Methods and Mixtures Pertaining to Analyte Determination Using Electrophilic Labeling Reagents

• US 20100112708A1
• Filed: 10/20/2009
• Published: 05/06/2010
• Est. Priority Date: 01/30/2003
• Status: Abandoned Application

• Current Assignee: DH Technologies Pte. Ltd.
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Pappin, Darryl J.C., Bartlet-Jones, Michael
• Application Number:
  US12/582,689
• Publication Number:
  US 20100112708A1
• Time in Patent Office: Days
• US Class Current: 436/86
• 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, mixtures, 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 method of proteomic analysis by mass spectrometry comprising:

a) reacting each of at least two peptide samples with a labeling reagent to produce two or more differentially labeled samples wherein each labeling reagent is comprised of;

one or more heavy atom isotopes and a reporter moiety comprising a substituted or nonsubstituted morphine, piperidine or piperazine compound or a salt thereof and, wherein the gross mass of each reporter is different for each reagent of the set;

b) mixing the two or more differentially labeled samples, and optionally one or more calibration standards to produce a sample mixture;

c) performing a first mass spectrometric analysis on the sample mixture or a fraction thereof

d) treating selected ions of labeled analytes from the first mass spectrometric analysis to dissociative energy levels to thereby form ionized reporter moieties and ionized daughter fragment ions of at least some of the selected ions;

e) performing a second mass analysis of the selected ions, the ionized reporter moieties and the daughter fragment ions, or a fraction thereof; and

f) determining the gross mass and relative amount of each reporter moiety in the second mass analysis and the gross mass of the daughter fragment ions.

4. The method of claim 1, further comprising repeating steps (d) through (f) one or more times on selected ions of labeled analytes at a different selected mass to charge ratio.

20. The method of claim 1, wherein the reactive group of each reagent of the set is an amine reactive active ester group.

21. The method of claim 20, wherein the active ester is a N-hydroxysuccinimidyl ester, a N-hydroxysulfosuccinimidyl ester, a pentafluorophenyl ester, a 2-nitrophenyl ester, a 4-nitrophenyl ester, a 2,4-dinitrophenylester or a 2,4-dihalophenyl ester.

36. The method of claim 1, wherein the one or more differentially labeled analytes each comprise an isobaric label that identifies the sample from which it originated.

37. The method of claim 36, wherein each of the labeling reagents is independently a 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 is linked through the carbonyl carbon of the N-alkyl acetic acid moiety, wherein each different label comprises one or more heavy atom isotopes.

38. The method of claim 37, wherein the isobarically labeled analytes in the sample mixture each comprise the formula:

40. The method of claim 38, wherein the sample mixture comprises one or more isobarically labeled analytes of the formula:

41. The method of claim 38, wherein the sample mixture comprises one or more isobarically labeled analytes of the formula:

42. The method of claim 38, wherein the labeling reagent comprises the formula:

43. The method of claim 38, wherein the sample mixture comprises one or more isobarically labeled analytes of the formula:

44. The method of claim 36, wherein the isobarically labeled analytes in the sample mixture each comprise the formula:

47. The method of claim 1, wherein each different labeling reagent of the set is support bound and is linked to the support through a cleavable linker such that each different sample is reacted with a support carrying a different labeling reagent;

and the method further comprises, before performing step (b);

i) optionally washing the resin to remove components of the sample that do not react with the reactive group of the labeling reagent; and

ii) collecting the two or more differentially labeled samples, each sample comprising one or more labeled analytes wherein the labeled analytes associated with a particular sample are identifiable and/or quantifiable.

48. The method of claim 47, wherein each different labeling reagent of the set is a solid support of the formula:

E-F-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 one or more of the reactive analytes of the samples;

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

iii) LK is a linker moiety that links the reactive group and the reporter group, wherein the mass of the linker compensates for the difference in gross mass between the reporters 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 and an atom of the linker;

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

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

vii) E is a solid support; and

viii) F is a cleavable linker linked to the solid support and cleavably linked to the reporter.

49. The method of claim 48, wherein the set of labeling reagents comprises one or more of the following support bound labeling reagents:

2-3. -3. (canceled)

5-19. -19. (canceled)

22-35. -35. (canceled)

39. (canceled)

45-46. -46. (canceled)

50-89. -89. (canceled)

90. A mixture comprising at least two labeled analytes, wherein each of the two labeled analytes originates from a different sample combined to form the mixture and each comprises the formula:

RP-X-LK-Y-Analyte

or a salt thereof wherein;

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

b) LK is a linker moiety that links the reactive group and the reporter group, wherein;

i) the mass of the linker compensates for the difference in gross mass between the reporters 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; and

ii) the linker comprises at least one heavy atom isotope and has the formula;

107. The mixture of claim 90, wherein the at least two labeled analytes each comprise an isobaric label.

108. The mixture of claim 107, wherein the at least two labeled analytes each comprise an isobaric label that is a 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 is linked through the carbonyl carbon of the N-alkyl acetic acid moiety, wherein each different label comprises one or more heavy atom isotopes.

91-106. -106. (canceled)

109-114. -114. (canceled)