Methods for the rapid preparation of labeled glycosylamines and for the analysis of glycosylated biomolecules producing the same

Patent metadata

Field	Value
Publication number	US-12158472-B2
Application number	US-202217741865-A
Country	US
Kind code	B2
Filing date	May 11, 2022
Priority date	Oct 30, 2014
Publication date	Dec 3, 2024
Grant date	Dec 3, 2024

Abstract

Methods of analyzing glycosylated biomolecules include the steps of producing a deglycosylation mixture of biomolecules deglycosylated by natural or synthetic enzymatic or chemical techniques; providing a reagent solution having a labeling reagent in a polar aprotic, non-nucleophilic organic solvent; and mixing the deglycosylation mixture with the reagent solution in an excess of labeling reagent to produce derivatized glycosylamines. The method steps can be carried out purposefully without depletion of protein matter. A quenching solution can be added to the reaction mixture so that the pH of the reaction mixture is shifted to above 10. The yield of derivatized glycosylamines can be in an amount of about 80 to about 100 mole percent of the reaction mixture with minimal overlabeling, less than 0.2 mole percent. The derivizated glycosylamines can be separated from the reaction mixture and detected by chromatographic detection, fluorescence detection, mass spectrometry (“MS”), or Ultra Violet (“UV”) detection and/or a combination thereof.

First claim

We claim:

1. A method of rapid derivatization of glycosylamines comprising the steps of:
providing a biological sample comprising a glycoprotein;
contacting the glycoprotein with an enzyme to produce a deglycosylation mixture; and
mixing (a) a reagent solution comprising a labeling reagent selected from an N-hydroxysuccinimide ester reagent and an N-hydroxysuccinimide carbamate reagent combined with a polar aprotic, non-nucleophilic organic solvent, (b) the deglycosylation mixture, and (c) a buffer solution to produce a reaction mixture, the reaction mixture being formed under conditions in which protein matter has not been depleted from the deglycosylation mixture, the reaction mixture comprising a molar excess of labeling reagent, the reaction mixture having the labeling reagent, released glycosylamines, proteinacious amines comprising lysine residues, and derivatized glycosylamines wherein the derivatized glycosylamines are yielded between about 80 to about 100 percent and with overlabeling of glycosylamines in an amount of less than 0.2 mole percent.

2. The method of claim 1 , further comprising the step of separating the derivatized glycosylamines by online solid phase extraction.

3. The method of claim 1 , wherein the polar aprotic, non-nucleophilic organic solvent is selected from the group consisting of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and acetonitrile.

4. The method of claim 3 , wherein the concentration of DMF in the reaction mixture is less than about 20 percent to about 30 percent by volume or DMSO in the reaction mixture is less than about 30 percent to about 50 percent.

5. The method of claim 3 , further comprising adding a quenching solution to the reaction mixture, wherein the quenching solution comprises ethylene diamine and the pH of the reaction mixture shift to greater than about 10.

6. The method of claim 5 , wherein the polar aprotic, non-nucleophilic organic solvent comprises acetonitrile and a ratio of ethylene diamine to water to acetonitrile is about 5 to about 90 by volume.

7. The method of claim 5 , wherein the quenching solution is added to the reaction mixture about 2 to about 10 minutes after the deglycosylation mixture is mixed with the reagent solution.

8. The method of claim 1 , wherein the deglycosylation mixture is mixed with the reagent solution in a volumetric ratio of about 2.5 to about 1.

9. The method of claim 1 , wherein the reagent solution has a temperature maintained at about ambient temperature to sub-ambient temperatures.

10. The method of claim 1 , further comprising the step of adding a buffer solution to the sample after the enzyme is combined with the biological sample.

11. The method of claim 10 , wherein the buffer solution is HEPES or sodium phosphate.

12. The method of claim 11 , wherein the buffer solution has a pH of about 7.9 to about 8.2 and a concentration of between about 5 mM to about 50 mM.

13. The method of claim 1 , wherein the enzyme is peptide N-glycosidase F.

14. The method of claim 1 , wherein the reaction mixture comprises a molar excess of labeling reagent in an amount of about 20 to 2000.

15. The method of claim 1 , wherein labeling is allowed to proceed for between 2 and 10 minutes before additional sample processing is performed.

16. The method of claim 1 , wherein the reagent solution has a temperature maintained at about ambient temperature to sub-ambient temperatures, wherein the reagent solution comprises dimethylformamide (DMF) in a concentration ranging from less than about 20 percent to about 30 percent by volume, wherein the buffer solution is selected from HEPES buffer and phosphate buffer, wherein the buffer solution has a pH of about 7.9 to about 8.2 and wherein the buffer solution has a concentration of between about 5 mM to about 50 mM.

Assignees

• Waters Technologies Corp

Inventors

• Lauber Matthew A
• Brousmiche Darryl W
• Koza Stephan M

Classifications

• G01N2333/98

  acting on amide bonds in linear amides (3.5.1) · CPC title

• C12P19/28

  N-glycosides · CPC title

• G01N2333/924

  acting on glycosyl compounds (3.2) · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N2440/38

  addition of carbohydrates, e.g. glycosylation, glycation · CPC title