Specific Analysis of α-2,3-Sialylated N-Glycan Linkage Isomers by Microchip Capillary Electrophoresis-Mass Spectrometry

Sialylated N-glycan isomers with alpha-2,3 and alpha-2,6 linkages play crucial and distinctive roles in diverse physiological and pathological processes. Changes of alpha-2,3-linked sialic acids in sialylated N-glycans are especially important in monitoring the initiation and progression of diseases. However, the specific analysis of alpha-2,3-sialylated N-glycan linkage isomers remains challenging due to their extremely low abundance and technical limitations in separation and detection. Herein, we designed an integrated strategy that combines linkage-specific derivatization and a charge-sensitive separation method based on microfluidic chip capillary electrophoresis-mass spectrometry (microchip CE-MS) for specific analysis of alpha-2,3-sialylated N-glycan linkage isomers for the first time. The alpha-2,6- and alpha-2,3-sialic acids were selectively labeled with methylamine (MA) and N,N-dimethylethylenediamine (DMEN), respectively, which selectively makes alpha-2,3-sialylated N-glycans positively charged and realizes online purification, concentration, and discrimination of alpha-2,3-sialylated N-glycans from other N-glycans in microchip CE-MS. This new approach was demonstrated with standard multisialylated N-glycans, and it was found that only the alpha-2,3-sialylated N-glycans migrated and were detected in order according to the number of alpha-2,3-sialic acids. Finally, this strategy was successfully applied in highly sensitive profiling and reproducible quantitation of the serum alpha-2,3-sialylated N-glycome from ovarian cancer (OC) patients, where 7 of 33 detected alpha-2,3-sialylated N-glycans significantly changed in the OC group compared with healthy controls.

Automated summary

A comprehensive strategy combining linkage-specific derivatization and charge-sensitive separation method was developed for specific analysis of alpha-2,3-sialylated N-glycan linkage isomers. This new approach demonstrated successful sensitive profiling and reproducible quantitation of serum alpha-2,3-sialylated N-glycome from ovarian cancer patients, identifying significant changes in certain glycan structures.