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Versatile fluorescent derivatization of glycans for glycomic analysis

Nature Methods volume 2, pages 845–850 (2005)Cite this article

Abstract

The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.

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Figure 1: The derivatization of free glycans with DAP.
Figure 2: Preparation and analyses of various GDAPs.
Figure 3: The derivatization of complex-type N-glycans with DAP and biotinylation of LNnT-DAP.
Figure 4: Functional recognition of GDAP conjugates by CBPs.
Figure 5: Preparation of a glycan-DAP-protein conjugate.
Figure 6: Preparation of a glycan-DAP microarray with selected GDAPs.

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Acknowledgements

This work was supported by US National Institutes of Health Grant HL065509 to G.P.S. and also supported in part by the Consortium for Functional Glycomics under National Institute of General Medical Sciences, US National Institutes of Health Grant GM62116. We thank A. Lee for help with glycan array analyses.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, 975 N.E. 10th St., BRC417, Oklahoma City, 73104, Oklahoma, USA

    Baoyun Xia, Ziad S Kawar, Tongzhong Ju, Richard A Alvarez & Richard D Cummings

  2. The Oklahoma Center for Medical Glycobiology, 975 N.E. 10th St., BRC417, Oklahoma City, 73104, Oklahoma, USA

    Baoyun Xia, Ziad S Kawar, Tongzhong Ju, Richard A Alvarez, Goverdhan P Sachdev & Richard D Cummings

  3. College of Medicine, University of Oklahoma Health Sciences Center, 975 N.E. 10th St., BRC417, Oklahoma City, 73104, Oklahoma, USA

    Baoyun Xia, Ziad S Kawar, Tongzhong Ju, Richard A Alvarez & Richard D Cummings

  4. College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, 73190, Oklahoma, USA

    Baoyun Xia & Goverdhan P Sachdev

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  1. Baoyun Xia
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Correspondence to Richard D Cummings.

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Supplementary information

Supplementary Fig. 1

Examples of glycans used in the study. (PDF 331 kb)

Supplementary Fig. 2

ESI-MS/MS spectra for GlcNAc2-DAP. (PDF 397 kb)

Supplementary Fig. 3

Identification and quantification of DAP on NHS-activated glass slides. (PDF 420 kb)

Supplementary Fig. 4

Free N-glycans analyzed by MALDI-TOF-MS before and after conjugation with DAP. (PDF 319 kb)

Supplementary Fig. 5

A mixture of free high mannose-type N-glycans Man5GlcNAc2 through Man9GlcNAc2. (PDF 329 kb)

Supplementary Fig. 6

Free hyaluronic acid-derived oligosaccharides analyzed by MALDI-TOF-MS before and after conjugation with DAP. (PDF 343 kb)

Supplementary Fig. 7

Free hyaluronic acid-derived oligosaccharides labeled with DAP or 2-AB and analyzed by HPLC. (PDF 270 kb)

Supplementary Fig. 8

Separation of mucin-derived O-glycan GDAPs by HPLC. (PDF 313 kb)

Supplementary Methods (PDF 117 kb)

Supplementary Data (PDF 70 kb)

Supplementary Discussion (PDF 61 kb)

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Xia, B., Kawar, Z., Ju, T. et al. Versatile fluorescent derivatization of glycans for glycomic analysis. Nat Methods 2, 845–850 (2005). https://doi.org/10.1038/nmeth808

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