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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.

No MeSH data available.


Repeatability of profiling O-acetylation in sialic acids by methylamidation for Neu5NAc2Hex5HexNAc4.The graph shows the average relative intensities of OAc0-6Hex5HexNAc4Neu5NAc2, with error bars for standard deviation. (a) Five methylamidated samples were prepared from the pooled serum in the same day; (b) The day-to-day repeatability analysis was performed for three different days; (c) Native glycans were measured by ESI-MS.
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f2: Repeatability of profiling O-acetylation in sialic acids by methylamidation for Neu5NAc2Hex5HexNAc4.The graph shows the average relative intensities of OAc0-6Hex5HexNAc4Neu5NAc2, with error bars for standard deviation. (a) Five methylamidated samples were prepared from the pooled serum in the same day; (b) The day-to-day repeatability analysis was performed for three different days; (c) Native glycans were measured by ESI-MS.

Mentions: Methylamidation has been previously proven to be an effective derivatization strategy for sialic acid-containing N-glycans32333435. Complete methylamidation of 2,6-sialyllactose could be achieved within 10 min, whereas the complete reaction for 2,3-sialyllactose took about 30 min32. So far, the applicability of methylamidation to profiling O-acetylation patterns of sialic acids has not been investigated. Because no O-acetylated N-glycan standards were available, we used the N-glycans from pooled serum samples of multiple crucian carp for repeatability testing. The glycans Neu5NAc2Hex5HexNAc4 with different O-acetyl groups were selected to investigate the stability of O-acetyl groups, by comparing ESI-MS analysis of native glycans and MALDI-MS analysis of methylamidated glycans. The intensities of native glycans from 5 technical replicates in ESI-MS experiments were summed and normalized to the sum of intensities of the highest peak (Fig. 2a). To investigate the stability of O-acetyl groups under methylamidation conditions, 5 samples from serum pool were individually PNGase-F digested and derivatized, followed by MALDI-MS analysis. The intensities of each glycan from 3 spots in MALDI target were summed and normalized to the sum of intensities of the highest peak (i.e. the glycan containing three O-acetyl groups). The average relative intensity values for six glycoforms are presented in Fig. 2b, with the CVs ranging from 1.9% to 25.4%. We then derivatized 5 N-glycan samples from the same serum pool collected on each day for two additional days to evaluate the day-to-day repeatability38. The relative intensity values of six glycoforms for three different days are illustrated in Fig. 2c, with the CVs ranging from 0.8 to 27.8%. While the profiles between native glycans (ESI-MS) and their methylamine derivatives (MALDI-MS) were similar, relatively lower intensities in ESI-MS spectra were observed for the glycans containing four, five and six O-acetyl groups, respectively. To further demonstrate the applicability of the approach, we analysed N-glycans from serum samples of additional six fish species.


Characterization of O -acetylation in sialoglycans by MALDI-MS using a combination of methylamidation and permethylation
Repeatability of profiling O-acetylation in sialic acids by methylamidation for Neu5NAc2Hex5HexNAc4.The graph shows the average relative intensities of OAc0-6Hex5HexNAc4Neu5NAc2, with error bars for standard deviation. (a) Five methylamidated samples were prepared from the pooled serum in the same day; (b) The day-to-day repeatability analysis was performed for three different days; (c) Native glycans were measured by ESI-MS.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Repeatability of profiling O-acetylation in sialic acids by methylamidation for Neu5NAc2Hex5HexNAc4.The graph shows the average relative intensities of OAc0-6Hex5HexNAc4Neu5NAc2, with error bars for standard deviation. (a) Five methylamidated samples were prepared from the pooled serum in the same day; (b) The day-to-day repeatability analysis was performed for three different days; (c) Native glycans were measured by ESI-MS.
Mentions: Methylamidation has been previously proven to be an effective derivatization strategy for sialic acid-containing N-glycans32333435. Complete methylamidation of 2,6-sialyllactose could be achieved within 10 min, whereas the complete reaction for 2,3-sialyllactose took about 30 min32. So far, the applicability of methylamidation to profiling O-acetylation patterns of sialic acids has not been investigated. Because no O-acetylated N-glycan standards were available, we used the N-glycans from pooled serum samples of multiple crucian carp for repeatability testing. The glycans Neu5NAc2Hex5HexNAc4 with different O-acetyl groups were selected to investigate the stability of O-acetyl groups, by comparing ESI-MS analysis of native glycans and MALDI-MS analysis of methylamidated glycans. The intensities of native glycans from 5 technical replicates in ESI-MS experiments were summed and normalized to the sum of intensities of the highest peak (Fig. 2a). To investigate the stability of O-acetyl groups under methylamidation conditions, 5 samples from serum pool were individually PNGase-F digested and derivatized, followed by MALDI-MS analysis. The intensities of each glycan from 3 spots in MALDI target were summed and normalized to the sum of intensities of the highest peak (i.e. the glycan containing three O-acetyl groups). The average relative intensity values for six glycoforms are presented in Fig. 2b, with the CVs ranging from 1.9% to 25.4%. We then derivatized 5 N-glycan samples from the same serum pool collected on each day for two additional days to evaluate the day-to-day repeatability38. The relative intensity values of six glycoforms for three different days are illustrated in Fig. 2c, with the CVs ranging from 0.8 to 27.8%. While the profiles between native glycans (ESI-MS) and their methylamine derivatives (MALDI-MS) were similar, relatively lower intensities in ESI-MS spectra were observed for the glycans containing four, five and six O-acetyl groups, respectively. To further demonstrate the applicability of the approach, we analysed N-glycans from serum samples of additional six fish species.

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.