Research Project 8:
Quantitative determination of the effect of transcription factors TFEB and TFE3 on the expression of lysosomal proteins
Studies investigating lysosomal function usually focus on single proteins or a group of representative proteins depending on their ease of detection by e.g. Western blot. This approach prevents analysis of the entire lysosome due to practical limitations. To allow analysis of the lysosome as a whole, we developed a mass spectrometry based strategy which is based on stable isotope labeled internal standard proteins termed QConCats in combination with targeted acquisition using multiple reaction monitoring (MRM). Once fully established, this approach will allow us to absolutely quantify the currently known lysosomal proteome in a single experiment. As part of this FOR, we will use this novel technique to quantify the effect of transcription factors TFEB and TFE3, which are known to regulate the expression of lysosomal proteins, on the composition of the lysosome. Furthermore, we will characterize the effect of combinations of different posttranslational modification on their transcriptional activity by mass spectrometry based analysis of PTM isoforms and correlation with lysosomal protein expression. Finally, we will assist the other members of the FOR with our expertise in qualitative and quantitative mass spectrometry based proteomics.
Research Project: Correlation of posttranslational modification of transcription factors with expression of lysosomal proteins.
Stimulation of cells results in different isoforms of posttranslational modifications (PTMs) which in turn influence expression of lysosomal proteins. Using our newly developed LysoQConCats we will quantify these changes using multiple reaction monitoring (MRM) mass spectrometry.