Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Glycome mapping on DNA sequencing equipment


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. Guttman, A. & Pritchett, T. Capillary gel electrophoresis separation of high-mannose type oligosaccharides derivatized by 1-aminopyrene-3,6,8-trisulfonic acid. Electrophoresis 16, 1906–1911 (1995).

    Article  CAS  Google Scholar 

  2. Jackson, P. The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. Biochem. J. 270, 705–713 (1990).

    Article  CAS  Google Scholar 

  3. Evangelista, R.A., Guttman, A. & Chen, F.T. Acid-catalyzed reductive amination of aldoses with 8-aminopyrene-1,3,6- trisulfonate. Electrophoresis 17, 347–351 (1996).

    Article  CAS  Google Scholar 

  4. Callewaert, N., Geysens, S., Molemans, F. & Contreras, R. Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment. Glycobiology 11, 275–281 (2001).

    Article  CAS  Google Scholar 

  5. Papac, D.I., Briggs, J.B., Chin, E.T. & Jones, A.J. A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Glycobiology 8, 445–454 (1998).

    Article  CAS  Google Scholar 

  6. Packer, N.H., Lawson, M.A., Jardine, D.R. & Redmond, J.W. A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J. 15, 737–747 (1998).

    Article  CAS  Google Scholar 

  7. Ludwiczak, P., Brando, T., Monsarrat, B. & Puzo, G. Structural characterization of Mycobacterium tuberculosis lipoarabinomannans by the combination of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 73, 2323–2330 (2001).

    Article  CAS  Google Scholar 

  8. Suzuki, H., Muller, O., Guttman, A. & Karger, B.L. Analysis of 1-aminopyrene-3,6,8-trisulfonate-derivatized oligosaccharides by capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Chem. 69, 4554–4559 (1997).

    Article  CAS  Google Scholar 

  9. Sandra, K. et al. Characterization of cellobiohydrolase I N-glycans and differentiation of their phosphorylated isomers by capillary electrophoresis-q-trap mass spectrometry. Anal. Chem. 76, 5878–5886 (2004).

    Article  CAS  Google Scholar 

  10. Anumula, K.R. High-sensitivity and high-resolution methods for glycoprotein analysis. Anal. Biochem. 283, 17–26 (2000).

    Article  CAS  Google Scholar 

  11. Raman, R., Raguram, S., Venkataraman, G., Paulson, J.C. & Sasisekharan, R. Glycomics: an integrated systems approach to structure-function relationships of glycans. Nat. Methods. 2, 817–824 (2005).

    Article  CAS  Google Scholar 

  12. Breitling, R. et al. Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr. Purif. 25, 209–218 (2002).

    Article  CAS  Google Scholar 

  13. Defrancq, L., Callewaert, N., Zhu, J., Laroy, W. & Contreras, R. DSA-FACE: high-throughput analysis of the N-glycans of NS0-cell secreted antibodies. Bioprocess Int. 6, 60–68 (2004).

    Google Scholar 

  14. Reeves, P.J., Callewaert, N., Contreras, R. & Khorana, H.G. Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. USA 99, 13419–13424 (2002).

    Article  CAS  Google Scholar 

  15. Vervecken, W. et al. In vivo synthesis of mammalian-like, hybrid-type N-glycans in Pichia pastoris. Appl. Environ. Microbiol. 70, 2639–2646 (2004).

    Article  CAS  Google Scholar 

  16. Callewaert, N. et al. Noninvasive diagnosis of liver cirrhosis using DNA sequencer-based total serum protein glycomics. Nat. Med. 10, 429–434 (2004).

    Article  CAS  Google Scholar 

  17. Maras, M. et al. Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-α-D-mannosidase. J. Biotechnol. 77, 255–263 (2000).

    Article  CAS  Google Scholar 

  18. He, Z., Aristoteli, L.P., Kritharides, L. & Garner, B. HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation. Biochem. Biophys. Res. Commun. 343, 496–503 (2006).

    Article  CAS  Google Scholar 

  19. O'Shea, M.G. & Morell, M.K. High resolution slab gel electrophoresis of 8-amino-1,3, 6-pyrenetrisulfonic acid (APTS) tagged oligosaccharides using a DNA sequencer. Electrophoresis 17, 681–686 (1996).

    Article  CAS  Google Scholar 

  20. Khandurina, J., Olson, N.A., Anderson, A.A., Gray, K.A. & Guttman, A. Large-scale carbohydrate analysis by capillary array electrophoresis: part 1. Separation and scale-up. Electrophoresis 25, 3117–3121 (2004).

    Article  CAS  Google Scholar 

  21. Vogel, K., Kuhn, J., Kleesiek, K. & Gotting, C. A novel ultra-sensitive method for the quantification of glycosaminoglycan disaccharides using an automated DNA sequencer. Electrophoresis 27, 1363–1367 (2006).

    Article  CAS  Google Scholar 

  22. Wing, D.R. et al. High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide. Anal. Biochem. 298, 207–217 (2001).

    Article  CAS  Google Scholar 

  23. Tretter, V., Altmann, F. & Marz, L. Peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached α1-3 to the asparagine-linked N-acetylglucosamine residue. Eur. J. Biochem. 199, 647–652 (1991).

    Article  CAS  Google Scholar 

  24. Elbers, I.J. et al. Influence of growth conditions and developmental stage on N-glycan heterogeneity of transgenic immunoglobulin G and endogenous proteins in tobacco leaves. Plant Physiol. 126, 1314–1322 (2001).

    Article  CAS  Google Scholar 

Download references


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).

Author information

Authors and Affiliations



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

Corresponding author

Correspondence to Wouter Laroy.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Laroy, W., Contreras, R. & Callewaert, N. Glycome mapping on DNA sequencing equipment. Nat Protoc 1, 397–405 (2006).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing