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Quantitative proteomics using stable isotope labeling with amino acids in cell culture

Abstract

Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d.

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Figure 1: Workflow of a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomic experiment.
Figure 2: Incorporation of heavy Lys (13C6) in the presence of complete serum.
Figure 3: Use of 5-plex stable isotope labeling with amino acids in cell culture experiment to monitor protein dynamics.
Figure 4: Liquid chromatography (LC) and mass spectrometry (MS) setup.
Figure 5: Distinguishing nonspecific and specific interactions using stable isotope labeling with amino acids in cell culture.
Figure 6: Mass resolution issues in ion trap mass spectrometers.

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Acknowledgements

This work was supported by National Institutes of Health Roadmap grant 'Technology Center for Networks and Pathways' (RR020839).

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Correspondence to Akhilesh Pandey.

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Harsha, H., Molina, H. & Pandey, A. Quantitative proteomics using stable isotope labeling with amino acids in cell culture. Nat Protoc 3, 505–516 (2008). https://doi.org/10.1038/nprot.2008.2

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