Glycome characterization is a major challenge because of the structural complexity and diversity of glycans, and the presence of numerous isomeric structures in biological samples. This necessitates the development of analytical methods that are capable of providing detailed structural information and compatible with on-line glycan separation. Although tandem mass spectrometry (MS/MS) has been broadly applied to biopolymer sequencing, the commonly used collision-induced dissociation method often fails to generate sufficient structural details at the MS<sup>2</sup> level for comprehensive glycan structural elucidation. Meanwhile, electron capture and transfer dissociation methods require multiply charged precursors and perform poorly on glycans labeled with a reducing-end chromophore. These limitations can be overcome by ion activation with higher-energy electron irradiation, as employed in electronic excitation dissociation (EED). We showed that EED can produce extensive glycosidic and cross-ring fragments for both native and derivatized glycans on the chromatographic timescale. Our theoretical investigation revealed that EED involves radical-induced dissociation of open-ring di-radicals produced by an ionization-electron recapture process.<br/>Here, we will present the recent progress in the development of an EED-based method for automated and comprehensive characterization of glycomes, with a particular focus on its potential towards analysis of isomeric glycan mixtures. We will demonstrate successful implementation of EED with a variety of on-line separation techniques, including reversed phase and porous graphitic carbon liquid chromatographies, and ion mobility spectrometry. We will discuss the EED fragmentation behaviors of metal-adducted, permethylated glycans, as well as native glycans with a reducing-end fixed charge label, and key spectral features that could facilitate both topology and linkage determinations. Finally, we will introduce a bioinformatics approach that can take advantage of the rich structural information in the EED tandem mass spectral data for automated, accurate glycan sequencing.

Carbohydrate sequencing by electronic excitation dissociation

256th National Meeting (2018)

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