17th International Mass Spectrometry Conference :: Prague, 2006
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|Presentation date:||Thu, Aug 31, 2006|
|Presentation time:||09:50 – 11:20|
Ilse Dohnal1, Jiri Veis2, Karin Grosstessner-Hain3, Elisabeth Roitinger2, Karl Mechtler3, Gustav Ammerer1,21 Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
Correspondence address: Ilse Dohnal, Max F. Perutz Laboratories, University of Vienna, Department of Biochemistry, Dr. Bohrgasse 9, Vienna, 1030 Austria.
Keywords: Fourier Transform ICR; Phosphorylation; Proteomic; Quantitative Analysis.
Novel aspect: Following the kinetics and the extent of protein phosphorylation upon environmental stress to identify new components of the signal transduction machinery by using a combined SILAC-IMAC-FT-ICR-MS approach.
The budding yeast, Saccharomyces cerevisiae, reacts to increased external osmolarity by activation of a Mitogen-activated Protein Kinase (MAPK) cascade, the HOG pathway (high osmolarity glycerol response). Such MAPK cascades are highly conserved signalling modules that are found from yeast to man. Upon hyperosmotic stress, the MAPK Hog1 coordinates a multitude of processes, either by direct phosphorylation or indirectly, through a change in the protein expression profile. Therefore, the HOG-pathway is an ideal model system to study some aspects of eukaryotic signal transduction. Moreover, it might be a well suited system to establish quantitative mass spectrometric techniques.
For a comprehensive picture and to identify new components of the pathway, it is necessary to follow global changes in the phosphorylation pattern of the yeast proteome upon osmotic challenge. An additional level of complexity is introduced by the fact that signal transduction through reversible protein phosphorylation works not solely in an on/off-like fashion but involves concentration-dependent effects. Therefore, a reliable quantitation of the phosphorylation level between stressed and unstressed samples is required. To achieve this goal we employed a combined SILAC-IMAC approach. The stable isotope labeling by amino acids in cell culture (SILAC) allows the mixing of two samples for relative quantitation at a very early timepoint, namely before cell-lysis. Therefore, errors introduced by sample-to-sample variations in the subsequent steps, e.g. protein extraction and proteolytic digestion, are eliminated.
Yeast cultures were stressed for 5, 15 and 30 minutes, respectively and mixed with the equal amount of cells grown in C-13 Lys/Arg medium, serving as unstressed control, prior to cell lysis. Proteins were extracted and digested with trypsin. The resulting peptide mixture was enriched for phosphorylated peptides by IMAC (immobilized metal affinity chromatography) and analyzed by nano-HPLC-ESI-FT-ICR-MS/MS/MS. The acquired data were evaluated with different search algorithms and the obtained results were compared to available data on known Hog1-kinase substrates and to datasets generated in similar experiments by Gruhler et al. in 2005 for the pheromone response pathway.
We identified several novel sites that showed a dramatic increase in phosphorylation upon stress-treatment, also on proteins that have so far not been directly connected to the osmostress response. We also monitored the region of the Hog1-kinase that is important for regulating its enzymatic activity. The observed kinetics of phosphorylation was found in accordance with previous immunological assays.
We are currently trying to improve the proteome coverage, for instance by prefractionation of the cell lysate by one dimensional gel-electrophoresis.