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Glycome mapping on DNA sequencing equipment

Abstract

Here we provide a detailed protocol for the analysis of protein-linked glycans on DNA sequencing equipment. This protocol satisfies the glyco-analytical needs of many projects and can form the basis of 'glycomics' studies, in which robustness, high throughput, high sensitivity and reliable quantification are of paramount importance. The protocol routinely resolves isobaric glycan stereoisomers, which is much more difficult by mass spectrometry (MS). Earlier methods made use of polyacrylamide gel–based sequencers, but we have now adapted the technique to multicapillary DNA sequencers, which represent the state of the art today. In addition, we have integrated an option for HPLC-based fractionation of highly anionic 8-amino-1,3,6-pyrenetrisulfonic acid (APTS)-labeled glycans before rapid capillary electrophoretic profiling. This option facilitates either two-dimensional profiling of complex glycan mixtures and exoglycosidase sequencing, or MS analysis of particular compounds of interest rather than of the total pool of glycans in a sample.

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Figure 1: Protocol workflow.
Figure 2: Comparison of gel- and CE-based glycan analysis on DNA sequencing equipment.
Figure 3: Exoglycosidase sequencing of a pure biantennary nonsialylated core fucosylated complex structure with a bisecting N-acetylglucosamine standard.
Figure 4: NP-HPLC separation of human serum N-glycans.

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Acknowledgements

We thank A. Van Hecke for technical assistance and A. Bredan for manuscript editing. W. Declercq critically read the protocol. We thank M. Aebi (ETH Zurich) for the use of the ABI 310 System. W.L. is a postdoctoral fellow with the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen; grant IWT/OZM/040636). N.C. is supported by a Marie Curie Excellence Grant of the European Union (Framework Programme 6). This work was further supported by the Fund for Scientific Research Flanders (FWO-Vlaanderen; G005201) and a grant from Ghent University (BOF No. 01106205).

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N.C. and W.L. designed and optimized the technology and wrote the manuscript. R.C. participated in the design of the method.

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Correspondence to Wouter Laroy.

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Laroy, W., Contreras, R. & Callewaert, N. Glycome mapping on DNA sequencing equipment. Nat Protoc 1, 397–405 (2006). https://doi.org/10.1038/nprot.2006.60

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