Summary
This is a joint proposal between Delft University of Technology Department of Biotechnology, both the Industrial Microbiology Section (IMB, J.H. de Winde, J.T. Pronk) and the Analytical Biotechnology Section (ABT, P.D.E.M. Verhaert, M.W.H. Pinkse), and with Utrecht University (Chemistry, A.J.R. Heck).

Systematic investigation of cellular processes often involves the quantification of protein abundance. While standard proteome analyses yield a ‘snap-shot’ view of the proteome, they do not provide information on protein turn-over, i.e. on the rate at which (specific) proteins are being synthesized and degraded at the time of proteome sampling. The lack of techniques capable of exploring the dynamics of protein synthesis and degradation at a proteome-wide scale explains the limited knowledge currently available on the mechanisms involved in the regulation of protein turn-over. While still technically challenging, peptidome analyses identify peptides released by cellular proteolysis and, thus de facto provide information on protein degradation. In combination with proteome analyses, information on both protein abundance and protein degradation is obtained. NPCII project form page 2/12
The first goal of this project is to investigate the potential of integrating proteomic and peptidomic analyses for the determination of protein turn-over. Quantitative proteome (and peptidome) analyses will be performed by using different approaches followed by the Utrecht and Delft partner. At Utrecht, 15N and/or 13C stable isotope metabolic (in vivo) labelings (SILAC-like or other) will be evaluated whereas Delft will focus on in-vitro isobaric mass-labeling of proteins (and peptides). Particularly the tandem mass tag (TMT) labels seem very suited for this approach due to its multiplexing capacity, allowing multiple samples to be simultaneously analyzed.
A second goal will be to investigate protein degradation and turn-over in cells adjusting to very low specific growth rates in retentostat cultures (doubling times larger than 1000 hours). The physiology of yeast cells under such conditions has long remained unexplored, but nevertheless is very important to interpret research on cellular aging as well as for yeast-based industrial processes. Recent research in the Kluyver Centre has indicated that turn-over of cellular macromolecules may be extremely important under these conditions. Additionally, the impact of oxygen (and therefore oxidative stress) on protein degradation and turn-over will be explored.