No abstract

Neutralization of carboxylic acid is an important means to avoid sialic acid dissociation when sialylated glycans are analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In this paper, we describe a simple and rapid method to modify the sialic acids of sialylated glycans in the presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate (PyAOP). After methylamidation, sialylated glycans can be analyzed by MALDI-MS without loss of the sialic acid moiety. The electrospray ionization mass spectrometry (ESI-MS) and MALDI-MS analysis of both 3'- and 6'-sialyllactose derivatives indicated that the quantitative conversion of sialic acids was achieved, regardless of their linkage types. This derivatization strategy was further validated with the N-glycans released from three standard glycoproteins (fetuin, human acid glycoprotein, and bovine acid glycoprotein) containing different types of complex glycans. Most importantly, this derivatization method enabled the successful characterization of N-glycans of sera from different species (human, mouse, and rat) by MALDI-MS. Because of the mild reaction conditions, the modification in sialic acid residues can be retained. This improvement makes it possible to detect sialylated glycans containing O-acetylated sialic acid moieties using MALDI-MS in positive-ion mode.