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Mining the O-glycoproteome using zinc-finger nuclease–glycoengineered SimpleCell lines

Nature Methods volume 8, pages 977–982 (2011)Cite this article

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A Corrigendum to this article was published on 29 January 2015

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Abstract

Zinc-finger nuclease (ZFN) gene targeting is emerging as a versatile tool for engineering of multiallelic gene deficiencies. A longstanding obstacle for detailed analysis of glycoproteomes has been the extensive heterogeneities in glycan structures and attachment sites. Here we applied ZFN targeting to truncate the O-glycan elongation pathway in human cells, generating stable 'SimpleCell' lines with homogenous O-glycosylation. Three SimpleCell lines expressing only truncated GalNAcα or NeuAcα2-6GalNAcα O-glycans were produced, allowing straightforward isolation and sequencing of GalNAc O-glycopeptides from total cell lysates using lectin chromatography and nanoflow liquid chromatography–mass spectrometry (nLC-MS/MS) with electron transfer dissociation fragmentation. We identified >100 O-glycoproteins with >350 O-glycan sites (the great majority previously unidentified), including a GalNAc O-glycan linkage to a tyrosine residue. The SimpleCell method should facilitate analyses of important functions of protein glycosylation. The strategy is also applicable to other O-glycoproteomes.

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Figure 1: Flow diagram depicting the use of SimpleCell lines to map the O-glycoproteome.
Figure 2: Characterization of wild-type and SimpleCell T3M4 isogenic cells by immunocytology with monoclonal antibodies to C1GalT enzyme and the resulting glycans.
Figure 3: ESI-Orbitrap-ETD-MS2 spectra of O-glycopeptides.
Figure 4: ESI-Orbitrap-MS2 spectra of O-glycopeptide identified from nucleobindin 2 (residues 387–399; precursor m/z 626.3097, z = +3).

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Change history

  • 08 January 2015

    In the version of this article initially published, the T3M4 cell line used for the experiments was mislabeled as Capan-1. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank R. Chalkley, K. Medzihradszky, and T. Schwientek for helpful discussion and advice, M.A. Hollingsworth for critical reading of the manuscript, and T. Ju and R. Cummings (Emory University School of Medicine) for providing the C1GalT1 enzyme for generation of the monoclonal antibody, S. Itzkowitz (Mount Sinai Medical Center) for cell lines, and R. Viner for valuable advice on implementing the HCD-triggered ETD experiment. This work was supported by Kirsten og Freddy Johansen Fonden, A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal, The Carlsberg Foundation, The Novo Nordisk Foundation, The Alfred Benzon Foundation, The Danish Research Councils and a program of excellence from the University of Copenhagen.

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  1. Catharina Steentoft and Sergey Y Vakhrushev: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Cellular and Molecular Medicine and School of Dentistry, Copenhagen Center for Glycomics, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark

    Catharina Steentoft, Sergey Y Vakhrushev, Malene B Vester-Christensen, Katrine T-B G Schjoldager, Yun Kong, Eric Paul Bennett, Ulla Mandel, Hans Wandall, Steven B Levery & Henrik Clausen

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Contributions

C.S., S.Y.V., S.B.L. and H.C. designed and performed experiments, analyzed data and wrote the paper. K.T.-B.G.S. and H.W. designed experiments. M.B.V.-C., Y.K., E.P.B. and U.M. performed experiments.

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Correspondence to Steven B Levery or Henrik Clausen.

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The authors declare no competing financial interests.

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Supplementary Figures 1–3, Supplementary Tables 1–2 and Supplementary Results (PDF 2293 kb)

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Steentoft, C., Vakhrushev, S., Vester-Christensen, M. et al. Mining the O-glycoproteome using zinc-finger nuclease–glycoengineered SimpleCell lines. Nat Methods 8, 977–982 (2011). https://doi.org/10.1038/nmeth.1731

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