Isobarically labeled analytes and fragment ions derived therefrom
First Claim
Patent Images
1. A method comprising:
- a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RG or a salt form and/or hydrate form thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels;
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein 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; and
c) digesting each differentially labeled sample with at least one enzyme to partially, or fully, degrade components of the sample, after performing step (a).
12 Assignments
0 Petitions
Accused Products
Abstract
This invention pertains to isobarically labeled analytes and fragment ions thereof.
65 Citations
18 Claims
-
1. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels;
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein RP;
i) has a gross mass of less than 250 daltons; and
/orii) 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
/oriii) is not a polymer or is not a biological polymer; and
c) digesting each differentially labeled sample with at least one enzyme to partially, or fully, degrade components of the sample, after performing step (a). - View Dependent Claims (6, 16, 17, 18)
-
-
2. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein the linker LK undergoes neutral loss under conditions of applied dissociative energy; and
c) digesting each differentially labeled sample with at least one enzyme to partially, or fully, degrade components of the sample, after performing step (a).
-
-
3. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein, under conditions of dissociative energy applied in a mass spectrometer, the fragmentation of one of bonds X or Y results in the fragmentation of the other of bonds X or Y; and
c) digesting each differentially labeled sample with at least one enzyme to partially, or fully, degrade components of the sample, after performing step (a).
-
-
4. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein;
i) under conditions of dissociative energy applied in a mass spectrometer, bond X is less prone to fragmentation as compared with bond Y; and
/orii) under conditions of dissociative energy applied in a mass spectrometer, bond X 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;
c) digesting each differentially labeled sample with at least one enzyme to partially, or fully, degrade components of the sample, after performing step (a).
-
-
5. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof wherein;
i) RG is a reactive group that is an electrophile and that is capable of reacting with one or more of the reactive analytes of the sample;
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;
a) 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
b) the linker comprises at least one heavy atom isotope and has the formula;
wherein R1 is the same or different and is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms;
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; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture; and
c) digesting each differentially labeled sample with at least one enzyme to partially, or fully, degrade components of the sample, after performing step (a).
-
-
7. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels;
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein RP;
i) has a gross mass of less than 250 daltons; and
/orii) 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
/oriii) is not a polymer or is not a biological polymer; and
c) immobilizing the analyte to a support by reaction of a functional group of the analyte with a functional group of the support.
-
-
8. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein the linker LK undergoes neutral loss under conditions of applied dissociative energy; and
c) immobilizing the analyte to a support by reaction of a functional group of the analyte with a functional group of the support.
-
-
9. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein, under conditions of dissociative energy applied in a mass spectrometer, the fragmentation of one of bonds X or Y results in the fragmentation of the other of bonds X or Y; and
c) immobilizing the analyte to a support by reaction of a functional group of the analyte with a functional group of the support.
-
-
10. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof, 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 sample;
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; and
vi) bonds X and Y fragment in at least a portion of the labeled analytes when subjected to dissociative energy levels; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture;
wherein;
i) under conditions of dissociative energy applied in a mass spectrometer, bond X is less prone to fragmentation as compared with bond Y; and
/orii) under conditions of dissociative energy applied in a mass spectrometer, bond X 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;
c) immobilizing the analyte to a support by reaction of a functional group of the analyte with a functional group of the support. - View Dependent Claims (12, 13, 14, 15)
-
-
11. A method comprising:
-
a) reacting two or more samples, each sample comprising one or more reactive analytes, with a different labeling reagent of a set of labeling reagents to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes wherein the different labeling reagents of the set each comprise the formula;
RP-X-LK-Y-RGor a salt form and/or hydrate form thereof wherein;
i) RG is a reactive group that is an electrophile and that is capable of reacting with one or more of the reactive analytes of the sample;
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;
a) 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
b) the linker comprises at least one heavy atom isotope and has the formula;
wherein R1 is the same or different and is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms;
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; and
b) mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture; and
c) immobilizing the analyte to a support by reaction of a functional group of the analyte with a functional group of the support.
-
Specification