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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.


Related in: MedlinePlus

MALDI-MS analysis of N-glycans from a representative serum sample of crucian carp (Crucian-1).Monosaccharides represented as galactose (), mannose (), N-acetylglucosamine (), N-acetylneuraminic () and fucose (). (a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
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f1: MALDI-MS analysis of N-glycans from a representative serum sample of crucian carp (Crucian-1).Monosaccharides represented as galactose (), mannose (), N-acetylglucosamine (), N-acetylneuraminic () and fucose (). (a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.

Mentions: Permethylation reaction removes O-linked modification, thus the resultant MALDI MS spectrum can be significantly simplified and used to derive the composition of N-glycans. A representative MALDI-MS spectrum of permethylated N-glycans from crucian carp (Carassius carassius) serum sample is presented in Fig. 1a. For simplicity, this serum sample was labelled as Crucian-1. The primary structures of N-glycans isolated from crucian carp serum are similar to that of salmon serum2627. Four major glycoforms, m/z 2431.1, 2792.2, 3241.4 and 3602.5, correspond to one, two and three sialic acid-containing oligosaccharides with chemical compositions of Neu5NAc1Hex5HexNAc4, Neu5NAc2Hex5HexNAc4, Neu5NAc2Hex6HexNAc5, and Neu5NAc3Hex6HexNAc5, respectively. Two high mannose structures were detected at m/z 1579.7 (Hex5HexNAc2) and m/z 1783.8 (Hex6HexNAc2). The non-sialylated biantennary structure was detected at m/z 2069.9 with a composition of Hex5HexNAc4. The detection of ions at m/z 4051.7 and 4412.9 suggests the presence of tetra-antennary structures with compositions of Neu5NAc3Hex7HexNAc6 and Neu5NAc4Hex7HexNAc6, respectively.


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 crucian carp (Crucian-1).Monosaccharides represented as galactose (), mannose (), N-acetylglucosamine (), N-acetylneuraminic () and fucose (). (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

f1: MALDI-MS analysis of N-glycans from a representative serum sample of crucian carp (Crucian-1).Monosaccharides represented as galactose (), mannose (), N-acetylglucosamine (), N-acetylneuraminic () and fucose (). (a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
Mentions: Permethylation reaction removes O-linked modification, thus the resultant MALDI MS spectrum can be significantly simplified and used to derive the composition of N-glycans. A representative MALDI-MS spectrum of permethylated N-glycans from crucian carp (Carassius carassius) serum sample is presented in Fig. 1a. For simplicity, this serum sample was labelled as Crucian-1. The primary structures of N-glycans isolated from crucian carp serum are similar to that of salmon serum2627. Four major glycoforms, m/z 2431.1, 2792.2, 3241.4 and 3602.5, correspond to one, two and three sialic acid-containing oligosaccharides with chemical compositions of Neu5NAc1Hex5HexNAc4, Neu5NAc2Hex5HexNAc4, Neu5NAc2Hex6HexNAc5, and Neu5NAc3Hex6HexNAc5, respectively. Two high mannose structures were detected at m/z 1579.7 (Hex5HexNAc2) and m/z 1783.8 (Hex6HexNAc2). The non-sialylated biantennary structure was detected at m/z 2069.9 with a composition of Hex5HexNAc4. The detection of ions at m/z 4051.7 and 4412.9 suggests the presence of tetra-antennary structures with compositions of Neu5NAc3Hex7HexNAc6 and Neu5NAc4Hex7HexNAc6, respectively.

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