Abstract
O-Acetylation of sialic acid in protein N-glycans is an important modification and can occur at either 4-, 7-, 8- or 9-position in various combinations. This modification is usually labile under alkaline reaction conditions. Consequently, a permethylation-based analytical method, which has been widely used in glycomics studies, is not suitable for profiling O-acetylation of sialic acids due to the harsh reaction conditions. Alternatively, methylamidation can be used for N-glycan analysis without affecting the base-labile modification of sialic acid. In this report, we applied both permethylation and methylamidation approaches to the analysis of O-acetylation in sialic acids. It has been demonstrated that methylamidation not only stabilizes sialic acids during MALDI processing but also allow for characterization of their O-acetylation pattern. In addition, LC-MS/MS experiments were carried out to distinguish between the O-acetylated glycans with potential isomeric structures. The repeatability of methylamidation was examined to evaluate the applicability of the approach to profiling of O-acetylation in sialic acids. In conclusion, the combination of methylamidation and permethylation methodology is a powerful MALDI-TOF MS-based tool for profiling O-acetylation in sialic acids applicable to screening of N-glycans.
Highlights
IntroductionMALDI-MS is the desired technique for profiling glycans[28,29,30,31]
Was most likely to be a product of immune reaction
This study aims at the development of MALDI-TOF MS for glycomics, especially the profiles of O-acetylation in sialic acids
Summary
MALDI-MS is the desired technique for profiling glycans[28,29,30,31]. Permethylation process removes acetyl esters, which precludes its application to the characterization of O-acetylation of sialic acids. An alternative strategy for stabilizing a sialic acid is the selective modification of its carboxyl group, including methylamidation[32,33,34,35], dimethylamidation[19], methyl esterification[36] and ethyl esterification[37,38]. This study aims at the development of MALDI-TOF MS for glycomics, especially the profiles of O-acetylation in sialic acids. The enzymatically released and purified fish serum N-glycans were subjected to methylamidation and permethylation derivatization and analysed using MALDI-TOF MS and LC-MS/MS. The tandem mass spectrometry experiments were performed to characterize glycan structures
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