Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging

Abstract

A major aim of proteomics is the identification of proteins in a given proteome at a given metabolic state. This protocol describes the step-by-step labeling, purification and detection of newly synthesized proteins in mammalian cells using the non-canonical amino acid azidohomoalanine (AHA). In this method, metabolic labeling of newly synthesized proteins with AHA endows them with the unique chemical functionality of the azide group. In the subsequent click chemistry tagging reaction, azide-labeled proteins are covalently coupled to an alkyne-bearing affinity tag. After avidin-based affinity purification and on-resin trypsinization, the resulting peptide mixture is subjected to tandem mass spectrometry for identification. In combination with deuterated leucine-based metabolic colabeling, candidate proteins can be immediately validated. Bioorthogonal non-canonical amino-acid tagging can be combined with any subcellular fractionation, immunopurification or other proteomic method to identify specific subproteomes, thereby reducing sample complexity and enabling the identification of subtle changes in a proteome. This protocol can be completed in 5 days.

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Figure 1: The BONCAT strategy for labeling, detection and identification of newly synthesized proteins.
Figure 2: Dot blot analysis of AHA versus Met-treated samples.
Figure 3: Western blot analysis of AHA versus Met-treated sample purification fractions.
Figure 4: Structure of AHA-based modifications.

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Acknowledgements

We thank S.A. Kim and E.H. Friedrich for critical reading of the manuscript. This work was supported by the Howard Hughes Medical Institute, the Beckman Institute at the California Institute of Technology and NIH (R21DA020589 to E.M.S.). MS analysis was performed at the MS facility of the laboratory of R.J. Deshaies (Howard Hughes Medical Institute, Caltech), which is supported by the Beckman Institute at Caltech and a grant from the Department of Energy to R.J.D. and Barbara J. Wold. D.C.D. is supported by the German Academy for Natural Scientists Leopoldina (BMBF-LPD9901/8-95). J.G. was supported by R.J. Deshaies through Howard Hughes Medical Institute funds. A.J.L. was supported by a National Science Foundation Graduate Research Fellowship.

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Correspondence to Daniela C Dieterich.

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