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Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells

Nature Methods volume 13, pages 81–86 (2016)Cite this article

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Abstract

Protein O-glycosylation has key roles in many biological processes, but the repertoire of O-glycans synthesized by cells is difficult to determine. Here we describe an approach termed Cellular O-Glycome Reporter/Amplification (CORA), a sensitive method used to amplify and profile mucin-type O-glycans synthesized by living cells. Cells convert added peracetylated benzyl-α-N-acetylgalactosamine to a large variety of modified O-glycan derivatives that are secreted from cells, allowing for easy purification for analysis by HPLC and mass spectrometry (MS). Relative to conventional O-glycan analyses, CORA resulted in an ∼100–1,000-fold increase in sensitivity and identified a more complex repertoire of O-glycans in more than a dozen cell types from Homo sapiens and Mus musculus. Furthermore, when coupled with computational modeling, CORA can be used for predictions about the diversity of the human O-glycome and offers new opportunities to identify novel glycan biomarkers for human diseases.

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Figure 1: Overview of CORA.
Figure 2: The chaperone Cosmc and active T-synthase are required for production of core 1– and core 2–based Bn-O-glycans.
Figure 3: Accuracy of CORA for profiling the O-glycome.
Figure 4: Sensitivity of CORA.
Figure 5: MALDI-TOF-MS/MS profiling of the O-glycome of primary cells.

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Acknowledgements

We thank J. Heimburg-Molinaro for help in editing and D.F. Smith and J. Zeng for critical reading of the manuscript. LOX cells were a kind gift from O. Fodstad (Oslo University Hospital, Oslo, Norway). MKN45, Colo205, MDA-MB-231 and MCF7 cells were a kind gift from H. Clausen (University of Copenhagen, Copenhagen, Denmark). pPSVE1-B1a was from J.D. Esko, UCSD, La Jolla, California, USA. BaGs6 anti-Tn was a kind gift from the late G. Springer (The Chicago Medical School, Chicago, Illinois, USA). This work was supported by the US National Institutes of Health (grants U01CA168930 to T.J. and R.D.C. and P41GM103694 to R.D.C.), a Georgia Cancer Coalition (now Georgia Research Alliance (GRA)) Award (to T.J.) and the Biotechnology and Biological Sciences Research Council (grant BB/K016164/1 to A.D. and S.M.H. for Core Support for Collaborative Research). A.D. is supported by a Wellcome Trust Senior Investigator Award. We also acknowledge support from the Emory Integrated Proteomics Core.

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Authors and Affiliations

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

    Matthew R Kudelka, Yingchun Wang, Duc M Duong, Xuezheng Song, Nicholas T Seyfried & Tongzhong Ju

  2. Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

    Matthew R Kudelka & Richard D Cummings

  3. Department of Life Sciences, Imperial College London, London, UK

    Aristotelis Antonopoulos, Anne Dell & Stuart M Haslam

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Contributions

M.R.K., R.D.C. and T.J. conceived of the project. M.R.K., Y.W., N.T.S., A.D., S.M.H., R.D.C. and T.J. designed experiments. M.R.K., A.A., Y.W., D.M.D. and X.S. performed experiments. M.R.K., A.A., Y.W., A.D., S.M.H., R.D.C. and T.J. analyzed the data. M.R.K., R.D.C. and T.J. wrote the manuscript. All authors edited the manuscript.

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Correspondence to Tongzhong Ju.

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Kudelka, M., Antonopoulos, A., Wang, Y. et al. Cellular O-Glycome Reporter/Amplification to explore O-glycans of living cells. Nat Methods 13, 81–86 (2016). https://doi.org/10.1038/nmeth.3675

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