Turnover of the Human Proteome: Determination of Protein Intracellular Stability by Dynamic SILAC

Abstract

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.

Analysis of proteome dynamics in the mouse brain

“…Typically, in vivo labeling is achieved by the introduction of food source in which 1 of 20 natural amino acids has been isotopically labeled (SILAC) (22,34,35). Here, we have chosen to instead use a ubiquitously labeled food source-an approach that was initially developed by Wu et al (23).…”

“…The advent of modern proteomics has enabled scientists to use mass spectrometry to detect the incorporation of stable isotopes into proteins (21,22). In measuring turnover rates, the latter approach offers three potential advantages.…”

Neuregulin/ErbB Signaling in Developmental Myelin Formation and Nerve Repair

“…However, new methods are now available that are based on a pulse-labeling of proteins with stable (nonradioactive) isotope-labeled amino acids and on quantification of labeled and unlabeled proteins by mass spectroscopy (Selbach et al, 2008;Doherty et al, 2009;Schwanhausser et al, 2011;Sheean et al, 2014). With these techniques it was possible to assess translational control mechanisms during myelination in the PNS and their dependence on MAPK/Erk (Sheean et al, 2014).…”

Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation

“…The general idea is to metabolically label a population of proteins with a short pulse and then to quantify how much of this population is left after different lengths of chase. We and others have previously used stable isotope labeling by amino acids in cell culture (SILAC) to study protein synthesis and turnover (Andersen et al, 2005;Doherty et al, 2009;Hinkson and Elias, 2011;Jovanovic et al, 2015;Kristensen et al, 2013;Larance et al, 2013;Schwanhäusser et al, 2011;Selbach et al, 2008). A disadvantage of these approaches is that they require rather long labeling times.…”