Quaternary amine containing isobaric tag for quantitative glycan profiling
AssigneesUniversity of Maryland, College Park, The Johns Hopkins University
InventorShuwei Li
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Abstract
The disclosure provides quaternary amine containing isobaric tag reagents useful in the analysis of biomolecules and methods of making and using the quaternary amine containing isobaric tag reagents. The quaternary amine containing isobaric tag reagents are particularly useful for glycan analysis, especially quantitative glycan profiling such as glycan quantitation by tandem mass spectrometry.
Claims22
A method of N-glycan analysis comprising labeling N-glycans with a quaternary amine containing isobaric tag reagent comprising an MS/MS scissionable bond and a reactive group capable of conjugating with N-glycans.
A quaternary amine containing isobaric tag reagent comprising the formula:
- reporter group—balancer group—reactive group,
- wherein the reagent has the structure:
- wherein at least one of positions 1-7 comprises an isotope atom, the reporter group and balancer group are linked by an MS/MS scissionable bond, and R is a reactive group capable of conjugating with glycans.
A method of making a quaternary amine containing isobaric tag reagent comprising the formula:
- reporter group—balancer group—reactive group,
- wherein the reagent has the structure:
- wherein the reporter group and balancer group are linked by an MS/MS scissionable bond, the reporter group has a mass in a range of from 176 to 179 Da, the reagent contains 2 or 3 isotope atoms independently selected from 13C and 2H, and the reactive group comprises a reactive primary amine capable of conjugating with glycans via reductive amination;
- the method comprising the steps of:
- (a) reacting Boc-(4-(aminomethyl)benzyl)-amine and triethylamine with 2-nitrobenzenesulfonyl chloride to obtain a first reaction product;
- (b) reacting the first reaction product with sodium carbonate and methyl iodide to obtain a second reaction product;
- (c) reacting the second reaction product with β-mercaptoacetic acid to obtain a third reaction product;
- (d) reacting the third reaction product with a first intermediate reaction product of a carboxybenzyl-protected 3-amino-1,2-propanediol and sodium periodate to form a fourth reaction product;
- (e) removing the tert-butyloxycarbonyl (Boc) protecting group from the fourth reaction product;
- (f) reacting the deprotected fourth reaction product with a second intermediate reaction product of propionic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to form a fifth reaction product;
- (g) reacting the fifth reaction product with methyl iodide to obtain a crude final product;
- and
- (h) removing the carboxybenzyl (Cbz) protecting group from the crude final product to obtain the quaternary amine-containing isobaric tag reagent;
- wherein the propionic acid and methyl iodide are selected from one of the following reactant pairs (i)-(iv) comprising isotope labeled reactant:
- (i) isotope-unlabeled propionic acid of the formula CH3CH2COOH, and isotope-labeled methyl iodide of the formula 13CHD2I;
- (ii) isotope-labeled propionic acid of the formula CH3CH213COOH, and isotope-labeled methyl iodide of the formula CHD2I;
- (iii) isotope-labeled propionic acid of the formula 13CH313CH2COOH, and isotope-labeled methyl iodide of the formula 13CH3I;
- and
- (iv) isotope-labeled propionic acid of the formula 13CH313CH213COOH, and isotope-unlabeled methyl iodide of the formula CH3I.
Dependencies are inferred from the claim text.