detect the mass label comprises one or more mass analysers, which mass analysers are capable of allowing ions of a particular mass, or range of masses, to pass through for detection and/or are capable of causing ions to dissociate.

30. A method according to claim 29, wherein ions of a particular mass or range of masses specific to one or more known mass labels are selected using the mass analyser, the selected ions are dissociated, and the dissociation products are detected to identify ion patterns indicative of the selected mass labels.

31. A method according to claim 29, wherein the mass spectrometer comprises three quadrupole mass analysers.

32. A method according to claim 31, wherein a first mass analyser is used to select ions of a particular mass or mass range, a second mass analyser is used to dissociate the selected ions, and a third mass analyser is used to detect resulting ions.

33. A method according to comprising:

(a) contacting one or more analytes with a set of probes, wherein the probes are as defined in claim 17,

(b) identifying an analyte, by detecting a probe relatable to that analyte.

34. A method according to claim 33, wherein the mass label is cleaved from the probe prior to detecting the mass label by mass spectrometry.

35. A method according to claim 33, which method comprises contacting one or more nucleic acids with a set of hybridisation probes.

36. A method of analysis comprising using mass label from a set of labels as defined in claim 1, in a method of analysis.

37. The method according to claim 36 in a method of 2-dimensional electrophoretic analysis.

38. The method according to claim 36 in a method of 2-dimensional mass spectrometric analysis.

39. The method according to claim 36 in a method of sequencing one or more nucleic acids.

40. The method according to claim 36 in a method of gene expression profiling.

41. The method according to claim 36 in a method of protein expression profiling.

42. The method according to claim 36 in a method of nucleic acid sorting.