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QuaNCAT: quantitating proteome dynamics in primary cells

Nature Methods volume 10, pages 343–346 (2013)Cite this article

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Abstract

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography–tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli.

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Figure 1: Schematic QuaNCAT workflow.
Figure 2: Global changes of protein expression in primary human CD4+ T cells after 2-h and 4-h activation.
Figure 3: Interaction network of differentially expressed proteins after activation of primary T cells for 2 h and 4 h.

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Acknowledgements

This work was supported by Wellcome Trust grant WT094296MA and EU-FP7 'Sybilla' number 201106 to O.A.; D.C.D. was supported by a Deutsche Forschungsgemeinschaft Emmy Noether grant DI1512/1-1. B.G.D. and O.A. are supported by Royal Society Wolfson Research Merit awards. We thank P. Charles, S. Taylor and E. Giannoulatou for advice on the use of bioinformatics and statistics software, W. Paster and K. Nika for helpful suggestions and for critical feedback on the manuscript, and M. Selbach for helpful suggestions. V.G. was supported by a Ph.D. fellowship from the Biotechnology and Biological Sciences Research Council, O.B. by a Marie Curie Intra European Fellowship, B.B. by a Rhodes scholarship. This paper is dedicated to Jamie.

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Author notes

  1. Andrew J M Howden, Omar Boutureira & David C Trudgian

    Present address: Present addresses: Division of Plant Sciences, College of Life Sciences, University of Dundee, Invergowrie, Dundee, UK (A.J.M.H.), Departament de Química Analítica i Química Orgànica, Universitat Rovira i Virgili, Tarragona, Spain (O.B.) and Biochemistry Department and Proteomics Core, University of Texas Southwestern Medical Center, Dallas, Texas, USA (D.C.T.).,

  2. Andrew J M Howden, Vincent Geoghegan, Kristin Katsch and Georgios Efstathiou: These authors contributed equally to this work.

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Andrew J M Howden, Vincent Geoghegan, Kristin Katsch, Georgios Efstathiou, Benjamin Thomas, David C Trudgian & Oreste Acuto

  2. Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, UK

    Bhaskar Bhushan, Omar Boutureira & Benjamin G Davis

  3. Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, UK

    Benedikt M Kessler

  4. Emmy Noether Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany

    Daniela C Dieterich

  5. Institute for Pharmacology and Toxicology, Otto von Guericke University, Magdeburg, Germany

    Daniela C Dieterich

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  1. Andrew J M Howden
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Contributions

A.J.M.H., B.T., D.C.T., V.G. and O.A. initially conceived the QuaNCAT strategy. A.J.M.H., V.G., K.K., G.E., B.T., D.C.T., O.B., B.B., B.G.D. and O.A. designed and optimized the QuaNCAT procedure. B.B. and O.B. synthesized reagents for the CuAAC reaction and associated cell labeling; D.C.D. provided the cleavable tag and an improved protocol for affinity purification; A.J.M.H., V.G., K.K. and G.E. performed cell stimulation, metabolic labeling, CuAAC reactions in T cell extracts, protein affinity purification; optimized CuAAC protocol was initially performed by B.B. and O.B. on A.J.M.H.'s initial cell extracts; K.K., G.E., B.M.K. and O.A. performed radioactive labeling; K.K. performed flow cytometry analysis. A.J.M.H., V.G., K.K., G.E. and B.T. carried out mass spectrometry experiments and data analysis; A.J.M.H., V.G., K.K., G.E., B.T., B.B., B.G.D. and O.A. wrote the manuscript.

Corresponding authors

Correspondence to Benjamin G Davis or Oreste Acuto.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1, Supplementary Note (PDF 2760 kb)

Supplementary Table 1

List of data set 1. (XLSX 125 kb)

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Howden, A., Geoghegan, V., Katsch, K. et al. QuaNCAT: quantitating proteome dynamics in primary cells. Nat Methods 10, 343–346 (2013). https://doi.org/10.1038/nmeth.2401

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