LAUSR

Sialic acids (Sia) are involved in various biological and pathological processes, and are often found attached to non-reducing ends of glycans through either α2,3- or α2,6-linkages. To quantitatively analyze glycan structures with these linkage isoforms by liquid chromatography-mass spectrometry (LC-MS), we established a linkage-specific two-step alkylamidation method for N-glycans. Using this method, carboxyl groups of α2,3- and α2,6-linked Sia are derivatized with two kinds of alkylamines with different mass values in a linkage-specific manner, allowing products to be easily distinguished. The reaction efficiencies for di-, tri-, and tetra-sialyl PA-N-glycans were >94%, with few by-products. Mixtures of 2-aminopyridine (PA)-tagged N-glycans from human α1-acid glycoprotein were subjected to the method, and products were analyzed by LC-MS and MS/MS, and simultaneously monitored with a fluorescence detector. The relative content of Siaα2-3Gal and Siaα2-6Gal was estimated from the integrated fluorescence intensity of each peak. Moreover, MS/MS data clearly indicated characteristic B-ion fragments of N-glycan branches, such as the sialyl Lex sequence, with Sia linkage-specific alkylamidation, suggesting that this method also provides useful information of branch sequences. We optimized the method with the aim of (1) enabling high-throughput analysis and (2) maximizing the analysis of glycans from various types of samples, including highly heterogeneous glycans.