Quantitation of Newly Synthesized Proteins by Pulse Labeling with Azidohomoalanine

  • Gertjan Kramer
  • Piotr T. Kasper
  • Luitzen de Jong
  • Chris G. de Koster
Part of the Methods in Molecular Biology book series (MIMB, volume 753)


Measuring protein synthesis and degradation rates on a proteomic scale is an important step toward modeling the kinetics in complicated cellular response networks. A gel-free method, able to quantify changes in the formation of new proteins on a 15 min timescale, compatible with mass spectrometry is described. The methionine analogue, azidohomoalanine (azhal), is used to label newly formed proteins during a short pulse-labeling period following an environmental switch in Escherichia coli. Following digestion a selective reaction against azhal-containing peptides is applied to enrich these peptides by diagonal chromatography. This technique enables quantitation of hundreds of newly synthesized proteins and provides insight into immediate changes in newly synthesized proteins on a proteomic scale after an environmental perturbation.

Key words

Pulse-chase labeling translational regulation azhal quantitative proteomics E. coli BONCAT COFRADIC 



The authors wish to acknowledge Dr. A. Bock and Dr. M. Thanbichler for providing E. coli strain MTD123 and M.A. Nessen for synthesis of azhal.


  1. 1.
    Hoper, D., Bernhardt, J., and Hecker, M. (2006) Salt stress adaptation of Bacillus subtilis: A physiological proteomics approach, Proteomics 6, 1550–1562.PubMedCrossRefGoogle Scholar
  2. 2.
    Pedersen, S., Bloch, P. L., Reeh, S., and Neidhardt, F. C. (1978) Patterns of protein synthesis in E. coli: A catalog of the amount of 140 individual proteins at different growth rates, Cell 14, 179–190.PubMedCrossRefGoogle Scholar
  3. 3.
    Bateman, R. J., Munsell, L. Y., Chen, X., Holtzman, D. M., and Yarasheski, K. E. (2007) Stable isotope labeling tandem mass spectrometry (SILT) to quantify protein production and clearance rates, J. Am. Soc. Mass Spectrom. 18, 997–1006.PubMedCrossRefGoogle Scholar
  4. 4.
    Cargile, B. J., Bundy, J. L., Grunden, A. M., and Stephenson, J. L., Jr. (2004) Synthesis/degradation ratio mass spectrometry for measuring relative dynamic protein turnover, Anal. Chem. 76, 86–97.PubMedCrossRefGoogle Scholar
  5. 5.
    Gustavsson, N., Greber, B., Kreitler, T., Himmelbauer, H., Lehrach, H., and Gobom, J. (2005) A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry, Proteomics 5, 3563–3570.PubMedCrossRefGoogle Scholar
  6. 6.
    Ong, S. E., Blagoev, B., Kratchmarova, I., Kristensen, D. B., Steen, H., Pandey, A., and Mann, M. (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics, Mol. Cell. Proteomics 1, 376–386.PubMedCrossRefGoogle Scholar
  7. 7.
    Pratt, J. M., Petty, J., Riba-Garcia, I., Robertson, D. H., Gaskell, S. J., Oliver, S. G., and Beynon, R. J. (2002) Dynamics of protein turnover, a missing dimension in proteomics, Mol. Cell. Proteomics 1, 579–591.PubMedCrossRefGoogle Scholar
  8. 8.
    Kramer, G., Sprenger, R. R., Back, J., Dekker, H. L., Nessen, M. A., van Maarseveen, J. H., de Koning, L. J., Hellingwerf, K. J., de Jong, L., and de Koster, C. G. (2009) Identification and quantitation of newly synthesized proteins in E. coli by enrichment of azido homoalanine-labeled peptides with diagonal chromatography, Mol. Cell. Proteomics 8, 1599–1611.PubMedCrossRefGoogle Scholar
  9. 9.
    Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A., and Schuman, E. M. (2006) Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT), Proc. Natl. Acad. Sci. USA 103, 9482–9487.PubMedCrossRefGoogle Scholar
  10. 10.
    Nessen, M. A., Kramer, G., Back, J., Baskin, J. M., Smeenk, L. E. J., de Koning, L. J., van Maarseveen, J. H., de Jong, L., Bertozzi, C. R., Hiemstra, H., and de Koster, C. G. (2009) Selective enrichment of azide-containing peptides from complex mixtures, J. Proteome Res. 8, 3702–3711.PubMedCrossRefGoogle Scholar
  11. 11.
    Meiring, H. D., van der Heeft, E., ten Hove, G. J., and de Jong, A. P. J. M. (2002) Nanoscale LC-MS(n): Technical design and applications to peptide and protein analysis, J. Sep. Sci. 25, 557–568.CrossRefGoogle Scholar
  12. 12.
    Boehm, A. M., Putz, S., Altenhofer, D., Sickmann, A., and Falk, M. (2007) Precise protein quantification based on peptide quantification using iTRAQ, BMC Bioinformatics 8, 214.PubMedCrossRefGoogle Scholar
  13. 13.
    Kiick, K. L., Saxon, E., Tirrell, D. A., and Bertozzi, C. R. (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, Proc. Natl. Acad. Sci. USA 99, 19–24.PubMedCrossRefGoogle Scholar
  14. 14.
    Gevaert, K., Van Damme, J., Goethals, M., Thomas, G. R., Hoorelbeke, B., Demol, H., Martens, L., Puype, M., Staes, A., and Vandekerckhove, J. (2002) Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins, Mol. Cell. Proteomics 1, 896–903.PubMedCrossRefGoogle Scholar
  15. 15.
    Back, J. W., David, O., Kramer, G., Masson, G., Kasper, P. T., de Koning, L. J., de Jong, L., van Maarseveen, J. H., and de Koster, C. G. (2005) Mild and chemoselective peptide-bond cleavage of peptides and proteins at azido homoalanine, Angew. Chem. Int. Ed. 44, 7946–7950.CrossRefGoogle Scholar
  16. 16.
    Kasper, P. T., Back, J. W., Vitale, M., Hartog, A. F., Roseboom, W., de Koning, L. J., van Maarseveen, J. H., Muijsers, A. O., de Koster, C. G., and de Jong, L. (2007) An aptly positioned azido group in the spacer of a protein cross-linker for facile mapping of lysines in close proximity, Chembiochem 8, 1281–1292.PubMedCrossRefGoogle Scholar
  17. 17.
    Thanbichler, M., Neuhierl, B., and Bock, A. (1999) S-methylmethionine metabolism in Escherichia coli, J. Bacteriol. 181, 662–665.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Gertjan Kramer
    • 1
  • Piotr T. Kasper
    • 1
  • Luitzen de Jong
    • 1
  • Chris G. de Koster
    • 1
  1. 1.Mass Spectrometry of Biomacromolecules of the Swammerdam Institute for Life Sciences, University of AmsterdamAmsterdamThe Netherlands

Personalised recommendations