Methods, compositions and kits pertaining to analyte determination

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.

1 . An active ester compound 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 alcohol moiety of the active ester is linked through the carbonyl carbon of the N-alkyl acetic acid moiety, wherein the compound is isotopically enriched with one or more heavy atom isotopes.  
     
     
         2 . The compound of  claim 1 , wherein the compound is isotopically enriched with three or more heavy atom isotopes.  
     
     
         3 . The compound of  claim 1 , wherein the heterocyclic ring is substituted with one or more substituents.  
     
     
         4 . The compound of  claim 3 , wherein the one or more substituents are alkyl, alkoxy or aryl groups.  
     
     
         5 . The compound of  claim 4 , wherein the one or more substituents are protected or unprotected amine groups, hydroxyl groups or thiol groups.  
     
     
         6 . The compound of  claim 1 , wherein the heterocyclic ring is aliphatic.  
     
     
         7 . The compound of  claim 1 , wherein the heterocyclic ring is aromatic.  
     
     
         8 . The compound of  claim 1 , wherein the heterocyclic ring comprises one or more additional nitrogen, oxygen or sulfur atoms.  
     
     
         9 . The compound of  claim 1 , wherein active ester is an N-hydroxysuccinimide ester.  
     
     
         10 . The compound of  claim 1 , wherein the compound is a salt.  
     
     
         11 . The compound of  claim 1 , wherein the compound is a mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl salt.  
     
     
         12 . The compound of  claim 1 , wherein each incorporated heavy atom isotope is present in at least 80 percent isotopic purity.  
     
     
         13 . The compound of  claim 1 , wherein each incorporated heavy atom isotope is present in at least 93 percent isotopic purity.  
     
     
         14 . The compound of  claim 1 , wherein each incorporated heavy atom isotope is present in at least 96 percent isotopic purity.  
     
     
         15 . A kit comprising a compound as claimed in  claim 1 , further comprising one or more reagents, containers, enzymes, buffers or instructions.  
     
     
         16 . The kit of  claim 15 , wherein the kit comprises a proteolytic enzyme.  
     
     
         17 . The kit of  claim 15 , wherein the kit comprises two or more compounds as claimed in  claim 1 , wherein said two or more compounds are related as a set of isomers and/or isobaric labeling reagents.  
     
     
         18 . The kit of  claim 17 , wherein each labeling reagent is independently linked to a solid support through a cleavable linker.  
     
     
         19 . The kit of  claim 15 , wherein the compound has the formula:  
       
         
           
           
               
               
           
         
       
       wherein; 
 a) RG is a reactive group that is an electrophile;  
 b) Z is O, S, NH or NR 1 ;  
 c) 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;  
 d) W is an atom or group that is located ortho, meta or para to the ring nitrogen and is selected from the group consisting of: NH, N—R 2 , P—R 2 , O or S; and  
 e) each carbon of the heterocyclic ring has the formula CJ 2 ;  
 f) 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  
 g) 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.  
 
     
     
         20 . A method comprising: 
 a) reacting an analyte with the compound as claimed in  claim 1  to thereby produce a labeled analyte; and    b) mixing the labeled analyte with one or more differentially labeled analytes.    
     
     
         21 . The method of  claim 20 , further comprising analyzing the mixture in a mass spectrometer.  
     
     
         22 . The method of  claim 20 , wherein the analysis in a mass spectrometer involves MS/MS analysis.