Oxidative release of natural glycans for functional glycomics

Journal name:
Nature Methods
Volume:
13,
Pages:
528–534
Year published:
DOI:
doi:10.1038/nmeth.3861
Received
Accepted
Published online

Abstract

Glycans have essential roles in biology and the etiology of many diseases. A major hurdle in studying glycans through functional glycomics is the lack of methods to release glycans from diverse types of biological samples. Here we describe an oxidative strategy using household bleach to release all types of free reducing N-glycans and O-glycan-acids from glycoproteins, and glycan nitriles from glycosphingolipids. Released glycans are directly useful in glycomic analyses and can be derivatized fluorescently for functional glycomics. This chemical method overcomes the limitations in glycan generation and promotes archiving and characterization of human and animal glycomes and their functions.

At a glance

Figures

  1. Comparison of ORNG by NaClO with traditional glycomics methods.
    Figure 1: Comparison of ORNG by NaClO with traditional glycomics methods.

    Traditional glycomic methods use different enzymes/chemicals and lengthy procedures to release glycans from miligram amounts of biological samples. Using ORNG, bleach serves as a single chemical reagent to quickly release N-, O-, and GSL-glycans with different reducing end modifications from kilogram amounts of biological samples.

  2. NaClO treatment of glycoproteins to release N-glycans.
    Figure 2: NaClO treatment of glycoproteins to release N-glycans.

    (a) Chemical scheme of NaClO treatment. (b) MALDI-TOF-MS profiles of N-glycans released from several glycoproteins by bleach treatment. (c) MALDI-TOF-MS profiles of permethylated glycans released from fetuin by PNGase F digestion (top) or bleach treatment (bottom). (d) Normal phase (NP) HPLC profiles of AEAB-conjugated N-glycans released from fetuin by PNGase F digestion (top) or bleach treatment (bottom). Peaks at 10–17 min were non-glycan peaks, presumably generated from oxidized peptide fragments. (e) MALDI-TOF-MS profiles of human plasma permethylated N-glycans released by PNGase F digestion (top) and bleach (bottom). (f) MALDI-TOF-MS profiles of N-glycans released by NaClO from egg white (top) and egg yolk (bottom). H, hexose; N, N-acetylhexosamine; F, fucose; S, sialic acid. The y-axis scales are not calibrated to reflect quantitative comparison.

  3. Lectin binding on the microarray of N-glycan-AEAB conjugates.
    Figure 3: Lectin binding on the microarray of N-glycan–AEAB conjugates.

    (ad) Binding of concanavalin A (ConA) (a), Aleuria aurantia lectin (AAL) (b), Sambucus nigra lectin (SNA) (c) and Ricinus communis agglutinin I (RCA I) (d) prepared from ovalbumin (1–25), egg yolk (26–60), FBS (61–64) and bovine IgG (65–67).

  4. NaClO treatment of glycoproteins to release O-glycans.
    Figure 4: NaClO treatment of glycoproteins to release O-glycans.

    (a) Chemical scheme of O-glycan release and labeling. (b) MALDI-TOF-MS profile of O-glycans released from porcine stomach mucin (PSM). (c) MALDI-TOF-MS profile of glycans from PSM and fetuin after NaClO treatment and permethylation. (d,e) MALDI-TOF-MS (d) and HPLC (e) profiles of O-glycans released from PSM by NaClO and labeled with mono-Fmoc ethylenediamine. Fluorescence excitation, 254 nm; emission, 340 nm.

  5. Release and tagging of glycans from GSLs by NaClO.
    Figure 5: Release and tagging of glycans from GSLs by NaClO.

    (a) Chemical scheme of GSL release and labeling. (b) MALDI-TOF-MS profile of porcine brain gangliosides (PBG) after NaClO treatment. (c) MALDI-TOF-MS profile of porcine brain tissue after NaClO treatment and permethylation. (d) MALDI-TOF-MS analysis of reduction products of glycan nitriles before (top) and after permethylation (bottom). (e) MALDI-TOF-MS profile of the Fmoc-protected glycan amines from PBG. (f) HPLC profile of Fmoc-protected glycan amines from PBG.

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Author information

Affiliations

  1. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA.

    • Xuezheng Song,
    • Hong Ju,
    • Yi Lasanajak,
    • Matthew R Kudelka &
    • David F Smith
  2. Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

    • Richard D Cummings

Contributions

X.S., D.F.S. and R.D.C. conceived the method; X.S., H.J., M.R.K. and Y.L. performed experiments; X.S., D.F.S. and R.D.C. analysed the data and wrote the paper.

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The authors declare no competing financial interests.

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