Systematic quantitation and analysis of protein modifications of transcription factors and signalling proteins
Understanding gene regulation requires knowledge of the constituents of the transcription complexes as well as their abundances at given cell states or at time-points during cellular processes. Since the function of signaling proteins and transcription complexes is often regulated by protein modification, analysis of the latter is also required. While several techniques exist for assessing qualitative differences among proteomes representing different cell states, information on how these differences are mediated is largely missing. A new approach, which was recently developed at the Max Planck Institute for Molecular Genetics (Gustavsson et al., 2004, submitted) involves time-based stable isotope metabolic incorporation in a single culture. This method is based on the growing of cultures in normal medium (e.g. 14N-medium), which at a defined time-point is exchanged for a heavy isotope enriched medium (e.g. 15N-medium). Proteins expressed after the medium change will incorporate the heavy isotope 15N, and can thus be quantitatively distinguished from proteins expressed prior to the change by mass spectrometric analysis. To examine changes in protein expression levels in response to a cellular signalling cue, a perturbation is introduced at the time-point for medium change. By sampling aliquots from the culture at selected time points, changes in the individual expression levels for all detected proteins within one single culture can be monitored over time by comparison of the 15N/14N signal intensity ratios. A non-perturbed culture serves as the control and will at the same time provide valuable information about the differences in protein turnover rates that occur naturally during control growth conditions. Several different analytical techniques will be employed for detection. For relatively high-abundant proteins, 2-dimensional polyacrylamide (PAGE) gel electrophoresis (2-DE) can provide snapshots of the cell state at different time points. This approach enables investigation of minute changes in protein expression that could not be detected as a change in protein spot intensity on the 2-DE gel, for example when the turnover rate for a protein is altered (e.g. during stress conditions) with a retained overall expression level. For low-abundant proteins such as kinases and transcription factors, rarely detected on 2-DE gels, nano LC-MALDI MS (Mirgorodskaya et al. 2004, Proteomics, in press) will be used. By combining both these techniques with immunoaffinity purification, determination of turnover rates as well as changes in expression levels at different cell states will be measured also for low-abundant proteins. Using these techniques, the cellular concentrations and modification states of 500 targeted proteins will be monitored at different cell states and at selected time-points following system perturbations, including, RNAi knock-down and heat stress.
