Methods and Mixtures Pertaining to Analyte Determination Using Electrophilic Labeling Reagents

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.

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.   
     
     
         2 - 3 . (canceled) 
     
     
         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. 
     
     
         5 - 19 . (canceled) 
     
     
         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. 
     
     
         22 - 35 . (canceled) 
     
     
         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: 
       
         
           
           
               
               
           
         
       
       wherein;
 a) Z is O, S, NH or NR 1 ; 
 b) each J is the same or different and is H, deuterium (D), R 1 , OR 1 , SR 1 , NHR 1 , N(R 1 ) 2 , fluorine, chlorine, bromine or iodine; 
 c) W is an atom or group that is located ortho, meta or para to the ring nitrogen and is NH, N—R 1 , N—R 1 , P—R 1 , P—R 1 , O or S; 
 d) each carbon of the heterocyclic ring has the formula CJ 2 ; 
 e) each R 1  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; and 
 f) R 2  is an amino alkyl, hydroxy alkyl, thio alkyl group or a cleavable linker that cleavably links the reagent to a solid support wherein the amino alkyl, hydroxy alkyl or thio alkyl group comprises one to eight carbon atoms, which may optionally contain a heteroatom or a substituted or unsubstituted aryl group, and wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms. 
 
     
     
         39 . (canceled) 
     
     
         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: 
       
         
           
           
               
               
           
         
       
       wherein each R 1  is the same or different and is an alkyl group. 
     
     
         43 . The method of  claim 38 , wherein the sample mixture comprises one or more isobarically labeled analytes of the formula: 
       
         
           
           
               
               
           
         
       
       wherein:
 a) G 1  is an amino alkyl, hydroxy alkyl or thio 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; 
 b) each carbon of the heterocyclic ring has the formula CJ 2 , wherein each J is the same or different and is selected from the group consisting of: H, deuterium (D), R 1 , OR 1 , SR 1 , NHR 1 , N(R 1 ) 2 , fluorine, chlorine, bromine and iodine; and 
 c) each R 1  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. 
 
     
     
         44 . The method of  claim 36 , wherein the isobarically labeled analytes in the sample mixture each comprise the formula: 
       
         
           
           
               
               
           
         
       
       wherein:
 a) Z is O, S, NH or NR 1 ; 
 b) each J is the same or different and is selected from the group consisting of: H, deuterium (D), R 1 , OR 1 , SR 1 , NHR 1 , N(R 1 ) 2 , fluorine, chlorine, bromine and iodine; 
 c) each R 1  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. 
 
     
     
         45 - 46 . (canceled) 
     
     
         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: 
       
         
           
           
               
               
           
         
       
       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) E is a solid support; 
 iii) F is a cleavable linker linked to the solid support and cleavably linked to the reporter; 
 iv) G is an amino alkyl, hydroxy alkyl or thio alkyl group, cleavably linked to the cleavable linker wherein the amino alkyl, hydroxy alkyl or thio alkyl group comprises one to eight carbon atoms, which may optionally contain a heteroatom or a substituted or unsubstituted aryl group, and wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms; 
 v) each carbon of the heterocyclic ring has the formula CJ2, wherein each J is the same or different and is selected from the group consisting of H, deuterium (D), R 1 , OR 1 , SR 1 , NHR 1 , N(R 1 ) 2 , fluorine, chlorine, bromine and iodine; and vi) each R 1  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. 
 
     
     
         50 - 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: 
 
 
       
         
           
           
               
               
           
         
       
       wherein R 1  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;
 c) X is a bond between an atom of the reporter and an atom of the linker; 
 d) Y is a bond between an atom of the linker and an atom of the analyte. 
 
     
     
         91 - 106 . (canceled) 
     
     
         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. 
     
     
         109 - 114 . (canceled)