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Gel-Free Proteomics

Volume 753 of the series Methods in Molecular Biology pp 169-181

Date:

Quantitation of Newly Synthesized Proteins by Pulse Labeling with Azidohomoalanine

  • Gertjan KramerAffiliated withMass Spectrometry of Biomacromolecules of the Swammerdam Institute for Life Sciences, University of Amsterdam
  • , Piotr T. KasperAffiliated withMass Spectrometry of Biomacromolecules of the Swammerdam Institute for Life Sciences, University of Amsterdam
  • , Luitzen de JongAffiliated withMass Spectrometry of Biomacromolecules of the Swammerdam Institute for Life Sciences, University of Amsterdam
  • , Chris G. de KosterAffiliated withMass Spectrometry of Biomacromolecules of the Swammerdam Institute for Life Sciences, University of Amsterdam

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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