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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References
Ghazalpour, A. et al. PLoS Genet. 7, e1001393 (2011).
Foss, E.J. et al. Nat. Genet. 39, 1369–1375 (2007).
Rogers, S. et al. Bioinformatics 24, 2894–2900 (2008).
Schwanhausser, B. et al. Nature 473, 337–342 (2011).
Mann, M. Nat. Rev. Mol. Cell Biol. 7, 952–958 (2006).
Geiger, T. et al. Nat. Protoc. 6, 147–157 (2011).
Schwanhausser, B., Gossen, M., Dittmar, G. & Selbach, M. Proteomics 9, 205–209 (2009).
Dieterich, D.C., Link, A.J., Graumann, J., Tirrell, D.A. & Schuman, E.M. Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).
Dieterich, D.C. et al. Nat. Protoc. 2, 532–540 (2007).
Van Kasteren, S.I., Kramer, H.B., Gamblin, D.P. & Davis, B.G. Nat. Protoc. 2, 3185–3194 (2007).
van Kasteren, S.I. et al. Nature 446, 1105–1109 (2007).
Szychowski, J. et al. J. Am. Chem. Soc. 132, 18351–18360 (2010).
Cox, J. & Mann, M. Nat. Biotechnol. 26, 1367–1372 (2008).
Smyth, G.K. in Bioinformatics and Computational Biology Solutions using R and Bioconductor (eds., Gentleman, R., Dudoit, S., Irizarry, R. & Huber, W.) 397–420 (Springer, New York, 2005).
Diehn, M. et al. Proc. Natl. Acad. Sci. USA 99, 11796–11801 (2002).
Eichelbaum, K., Winter, M., Diaz, M.B., Herzig, S. & Krijgsveld, J. Nat. Biotechnol. 30, 984–990 (2012).
The UniProt Consortium. Nucleic Acids Res. 40, D71–D75 (2012).
Cox, J. et al. J. Proteome Res. 11, 1794–1805 (2011).
Perkins, D.N., Pappin, D.J., Creasy, D.M. & Cottrell, J.S. Electrophoresis 20, 3551–3567 (1999).
Ihaka, R. & Gentleman, R.R. J. Comput. Graph. Statist. 5, 299–314 (1996).
Carrillo, B., Yanofsky, C., Laboissiere, S., Nadon, R. & Kearney, R.E. Bioinformatics 26, 98–103 (2010).
Smyth, G.K. & Speed, T.P. Methods 31, 265–273 (2003).
Smyth, G.K. Stat. Appl. Genet. Mol. Biol. 3, 3 (2004).
Benjamini, Y. & Hochberg, Y. J. R. Stat. Soc., B 57, 289–300 (1995).
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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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.
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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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DOI: https://doi.org/10.1038/nmeth.2401
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