# US 2016/0252518 A1 · Quaternary amine containing isobaric tag for quantitative glycan profiling > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2016/0252518 A1 - Document ID: US20160252518A1 - Status: Published application - Kind: A1 - Application number: 14/880,972 - Filed: 2015-10-12 - Published: 2016-09-01 - Earliest claimed: 2014-10-13 - Assignees: University of Maryland, College Park; The Johns Hopkins University - Applicants: University of Maryland, College Park - Inventors: Shuwei Li - Web page: https://patentgrep.com/patent/US20160252518A1 ## CPC - G01N 33/6848: {Methods of protein analysis involving mass spectrometry} - G01N 2458/15: Non-radioactive isotope labels, e.g. for detection by mass spectrometry - G01N 2560/00: Chemical aspects of mass spectrometric analysis of biological material - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## 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. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the quaternary amine containing isobaric tag reagent comprises the formula: reporter group—balancer group—reactive group, wherein the reporter group and balancer group are linked by the MS/MS scissionable bond. ### Claim 3 (depends on 1) The method of claim 1, wherein the quaternary amine containing isobaric tag reagent comprises 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 the MS/MS scissionable bond, and R is the reactive group capable of conjugating with glycans. ### Claim 4 (depends on 3) The method of claim 3, wherein the reporter group has a mass in a range from 176 to 179 Da and the reagent contains 2 or 3 isotope atoms independently selected from 13C and 2H. ### Claim 5 (depends on 2) The method of claim 2, wherein the reactive group comprises a reactive primary amine capable of conjugating with glycans via reductive amination. ### Claim 6 (depends on 2) The method of claim 2, further comprising quantitatively analyzing the labeled N-glycans. ### Claim 7 (depends on 2) The method of claim 2, wherein the N-glycans are obtained by (a) immobilizing glycoproteins comprising N-glycans on a solid support; (b) chemically modifying the N-glycans; and (c) releasing the N-glycans from the solid support. ### Claim 8 (depends on 7) The method of claim 7, wherein the glycoproteins are obtained from human or nonhuman animal serum. ### Claim 9 (depends on 7) The method of claim 7, wherein the N-glycans carry sialic acids and the chemical modification of the N-glycans comprises carbodiimide coupling of the sialic acids. ### Claim 10 (depends on 6) The method of claim 6, wherein the quantitatively analyzing step comprises fragmenting the reagent and quantitating the N-glycans. ### Claim 11 (depends on 10) The method of claim 10, wherein fragmenting the reagent comprises MS/MS scission of the MS/MS scissionable bond. ### Claim 12 (depends on 6) The method of claim 6, wherein Na+ is not used in quantitatively analyzing the labeled glycans. ### Claim 13 (depends on 1) The method of claim 1, wherein the quaternary amine containing isobaric tag reagent comprises the formula: reporter group—balancer group—reactive group, wherein the reagent has the structure: wherein at least one of positions 1-4 comprises an isotope atom, the reporter group and balancer group are linked by the MS/MS scissionable bond, and R is the reactive group capable of conjugating with glycans. ### Claim 14 (depends on 1) The method of claim 1, wherein the quaternary amine containing isobaric tag reagent comprises the formula: reporter group—balancer group—reactive group, wherein the reagent has the structure: wherein at least one of positions 1-4 comprises an isotope atom and the reporter group and balancer group are linked by the MS/MS scissionable bond. ### Claim 15 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. ### Claim 16 (depends on 15) The quaternary amine containing isobaric tag reagent of claim 15, wherein 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 R comprises a reactive primary amine capable of conjugating with glycans via reductive amination. ### Claim 17 (depends on 16) The quaternary amine containing isobaric tag reagent of claim 16 wherein the quaternary amine containing isobaric tag has the structure (I): and wherein the methyl group at position 4 is 13CHD2. ### Claim 18 (depends on 16) The quaternary amine containing isobaric tag reagent of claim 16 wherein the quaternary amine containing isobaric tag has the structure (I): and wherein the methyl group at position 4 is CHD2 and the carbon at position 3 is 13C. ### Claim 19 (depends on 16) The quaternary amine containing isobaric tag reagent of claim 16 wherein the quaternary amine containing isobaric tag has the structure (I): and wherein the carbon at each of positions 1, 2 and 4 is 13C. ### Claim 20 (depends on 15) The quaternary amine containing isobaric tag reagent of claim 15 wherein the quaternary amine containing isobaric tag has the structure (I): and wherein the carbon at each of positions 1, 2 and 3 is 13C. ### Claim 21 (depends on 15) The quaternary amine containing isobaric tag reagent of claim 15, wherein the reagent has the structure: wherein at least one of positions 1-4 comprises an isotope atom and the reporter group and balancer group are linked by the MS/MS scissionable bond. ### Claim 22 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.