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Characterization of O -acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation

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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.

No MeSH data available.


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MALDI-MS analysis of N-glycans from a representative serum sample of grass carp (Grass-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
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f4: MALDI-MS analysis of N-glycans from a representative serum sample of grass carp (Grass-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.

Mentions: Three biological replicates of grass carp (Ctenopharyngodon idella) serum samples were analysed. The permethylated and methylamidated N-glycans profiles from a representative serum sample, labelled as Grass-1, are shown in Fig. 4, in which two major ions at m/z 2723.3 and 3084.5 correspond to triantennary oligosaccharides with chemical compositions of Hex7HexNAc5 and Neu5NAc1Hex7HexNAc5 (Fig. 4a). In the high mass region, the ions at m/z 3737.8, 3941.8, 4098.9 and 4460.1 correspond to tetraantennary oligosaccharides with different numbers of terminal Hex residues and sialic acid residues.


Characterization of O -acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation
MALDI-MS analysis of N-glycans from a representative serum sample of grass carp (Grass-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384204&req=5

f4: MALDI-MS analysis of N-glycans from a representative serum sample of grass carp (Grass-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
Mentions: Three biological replicates of grass carp (Ctenopharyngodon idella) serum samples were analysed. The permethylated and methylamidated N-glycans profiles from a representative serum sample, labelled as Grass-1, are shown in Fig. 4, in which two major ions at m/z 2723.3 and 3084.5 correspond to triantennary oligosaccharides with chemical compositions of Hex7HexNAc5 and Neu5NAc1Hex7HexNAc5 (Fig. 4a). In the high mass region, the ions at m/z 3737.8, 3941.8, 4098.9 and 4460.1 correspond to tetraantennary oligosaccharides with different numbers of terminal Hex residues and sialic acid residues.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus