Abstract
Although the classification of cell types often relies on the identification of cell surface proteins as differentiation markers, flow cytometry requires suitable antibodies and currently permits detection of only up to a dozen differentiation markers in a single measurement. We use multiplexed mass-spectrometric identification of several hundred N-linked glycosylation sites specifically from cell surface–exposed glycoproteins to phenotype cells without antibodies in an unbiased fashion and without a priori knowledge. We apply our cell surface–capturing (CSC) technology, which covalently labels extracellular glycan moieties on live cells, to the detection and relative quantitative comparison of the cell surface N-glycoproteomes of T and B cells, as well as to monitor changes in the abundance of cell surface N-glycoprotein markers during T-cell activation and the controlled differentiation of embryonic stem cells into the neural lineage. A snapshot view of the cell surface N-glycoproteins will enable detection of panels of N-glycoproteins as potential differentiation markers that are currently not accessible by other means.
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Change history
09 September 2009
In the version of this article initially published, in Methods, p.385, line 5, the concentration of MgCl2, given as 0.5 M, is incorrect. The correct concentration is 0.5 mM MgCl2.The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
This work has been funded in part with federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health (NIH), under contract no. N01-HV-28179 (to R.A.), from NIH RO1-AI51344-01 (to J.D.W.), from NIH N01-HV-28179-22 (to B.W.) and from National Center of Competence in Research (NCCR) Neural Plasticity and Repair (to B.W.). Thanks to Anne-Claude Gingras and Peter Zandstra for critical reading of the manuscript; to Andreas Hofmann and Thomas Bock for supplying information and graphics support; to Alexander Schmidt for LTQ-FT performance; and to Jimmy Eng, Andy Keller, Alexey Nesvizhskii, David Shteynberg, Luis Mendoza, Josh Tasman, James Eddes, Andreas Panagiotidis and Patrick Pedrioli for bioinformatic support.
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Authors and Affiliations
Contributions
B.W., R.A. and J.D.W. planned the project. B.W., C.H., D.B.-F., M.B. and R.O. carried out experimental work. R.S. carried out the Drosophila experiments. B.W. and D.B.-F. analyzed the data. B.W., R.A. and J.D.W. wrote the paper. All authors discussed the results and commented on the manuscript.
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Supplementary information
Supplementary Figures
Supplementary Figures 1–4 (PDF 1152 kb)
Supplementary Table 1
CSC identified Jurkat T lymphocyte proteins (XLS 45 kb)
Supplementary Table 2
CSC identified Jurkat T lymphocyte glycoproteins upon initial Neuramidase treatment (XLS 49 kb)
Supplementary Table 3
CSC identified Kc167 cell proteins (XLS 38 kb)
Supplementary Table 4
CSC identified Kc167 cell peptides (XLS 50 kb)
Supplementary Table 5
CSC identified Jurkat T lymphocyte peptides (XLS 70 kb)
Supplementary Table 6
CSC identified mouse splenocyte proteins (XLS 39 kb)
Supplementary Table 7
CSC identified mouse splenocyte peptides (XLS 68 kb)
Supplementary Table 8
CSC identified and quantified human Ramos and Jurkat lymphocyte proteins (XLS 47 kb)
Supplementary Table 9
CSC identified human Ramos and Jurkat lymphocyte peptides (XLS 63 kb)
Supplementary Table 10
CSC identified and quantified proteins from unstimulated and CD3/CD28 stimulated human Jurkat T lymphopcytes (XLS 55 kb)
Supplementary Table 11
CSC identified peptides from unstimulated and CD3/CD28 stimulated human Jurkat T lymphopcytes (XLS 187 kb)
Supplementary Table 12
CSC identified proteins from embryonic stem cell, embroid bodies and neural precursors (XLS 185 kb)
Supplementary Table 13
CSC identified peptides from embryonic stem cell, embroid bodies and neural precursors (XLS 322 kb)
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Wollscheid, B., Bausch-Fluck, D., Henderson, C. et al. Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol 27, 378–386 (2009). https://doi.org/10.1038/nbt.1532
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DOI: https://doi.org/10.1038/nbt.1532
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