Limits...
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.

No MeSH data available.


Related in: MedlinePlus

MALDI-MS analysis of N-glycans from a representative serum sample of bream carp (Bream-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384204&req=5

f6: MALDI-MS analysis of N-glycans from a representative serum sample of bream carp (Bream-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.

Mentions: The N-glycans from a representative serum sample of bream carp, labelled as Bream-1, were initially analysed using permethylation method (Fig. 6a). The most abundant structures are the triantennary glycoforms with different numbers of sialic acids and Hex terminal motifs. For example, the ion detected at m/z 2723.3 is a triantennary structure with the chemical composition of Hex7HexNAc5. The ions at m/z 3084.4 correspond to the composition of Neu5NAc1Hex7HexNAc5, with the addition of a sialic acid group. More complex structures were observed as the tetraantennary glycans with ions at m/z 3894.8 and 4255.9, respectively. Methylamidation was used to investigate the O-acetyl modification in bream carp serum N-glycans as well (Fig. 6b). The monosialylated glycans at m/z 2212.7, 2374.7 and 2536.7 can be assigned to the addition of an O-acetyl group to the oligosaccharides with m/z 2170.8, 2332.7 and 2494.7, respectively. The disialylated glycans, with the addition of two O-acetyl groups, were detected at m/z 2720.8 (OAc2Neu5NAc2Hex6HexNAc5) and 2882.8 (OAc2Neu5NAc2Hex7HexNAc5). This observation suggests that the O-acetylation patterns of serum N-glycans from bream carp are similar for silver carp and grass carp. The representative MS/MS spectra are presented in Figure S9 in Supporting Information. Similar glycoforms were detected in two additional biological replicates (Bream-2 and Bream-3) as shown in Figure S10b, Figure S10c, Figure S10e and Figure S10f in Supporting Information, although relative intensities varied among the three replicates. The proposed compositions for most detected ions are presented in Table S5 in Supporting Information.


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 bream carp (Bream-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

f6: MALDI-MS analysis of N-glycans from a representative serum sample of bream carp (Bream-1).(a) MS spectrum of permethylated N-glycans; (b) MS spectrum of methylamidated N-glycans.
Mentions: The N-glycans from a representative serum sample of bream carp, labelled as Bream-1, were initially analysed using permethylation method (Fig. 6a). The most abundant structures are the triantennary glycoforms with different numbers of sialic acids and Hex terminal motifs. For example, the ion detected at m/z 2723.3 is a triantennary structure with the chemical composition of Hex7HexNAc5. The ions at m/z 3084.4 correspond to the composition of Neu5NAc1Hex7HexNAc5, with the addition of a sialic acid group. More complex structures were observed as the tetraantennary glycans with ions at m/z 3894.8 and 4255.9, respectively. Methylamidation was used to investigate the O-acetyl modification in bream carp serum N-glycans as well (Fig. 6b). The monosialylated glycans at m/z 2212.7, 2374.7 and 2536.7 can be assigned to the addition of an O-acetyl group to the oligosaccharides with m/z 2170.8, 2332.7 and 2494.7, respectively. The disialylated glycans, with the addition of two O-acetyl groups, were detected at m/z 2720.8 (OAc2Neu5NAc2Hex6HexNAc5) and 2882.8 (OAc2Neu5NAc2Hex7HexNAc5). This observation suggests that the O-acetylation patterns of serum N-glycans from bream carp are similar for silver carp and grass carp. The representative MS/MS spectra are presented in Figure S9 in Supporting Information. Similar glycoforms were detected in two additional biological replicates (Bream-2 and Bream-3) as shown in Figure S10b, Figure S10c, Figure S10e and Figure S10f in Supporting Information, although relative intensities varied among the three replicates. The proposed compositions for most detected ions are presented in Table S5 in Supporting Information.

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