Structural identification of N-glycan isomers using logically derived sequence tandem mass spectrometry

Abstract

N-linked glycosylation is one of the most important protein post-translational modifications. Despite the importance of N-glycans, the structural determination of N-glycan isomers remains challenging. Here we develop a mass spectrometry method, logically derived sequence tandem mass spectrometry (LODES/MSn), to determine the structures of N-glycan isomers that cannot be determined using conventional mass spectrometry. In LODES/MSn, the sequences of successive collision-induced dissociation are derived from carbohydrate dissociation mechanisms and apply to N-glycans in an ion trap for structural determination. We validate LODES/MSn using synthesized N-glycans and subsequently applied this method to N-glycans extracted from soybean, ovalbumin, and IgY. Our method does not require permethylation, reduction, and labeling of N-glycans, or the mass spectrum databases of oligosaccharides and N-glycan standards. Moreover, it can be applied to all types of N-glycans (high-mannose, hybrid, and complex), as well as the N-glycans degraded from larger N-glycans by any enzyme or acid hydrolysis.

Identification of Anomericity and Linkage of Arabinose and Ribose through Collision-Induced Dissociation

“…As demonstrated in the example of disaccharide linkage determination, understanding these dissociation mechanisms is useful when applying CID mass spectrometry to the structural determination. The findings in this work can be integrated with the mass spectrometry method we recently developed ,− to determine the structures of oligosaccharide, a long-standing bottleneck in carbohydrate research.…”

“…We showed that the CID mass spectra of isomers α-arabinose pyranose (α-Ara p ), β-arabinose pyranose (β-Ara p ), α-arabinose furanose (α-Ara f ), β-arabinose furanose (β-Ara f ), α-ribose pyranose (α-Rib p ), β-ribose pyranose (β-Rib p ), α-ribose furanose (α-Rib f ), and β-ribose furanose (β-Rib f ), the structures of which are illustrated in Figure , were markedly distinct. The results of this study can be applied to current mass spectrometry methods ,− for identifying pentoses in oligosaccharides.…”

Electrospray ionization in‐source decay of N‐glycans and the effects on N‐glycan structural identification

“…Sialic acid is known to be easily dissociated from oligosaccharides using CID in tandem mass spectrometry (MS/MS). 35,37 The chromatograms of tri-antennary N-glycan consisting of an α-2-3 linked sialic acid at different ion transferring capillary temperatures are shown in Figures 3A and 3B intensities, indicating ESI in-source decay of 30% solution occurs easier than the other compositions of solution. Previous study used thermometer ion and showed that ions generated from the solvent with small heat of evaporation contain smaller internal energy than the ions generated from the solvent with large heat of evaporation.…”

“…Triantennary N ‐glycan consisting of a sialic acid was gifted by Dr. Chung‐Yi Wu (Genomics Research Center, Academia Sinica, Taiwan). Details of extraction method have been described in our previous report 35 …”

“…N ‐Glycan is one major component of carbohydrate; it plays an important role in post‐translational modifications of proteins. Most small N ‐glycan ions generated by ESI are singly charged, but doubly charged ions start to dominate as the size of the N ‐glycans increases 35 . The fragmentation of carbohydrate ions during the ESI process has not been investigated.…”