Dynamic SILAC for global assessment of proteome turnover
Doherty, M.K., Hammond, D., Clague, M. J., Gaskell, S.J. & Beynon, R.J. (2009) Turnover of the human proteome: determination of protein intracellular stability by dynamic SILAC. J. Proteome Res. 8, 104-112 [PDF] [PubMed]
The proteome of any system is a dynamic entity, such that the intracellular concentration of a protein is dictated by the relative rates of synthesis and degradation. In this work, we have analyzed time- dependent changes in the incorporation of a stable amino acid resolved precursor, a protocol we refer to as “dynamic SILAC”, using 1-D gel separation followed by in-gel digestion and LC-MS/MS analyses to profile the intracellular stability of almost 600 proteins from human A549 adenocarcinoma cells, requiring multiple measures of the extent of labeling with stable isotope labeled amino acids in a classic label-chase experiment. As turnover rates are acquired, a profile can be built up that allows exploration of the ‘dynamic proteome’ and of putative features that predispose a protein to a high or a low rate of turnover. Moreover, measurement of the turnover rate of individual components of supramolecular complexes provides a unique insight in processes of protein complex assembly and turnover.
The proteome of any system is a dynamic entity, such that the intracellular concentration of a protein is dictated by the relative rates of synthesis and degradation. In this work, we have analyzed time- dependent changes in the incorporation of a stable amino acid resolved precursor, a protocol we refer to as “dynamic SILAC”, using 1-D gel separation followed by in-gel digestion and LC-MS/MS analyses to profile the intracellular stability of almost 600 proteins from human A549 adenocarcinoma cells, requiring multiple measures of the extent of labeling with stable isotope labeled amino acids in a classic label-chase experiment. As turnover rates are acquired, a profile can be built up that allows exploration of the ‘dynamic proteome’ and of putative features that predispose a protein to a high or a low rate of turnover. Moreover, measurement of the turnover rate of individual components of supramolecular complexes provides a unique insight in processes of protein complex assembly and turnover.