Proteome wide determination of absolute turnover rates in mice

Introduction:

The administration of labeled amino acids has become the preferred method to measure the dynamics of protein turnover in living animals. Stable isotopes have been used as tracers to explore the amino acid flux in different metabolic pathways. Recently, the stable isotope labeling of amino acids in cell culture (SILAC), has been used to monitor individual protein turnover rates in cell culture and intact animals. This method is based on the incorporation of labeled essential amino acids resulting in appearance of labeled end products that can be quantified by mass spectrometry. We have performed an pulsed SILAC labeling of mice to calculate the protein turnover via the incorporation rate of labeled amino acid in post mitotic cardiomyocytes and could easily be applied to tissue from Lung.

Methods:

Mice fed with the heavy 13C6 -lysine containing diet will incorporate the heavy amino acid into newly synthesized proteins. This allows a distinction from natural isotope 12C6-lysine (light) forms by the 6 Da mass difference after mass spectrometric analysis. In order to track the 13C6 -lysine incorporation into the proteome, we analyzed postmitotic cardiomyocytes each week over a time period of 6 weeks. To reduce sample complexity, we used 1D-gels or isoelectric focussing of peptides and samples were analyzed by LC-MS/MS using a combination of nanoflow-HPLC system and high resolution mass spectrometry (LTQ-Orbitrap).

Preliminary Data:

One challenging parameter for the calculation of absolute protein turnover in intact animals is the characterization of the isotope enrichment of the precursor pool. We used an exponential function which we fitted to proteins with the highest incorporation rate to describe the precursor pool of the natural and labeled lysine. Next, a differenzial equation was used to calculate the absolute turnover for each protein. This approach allowed us to calculate the absolute turnover of more than 3000 proteins derived from cardiomyocytes. Comprehensive bio-computational analysis enabled us to correlate turnover rates to cellular structures, such as the nucleus and mitochondria. Here we extend the in vivo SILAC method based on 13C6 lysine incorporation to determine absolute turnover rates of individual proteins.