Isobaric Multiplex Reagents for Carbonyl Containing Compound High-Throughput Quantitative Analysis

Abstract

The present invention provides a set of novel isobaric chemical tags, also referred herein as SUGAR (Isobaric Multiplex Reagents for Carbonyl Containing Compound). These labeling tags are compact and easy to synthesize at high yield and purity in just a few steps using commercially available starting materials. The tagging reagents of the present invention comprise: a) a reporter group, having at least one atom that is optionally isotopically labeled; b) a balancing group, also having at least one atom that is optionally isotopically labeled, and c) an aldehyde, ketone, or carboxylic acid reactive group. The multiplex SUGAR tags are able to react with an aldehyde, ketone, or carboxylic acid group of the molecule to be tagged, which offers the capability for labeling and quantitation of glycans, proteins/peptides, and fatty acids.

We claim: 
     
         1 . A mass spectrometry tagging reagent comprising a compound having the formula of: 
       
         
           
           
               
               
           
         
         wherein, 
         R 1  is an aldehyde reactive group, ketone reactive group, or carboxylic acid reactive group; 
         R 2 , R 3 , R 4 , R 5  and R 6 , independently of one another, are selected from the group consisting of hydrogen, deuterium, branched and unbranched C 1  to C 12  alkyl groups, C 4  to C 12  cycloalkyl groups, C 2  to C 12  alkenyl groups, C 5  to C 12  cycloalkenyl groups, C 6  to C 12  aryl groups and C 7  to C 12  arylalkyl groups, wherein each of R 2 , R 3 , R 4 , R 5  and R 6  optionally contain one or more  13 C atoms and one or more deuterium atoms;
 C V  and C x , independently of one another, are  12 C or  13 C, 
 O U  and O y , independently of one another, are  16 O or  18 O; and 
 N z  and N W , independently of one another, are  14 N or  15 N. 
 
       
     
     
         2 . The method of  claim 1  wherein R 1  is a hydrazide, hydrazine, amine, or oxyamine. 
     
     
         3 . The tagging reagent of  claim 1  wherein R 2  and R 3 , independently of one another, are CH 3 ,  13 CH 3 , CDH 2 ,  13 CDH 2 , CD 2 H,  13 CD 2 H, CD 3  or  13 CD 3 . 
     
     
         4 . The tagging reagent of  claim 1  wherein R 6  is hydrogen or deuterium. 
     
     
         5 . The tagging reagent of  claim 1  wherein at least one of: a) R 2  or R 3  contains a deuterium atom, b) N z  is  15 N, c) or N W  is  15 N. 
     
     
         6 . The tagging reagent of  claim 1  wherein R 6  is hydrogen or deuterium and R 5  is selected from the group consisting of:
 a) a methyl group containing one or more deuterium atoms and wherein the carbon is  12 C or  13 C; 
 b) hydrogen; 
 c) deuterium; 
 d) an isopropyl group containing one or more deuterium atoms and one or more  13 C atoms; and 
 e) a butyl group containing one or more deuterium atoms and one or more  13 C atoms. 
 
     
     
         7 . The tagging reagent of  claim 1  wherein the tagging reagent has a formula selected from the following: 
       
         
           
           
               
               
           
         
       
     
     
         8 . A kit comprising two or more tagging reagents, wherein each tagging reagent comprises a reporter group, an aldehyde, ketone, or carboxylic acid reactive group, and a balancing group located between the reporter group and aldehyde, ketone, or carboxylic acid reactive group, wherein one or more atoms in the reporter group, balancing group, or both, are isotopically heavy versions of the atom,
 wherein the reporter group of each tagging reagent has a mass different than the reporter groups of the other tagging reagents, the balancing group of each tagging reagent has a mass different than the balancing groups of other tagging reagents, and the aggregate mass of the reporter group plus the balancing group for each tagging reagent is the same;   wherein the balancing group of each tagging reagent has the formula:   
       
         
           
           
               
               
           
         
       
       wherein R 5  and R 6 , independently from one another, are selected from the group consisting of hydrogen, deuterium, branched and unbranched C 1  to C 12  alkyl groups, C 4  to C 12  cycloalkyl groups, C 2  to C 12  alkenyl groups, C 5  to C 12  cycloalkenyl groups, C 6  to C 12  aryl groups and C to C 12  arylalkyl groups, wherein each of R 5  and R 6  optionally contains one or more  13 C atoms and one or more deuterium atoms;
 C V  and C x , independently of one another, are  12 C or  13 C, 
 O U  and O y , independently of one another, are  16 O or  18 O; 
 N W  is  14 N or 15N; 
 wherein the reporter group of each tagging reagent has the formula: 
 
       
         
           
           
               
               
           
         
       
       R 2 , R 3  and R 4 , independently of one another, are selected from the group consisting of hydrogen, deuterium, branched and unbranched C 1  to C 12  alkyl groups, C 4  to C 12  cycloalkyl groups, C 2  to C 12  alkenyl groups, C 5  to C 12  cycloalkenyl groups, C 6  to C 12  aryl groups and C 7  to C 12  arylalkyl groups, wherein each of R 2 , R 3  and R 4  optionally contains one or more  13 C atoms and one or more deuterium atoms; and N z  is  14 N or  15 N. 
     
     
         9 . The kit of  claim 8  wherein the aldehyde, ketone, or carboxylic acid reactive group is a hydrazide, hydrazine, amine, or oxyamine. 
     
     
         10 . The kit of  claim 8  wherein R 6  is hydrogen or deuterium. 
     
     
         11 . The kit of  claim 8  wherein R 2  and R 3 , independently of one another, are CH 3 ,  13 CH 3 , CDH 2 ,  13 CDH 2 , CD 2 H,  13 CD 2 H, CD 3  or  13 CD 3 . 
     
     
         12 . The kit of  claim 8  wherein at least one of R 2  or R 3  contains a deuterium atom, and N z  is 15 or N W  is  15 N. 
     
     
         13 . The kit of  claim 8  wherein the reporter group of each tagging reagent has a mass that differs from one another by one or more Daltons. 
     
     
         14 . The kit of  claim 8  wherein the reporter group of each tagging reagent has a mass that differs from one another by less than 25 mDa.