Examining the dynamics of the proteome using stable isotopes as metabolic tracers

Abstract

The primary objective of this study was to examine the turnover of different proteins using stable isotopes as metabolic tracers. Two different labelling methods were used during the course of this investigation; [2H]20 and [2H8] valine. Heavy water labelling resulted in the incorporation of deuterium into non-essential amino acids, and was primarily used to assess the turnover of mouse urinary proteins (MUPs). MUP’s showed rapid deuterium incorporation, with equilibrium reached at day two, indicating high turnover. This result was anticipated as it had already been established that MUPs are made by the liver and rapidly excreted in the urine. Heavy valine was used to perform a more comprehensive study of turnover. Mice were provided with a diet in which 50% of the valine was in heavy. Heavy valine was incorporated into proteins via protein synthesis and labelling was monitored using mass spectrometry. As with the heavy water labelling, MUP’s showed rapid labelling, with other proteins showing a range of different labelling rates. The most interesting observation in the heavy valine study was made when monitoring label incorporation in sperm and seminal vesicle secretion (SVS) proteins. SVS proteins showed rapid label incorporation from day two onwards, indicating high protein turnover. However the sperm proteins showed a distinct delay in labelling relative to the SVS proteins. It is hoped that this delay in the labelling of sperm proteins relative to SVS proteins could be exploited to selectively label specific proteins.