Methylamidation for Sialoglycomics by MALDI-MS: A Facile Derivatization Strategy for Both α2,3- and α2,6-Linked Sialic Acids

Abstract

Neutralization of carboxylic acid is an important means to avoid sialic acid dissociation when sialylated glycans are analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). In this paper, we describe a simple and rapid method to modify the sialic acids of sialylated glycans in the presence of methylamine and (7-azabenzotriazol-1-yloxy) trispyrrolidinophosphonium hexafluorophosphate (PyAOP). After methylamidation, sialylated glycans can be analyzed by MALDI-MS without loss of the sialic acid moiety. The electrospray ionization mass spectrometry (ESI-MS) and MALDI-MS analysis of both 3'- and 6'-sialyllactose derivatives indicated that the quantitative conversion of sialic acids was achieved, regardless of their linkage types. This derivatization strategy was further validated with the N-glycans released from three standard glycoproteins (fetuin, human acid glycoprotein, and bovine acid glycoprotein) containing different types of complex glycans. Most importantly, this derivatization method enabled the successful characterization of N-glycans of sera from different species (human, mouse, and rat) by MALDI-MS. Because of the mild reaction conditions, the modification in sialic acid residues can be retained. This improvement makes it possible to detect sialylated glycans containing O-acetylated sialic acid moieties using MALDI-MS in positive-ion mode.

Addressing the glycan complexity by using mass spectrometry: In the pursuit of decoding glycologic

“…Unlike the other deoxy sugars, sialic acid has a unique structure with nine carbon atoms, a carboxyl group at the C-1 position, and a carbonyl group at C-2 forming a glycosidic linkage. (Figure 1) Although this particular linkage is as labile as those of the other deoxy sugars, it becomes as stable as those of the other ordinary monosaccharides after esterification [60,61] or amidation [62,63] of a carboxyl group. Another important feature of sialic acid is that if linked to 3-position of adjacent galactose could form lactone structure, which can be used to distinguish the positional isomers of this particular monosaccharide [64].…”

Sensitive and Structure-Informative <i>N</i>-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization

“…61) Miura et al introduced a unique procedure for methyl esteri cation for derivatizing glycans attached to a solid support. 62) As another type of derivatization, the amidation of sialic acid residues using condensing reagent(s) was introduced by Sekiya et al 63) Amidation with methylamine 64) and acetohydrazide 65) was then investigated as more reliable methods for derivatizing sialic acid residues, regardless of their linkage types.…”

Solid-phase methylamidation for sialoglycomics by MALDI-MS

“…The resin was then dissolved in 100 μL DMSO solution containing 1 M methylamine hydrochloride and 0.5 M Nmethylmorpholine, followed by the addition of 100 μL PyAOP (50 mM in DMSO) solution [20]. The glycan derivatization reaction lasted for 30 min at room temperature before the chemicals were washed off from the solid support with 400 μL DMSO for three times and then 400 μL water for three times.…”

“…Some alternative approaches have been developed to stabilize the sialic acid moieties, including esterification [17], and solid-phase esterification [18], amidation [19], methylamidation [20]. All of these methods undergo two steps of purification before and after derivatization which would bring impurities, such as, derivative reagents, into the samples interfering the profile of MALDI-MS detection.…”

“…This activator was chosen for its ability to overcome the steric hindrance that resulted in incomplete conversions of α (2,3)-linked sialic acids. The sialic acid groups were stable and decomposition could be avoided in the positive-mode MALDI condition [20].…”