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Quantitation of Newly Synthesized Proteins by Pulse Labeling with Azidohomoalanine

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

Abstract

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 

Notes

Acknowledgments

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.

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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

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