Current Bioinformatics Challenges in Proteome Dynamics using Heavy Water-based Metabolic Labeling.

Protein turnover in living systems has been measured with the use of stable isotope labeled tracers for over half a century [1]. Recent advances, in mass spectrometry, sample preparation and separation sciences have made it possible for this approach to become applicable at a global proteomics level, permitting analysis of the turnover of many proteins instead of single proteins or an aggregated protein pool [2-4]. Different stable isotope based tracers, including exogenously labeled amino acids [U-13C], glucose and 15N-labeled diet were used to assess global proteome dynamics. Among the available stable isotope precursors, heavy water, D2O, has advantages in safety, higher sensitivity (due to incorporation of multiple copies of 2H into analyzed peptide) and cost [5]. In addition, the intake of heavy water leads to universal 2H incorporation into all biomolecules, potentially permitting turnover analyses of nucleic acids, carbohydrate, or lipids and comparing them with protein turnover. Since heavy water can be administered in drinking water and does not require iv infusion, proteome dynamics studies can be conducted in free leaving organisms, including humans. The key assumptions for the heavy water-based metabolic labeling method are that the labeling of endogenous amino acids is very rapid and there is no post-synthetic labeling of a protein. Both assumptions have been tested experimentally; we have confirmed that there is no post-secretory labeling of plasma proteins in rodents [6]. In addition, we and others have demonstrated that most of the amino acids are labeled after 10-20 min of D2O administration [7,8]. Experimental procedure for heavy water labeling starts with bolus loading of heavy water followed by regular supply of 0.5-5% D2O enriched water for varying durations depending of the half-life of the proteins to be analyzed. As the heavy water is administered, total body water is rapidly labeled followed by extensive labeling of nonessential amino acids and minor labeling essential amino acids through de novo synthesis and/or transaminase reactions. The time course of 2H-incorporation into a protein allows the calculation of the rate of protein synthesis. Note, that longer durations of heavy water intake allow more accurate measurements of the kinetics of proteins with longer half-lives. The average protein turnover rate in a human cell line is about 20 h [9]. One of the drawbacks of the heavy water labeling method is that it cannot be applied to measure the synthesis rates of proteins with the half-life shorter than 1 hour.