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

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.

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MALDI-MS analysis of N-glycans from a representative serum sample of bighead carp (Bighead-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
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f7: MALDI-MS analysis of N-glycans from a representative serum sample of bighead carp (Bighead-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.

Mentions: The N-glycan profiles of a representative serum sample from bighead carp (Hypophthalmichthys nobilis) were also investigated using both permethylation and methylamidation methods (Fig. 7). For permethylated glycans (Fig. 7a), the most abundant ions corresponded to triantennary glycoforms at m/z 2723.4, 2880.4 and 3084.4, which have the chemical compositions of Hex7HexNAc5, Neu5NAc1Hex6HexNAc5 and Neu5NAc1Hex7HexNAc5, respectively. High-mannose structures, m/z 1579.8 and 1783.9, were also detected. In the high mass region, several tetraantennary structures were observed, e.g. m/z 3533.7, 3894.9, 4051.9 and 4256.1, corresponding to different number of sialic acids and terminal Hex motifs.


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 bighead carp (Bighead-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

f7: MALDI-MS analysis of N-glycans from a representative serum sample of bighead carp (Bighead-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
Mentions: The N-glycan profiles of a representative serum sample from bighead carp (Hypophthalmichthys nobilis) were also investigated using both permethylation and methylamidation methods (Fig. 7). For permethylated glycans (Fig. 7a), the most abundant ions corresponded to triantennary glycoforms at m/z 2723.4, 2880.4 and 3084.4, which have the chemical compositions of Hex7HexNAc5, Neu5NAc1Hex6HexNAc5 and Neu5NAc1Hex7HexNAc5, respectively. High-mannose structures, m/z 1579.8 and 1783.9, were also detected. In the high mass region, several tetraantennary structures were observed, e.g. m/z 3533.7, 3894.9, 4051.9 and 4256.1, corresponding to different number of sialic acids and terminal Hex motifs.

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