Isobaric tags for analyte detection and quantification
Isobaric reagents for labeling analytes are provided. The isobaric reagents have facile design and synthesis that allows for differential labeling of an unlimited number of analyte samples.
1. A reagent for labeling an analyte, the reagent represented by Formula I:
wherein:
R is a hydrocarbon group or a hydrocarbon group containing one or more heteroatoms;
R′ is an isotopomer of R; and
X is a reactive group capable of coupling to the analyte.
2. The reagent of claim 1 , wherein the one or more heteroatoms is oxygen.
3. The reagent of claim 1 , wherein the one or more heteroatoms is nitrogen.
4. The reagent of claim 1 , wherein X is selected from the group consisting of active esters, mixed anhydrides, and hydroxy reactive groups.
5. The reagent of claim 1 , wherein X is a thiol group.
6. The reagent of claim 1 , wherein the analyte is selected from the group consisting of proteins, peptides, nucleic acids, lipids, carbohydrates, steroids, and small molecules.
7. The reagent of claim 1 , wherein at least one of R and R′ is synthesized using isotopic substitution.
8. The reagent of claim 7 , wherein the isotopic substitution includes at least one isotope selected from the group consisting of 1 H, 2 H, 3 H, 12 C, 13 C, 14 C, 14 N, 15 N, 16 O, 17 O, and 18 O.
9. The reagent of claim 1 , wherein the hydrocarbon group is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, arylalkyl, alkaryl, ether, and hetero-substituted hydrocarbon groups.
10. The reagent of claim 1 , wherein the hydrocarbon group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, allyl, and benzyl.
11. The reagent of claim 1 , wherein the hydrocarbon group is polyethylene glycol.
12. A method of detecting at least one analyte comprising:
forming a labeled analyte by labeling the at least one analyte with a reagent of claim 1 ;
separating the labeled analyte by chromatography;
ionizing the separated, labeled analyte to obtain an analyte ion;
isolating the analyte ion;
activating the isolated analyte ion to obtain a reporter ion; and
detecting the mass of the reporter ion by tandem mass spectrometry.
13. The method of claim 12 , wherein the chromatography step comprises liquid chromatography-mass spectrometry (LC-MS); the ionizing step comprises matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), or fast atom bombardment (FAB); and the activating step comprises pulsed Q dissociation (PQD), collision induced dissociation (CID), higher energy C-trap dissociation (HCD), or CID-HCD.
14. A kit comprising (m) number of reagents for labeling (m) number of analytes, the kit comprising:
a first reagent; and
at least a second reagent, wherein the first reagent and the at least a second reagent are, independently represented by Formula I:
wherein:
R is a hydrocarbon group or a hydrocarbon group containing one or more heteroatoms;
R′ is an isotopomer of R; and
X is a reactive group capable of coupling to the analytes,
wherein for each reagent, the sum of the mass of R and the mass of R′ is constant, and wherein the mass of R of each reagent differs from the mass of every other R of the reagents in the kit, and the mass of R′ of each reagent differs from the mass of every other R′ of the reagents in the kit.
15. The kit of claim 14 , wherein the one or more heteroatoms is oxygen.
16. The kit of claim 14 , wherein the one or more heteroatoms is nitrogen.
17. The kit of claim 14 , wherein X is selected from the group consisting of active esters, mixed anhydrides, and hydroxy reactive groups.
18. The kit of claim 14 , wherein X is a thiol group.
19. The kit of claim 14 , wherein the at least one analyte is selected from the group consisting of proteins, peptides, nucleic acids, lipids, carbohydrates, steroids, and small molecules.
20. The kit of claim 14 , wherein at least one of R and R′ is synthesized using isotopic substitution.
21. The kit of claim 20 , wherein the isotopic substitution includes at least one isotope selected from the group consisting of 1 H, 2 H, 3 H, 12 C, 13 C, 14 C, 14 N, 15 N, 16 O, 17 O, and 18 O.
22. The kit of claim 14 , wherein the hydrocarbon group is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aralkyl, arylalkyl, alkaryl, ether, and hetero-substituted hydrocarbon groups.
23. The kit of claim 14 , wherein the hydrocarbon group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, allyl, and benzyl.
24. The kit of claim 14 , wherein the hydrocarbon group is polyethylene glycol.
25. A method of detecting (m) number of analytes comprising:
forming (m) number of labeled analytes by labeling the (m) number of analytes with the kit comprising (m) number of reagents of claim 14 ;
separating the labeled analytes by chromatography;
ionizing the separated, labeled analytes to obtain analyte ions;
isolating the analyte ions;
activating the isolated analyte ions to obtain a reporter ions; and
detecting the mass of the reporter ions by tandem mass spectrometry.
26. A kit comprising (m) number of reagents for labeling (m) number of analytes, the kit comprising:
a first reagent; and
at least a second reagent, wherein the first reagent and the at least a second reagent are, independently represented by Formula X:
wherein:
R is a hydrocarbon group or a hydrocarbon group containing one or more heteroatoms;
R′ is an isotopomer of R;
M is a hydrocarbon linker group; and
X is a reactive group capable of coupling to the analyte;
wherein for each reagent, the sum of the mass of the Reporter Region and the mass of Mass Balancing Region is constant, and wherein the mass of the Reporter Region of each reagent differs from the mass of every other Reporter Region of the reagents in the kit, and the mass of the Mass Balancing Region of each reagent differs from the mass of every other Mass Balancing Region of the reagents in the kit.
27. A method of detecting (m) number of analytes comprising:
forming (m) number of labeled analytes by labeling the (m) number of analytes with the kit comprising (m) number of reagents of claim 26 ;
separating the labeled analytes by chromatography;
ionizing the separated, labeled analytes to obtain analyte ions;
isolating the analyte ions;
activating the isolated analyte ions to obtain a reporter ions; and
detecting the mass of the reporter ions by tandem mass spectrometry.