IMPROVED METHOD OF MAPPING GLYCANS OF GLYCOPROTEINS

摘要

The use of anthranilic acid (2-AA) to label N-glycans prior to separation using a reversed-phase liquid chromatography (RP-LC) column under acidic conditions using formic acid is described herein. Negatively charged 2-AA offers stronger retention on the reversed phase column than 2-aminobenzamide (2-AB) in RP-LC and allows efficient ionization and detection of 2-AA labeled N-glycans. The acidic conditions used for the RP-LC leads to an efficient separation of acidic 2-AA N-glycans carrying terminal sialylation without the need for an ion-pairing reagent. The method and compositions described herein may be used with RP-nano-LC-MS and a 96-well plate sample preparation, which allows attomolar sensitivity and high throughput.

主权项

1. A method of analyzing the glycans of a glycoprotein, comprising
(i) labeling the glycans with anthranilic acid (2-AA); and (ii) separating the labeled glycans using reversed-phase liquid chromatography (RP-LC), wherein
(a) the mobile phase used during RP-LC is acidic; and/or(b) the reversed-phase liquid chromatography (RP-LC) is performed under conditions under which the carboxyl group of the 2-AA label attached to the glycans is neutral;and optionally (iii) subjecting the labeled glycans to mass spectrometry (MS) after the separation using RP-LC.