Sets and Compositions Pertaining to Analyte Determination

• US 20070141659A1
• Filed: 02/12/2007
• Published: 06/21/2007
• Est. Priority Date: 01/30/2003
• Status: Active Grant

• Current Assignee: DH Technologies Pte. Ltd.
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Bartlet-Jones, Michael, Pappin, Darryl
• Granted Patent: US 7,947,513 B2
• Time in Patent Office: Days
• US Class Current: 435/23
• CPC Class Codes: A61K 51/04 Organic compounds
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• 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 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: Y10T 436/142222 Hetero-O [e.g., ascorbic acid, etc.]
• CPC Class Codes: Y10T 436/145555 Hetero-N
• CPC Class Codes: Y10T 436/147777 Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

This invention pertains to methods, kits and/or compositions for the determination of analytes by mass analysis using unique labeling reagents or sets of unique labeling reagents. The labeling reagents can be isomeric or isobaric and can be used to produce mixtures suitable for multiplex analysis of the labeled analytes.

Claims

1. A set of two or more reagent compounds, or a salt thereof, of the formula RP-X-LK-Y-RG, wherein:

i) RG is a reactive group that is a nucleophile or an electrophile and that is capable of reacting with a functional group of one or more reactive analytes of a sample;

ii) RP is a reporter moiety that comprises a fixed charge or that is ionizable, wherein the gross mass of each reporter moiety is different for each reagent of the set;

iii) LK is a linker moiety that links the reactive group to the reporter moiety, wherein the mass of the linker compensates for the difference in gross mass between the reporter moieties for the different labeling reagents of the set such that the aggregate gross mass of the reporter and linker combination is the same for each reagent of the set;

iv) X is a bond between an atom of the reporter moiety and an atom of the linker moiety;

v) Y is a bond between an atom of the linker moiety and an atom of the reactive group, wherein, once the labeling reagent is reacted with the reactive analyte, bond Y links the linker to the analyte; and

vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels.

2. The compound set of claim 1, wherein the reporter moiety, RP:

i) has a gross mass of less than 250 daltons; and

/or

ii) does not substantially sub-fragment under conditions of dissociative energy applied to cause fragmentation of at least a portion of both bonds X and Y of a labeled analyte in a mass spectrometer; and

/or

iii) is not a polymer or is not a biological polymer.

3. The compound set of claim 1, wherein the linker moiety, LK, undergoes neutral loss under conditions of applied dissociative energy.

4. The compound set of claim 1, wherein under conditions of dissociative energy applied in a mass spectrometer, the fragmentation of one of bonds X or Y of the reagents results in the fragmentation of the other of bonds X or Y.

5. The compound set of claim 1, wherein:

i) under conditions of dissociative energy applied in a mass spectrometer, bond X of the reagents is less prone to fragmentation as compared with bond Y; and

/or

ii) under conditions of dissociative energy applied in a mass spectrometer, bond X of the reagents is less prone to fragmentation as compared with the peptide bond of a Z″

-pro amino acid dimer or Z″

-asp amino acid dimer, wherein Z″

is any natural amino acid, pro is proline and asp is aspartic acid.

6. The compound set of claim 1, wherein compounds of the set are isotopically enriched with one or more heavy atom isotopes.

7. The compound set of claim 1, wherein compounds of the set are isotopically enriched with three or more heavy atom isotopes.

8. The compound set of claim 1, wherein the reporter moiety, RP, of the reagents is a substituted or unsubstituted, aliphatic or aromatic, 5, 6 or 7 membered heterocyclic ring comprising a ring nitrogen atom that is N-alkylated with a substituted or unsubstituted acetic acid moiety to which the analyte can be linked, by reaction of the reactive group with a functional group of the analyte, through the carbonyl carbon of the N-alkyl acetic acid moiety, wherein each different label comprises one or more heavy atom isotopes.

9. The compound set of claim 8, wherein the heterocyclic ring comprises one or more additional nitrogen, oxygen or sulfur atoms.

10. The compound set of claim 1, wherein the linker moiety comprises at least one heavy atom isotope and has the formula:

11. The compound set of claim 1, wherein the reactive group is an active ester.

12. The compound set of claim 1, wherein the reactive group is a mixed anhydride.

13. The compound set of claim 1, wherein each reagent is a mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt.

14. The compound set of claim 1, wherein each incorporated heavy atom isotope of each reagent is present in at least 80 percent isotopic purity.

15. The compound set of claim 1, wherein each incorporated heavy atom isotope of each reagent is present in at least 93 percent isotopic purity.

16. The compound set of claim 1, wherein each incorporated heavy atom isotope of each reagent is present in at least 96 percent isotopic purity.

17. A compound of formula RP-X-LK-Y-RG, or a salt thereof, wherein:

i) RG is a reactive group that is a nucleophile or an electrophile and that is capable of reacting with a functional group of one or more reactive analytes of a sample;

ii) RP is a reporter moiety that comprises a fixed charge or that is ionizable;

iii) LK is a linker moiety that links the reactive group to the reporter moiety;

iv) X is a bond between an atom of the reporter moiety and an atom of the linker moiety;

v) Y is a bond between an atom of the linker moiety and an atom of the reactive group, wherein, once the labeling reagent is reacted with the reactive analyte, bond Y links the linker to the analyte; and

vi) bonds X and Y fragment in at least a portion of labeled analytes, which labeled analytes can be formed by reacting the compound of formula RP-X-LK-Y-RG with a sample comprising one or more reactive analytes, when subjected to dissociative energy levels;

wherein, the linker moiety of the compound is isotopically enriched with at least one heavy atom isotope and the reporter moiety of the compound is isotopically enriched with at least one heavy atom isotope.

18. The compound of claim 17, wherein the linker moiety is capable of undergoing neutral loss when the compound is subjected to dissociative energy.

19. A compound of formula RP-X-LK-Y-Analyte, or a salt thereof, wherein:

i) Analyte is any analyte;

ii) RP is a reporter moiety that comprises a fixed charge or that is ionizable;

iii) LK is a linker moiety that links the Analyte to the reporter moiety;

iv) X is a bond between an atom of the reporter moiety and an atom of the linker moiety;

v) Y is a bond between an atom of the linker moiety and an atom of the analyte; and

vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels;

wherein, the linker moiety of the compound is isotopically enriched with at least one heavy atom isotope and the reporter moiety of the compound is isotopically enriched with at least one heavy atom isotope.

20. The compound of claim 19, wherein the linker moiety is capable of undergoing neutral loss when the compound is subjected to dissociative energy.