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Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS

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Journal of The American Society for Mass Spectrometry

Abstract

Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure–function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.

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Acknowledgment

The authors acknowledge Dr. Niclas Karlsson and Dr. Catherine Hayes for their ongoing collaboration with the UniCarb-DB database project. This research project was facilitated by access to the Australian Proteomics Analysis Facility (APAF) established under the Australian Government NCRIS program.

This work was funded by Macquarie University Research Excellence Scheme postgraduate scholarship and Cystic Fibrosis Australia postgraduate studentship grant, Australian National Data Service (ANDS) grant DC12A and National eResearch Collaboration Tools and Resources (NeCTAR) grant RT016.

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Authors and Affiliations

  1. Biomolecular Frontiers Research Centre, Macquarie University, North Ryde, NSW, Australia

    Arun V. Everest-Dass, Jodie L. Abrahams, Nicolle H. Packer & Matthew P. Campbell

  2. Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany

    Daniel Kolarich

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  1. Arun V. Everest-Dass
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  2. Jodie L. Abrahams
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  4. Nicolle H. Packer
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Correspondence to Nicolle H. Packer.

Electronic Supplementary Material

Figure S1

Workflow for glycan analysis including bioinformatic support for the annotation and interpretation of the generated MS/MS spectra. N- and O-linked glycans were released sequentially from salivary proteins dot blotted on to PVDF membranes, by PNGase F and reductive β-elimination and subsequently analyzed by PGC LC-ESI-IT MS/MS (PNG 158 kb)

Figure S2

Identification and confirmation of sialic acid linkage and arm location of monosialyated biantennary N-glycan isomers. MS/MS spectra were obtained from the doubly charged negative ion parent ions of m/z 965.82– (NeuAc1Hex5HexNAc4) eluting at 44.5 min (a), 45.5 min (b), 52.5 min (c), and 53.5 min (d); (e) shows the extracted ion chromatogram of the four m/z 965.82- isomers (JPEG 243 kb)

High resolution image (TIFF 1820 kb)

Figure S3

Identification and characterization of sialylated O-glycan isomers. (a) and (b) The negative ion fragmentation spectra of m/z 878.51– (NeuAc1Hex1HexNAc2) isomers eluting at 25 and 26.7 min, respectively. The extracted ion chromatogram of the m/z 878.52– isomers is shown in (c) (JPEG 86 kb)

High resolution image (TIFF 1167 kb)

Figure S4

Identification and characterization of sulphated O-glycan isomers. Negative ion fragmentation spectra of m/z 8131– (Sulph1Hex1HexNAc2) isomers eluting at 21.8 min (a) and 22.5 min (b) are shown; (c) shows an extracted ion chromatogram of m/z 8131– isomers (JPEG 90 kb)

High resolution image (TIFF 1262 kb)

Figure S5

Extracted ion chromatograms of m/z 1039.42– (Hex5HexNAc4dHex3) N-glycan isomers. The glycan structural and linkage isomers were orthogonally confirmed by exoglycosidase treatments. ABS: Arthrobacter ureafaciens sialidase (α2–3,6,8). BTG: bovine testes β-galactosidase (β1–3,4,6). AMF: Almond meal α-fucosidase (α1–3,4). BKF: bovine kidney α-fucosidase (α1–2, 6) (JPEG 64 kb)

High resolution image (TIFF 1077 kb)

Figure S6

Glycan annotation report generated by GlycoWorkBench tool [20] of monosialyated biantennary N-glycan isomers of m/z 965.82– (NeuAc1Hex5HexNAc4) differing in α2-6 sialylation of the antennae. (a) MS/MS spectra of the doubly charged negative ion eluting at 44.5 min and its GlycoWorkBench generated annotation based on in silico fragmentation (b) MS/MS spectra of the doubly charged negative ion eluting at 45.5 min and its Glycoworkbench generated annotation based on in silico fragmentation. To simplify the figure, only singly charged glycan fragment masses above a 3 % intensity threshold are displayed. (c) Statistical coverage from the two isomers generated by Glycoworkbench (JPEG 207 kb)

High resolution image (TIFF 2005 kb)

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Everest-Dass, A.V., Abrahams, J.L., Kolarich, D. et al. Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS. J. Am. Soc. Mass Spectrom. 24, 895–906 (2013). https://doi.org/10.1007/s13361-013-0610-4

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