Research Projects
pSILAC and microRNAs
Regulation of gene expression occurs at all stages from mRNA transcription to protein synthesis. It is now clear that translation itself is a regulated process with a central role in cellular physiology and a growing catalogue of human diseases. A prominent example are microRNAs: These small non-coding RNAs repress target genes by inhibiting translation or by inducing degradation of target mRNAs. In mammals, microRNAs are predicted to control the activity of ~30% of all protein-coding genes. They have been shown to be involved in regulation of almost every cellular process investigated so far. In order to understand microRNA function it is crucial to investigate how they regulate protein production. We developed pulsed SILAC (pSILAC) as a novel method to directly compare protein translation rates between two samples (Fig. 2). Cells are first cultivated in standard growth medium with the normal light (L) amino acids. Concomitantly with differential treatment, cells are transferred to culture medium containing heavy (H) or medium-heavy (M) amino acids. All newly synthesized proteins will be made in the H or M form, respectively. Subsequently, both samples are combined and analyzed together. The abundance ratio of H versus M peptides reflects differences in translation of the corresponding proteins integrated over the pulse labeling incubation time. We employed pSILAC to measure changes in synthesis of several thousand proteins after misexpression of microRNAs (collaboration with the group of Nikolaus Rajewsky). Bioinformatic analysis of the data showed that a single microRNA can directly repress hundreds of targets and that this repression is typically rather mild. By comparing the pSILAC and the microarray data we were able to show that microRNAs directly repress translation of hundreds of mRNAs. We are currently extending this analysis to microRNAs involved in carcinogenesis. (Olivia Ebner, Björn Schwanhäusser)
Fig. 1: The principle standard SILAC (left) and pulsed SILAC (right). In standard SILAC, cells are completely labeled by cultivating them in growth medium containing light (L) or heavy (H) stable (i.e. non-radioactive) amino acids. After several cell generations, all proteins have uniformly incorporated the isotope label. Differentially labeld cells can be combined and analyzed together by mass spectrometry. The ratio of peak intensities reflects differences in protein abundance between both samples. In pSILAC, cells are growing in normal (i.e. light) medium. Upon differential treatment, cells are transferred to medium-heavy (M) of heavy (H) medium for pulse labelling. The ratio of heavy and medium-heavy peaks is a direct measure of the differences in protein synthesis between both experimental conditions.
Protein-protein interaction in neurodegenerative diseases
Identifying interaction partners is the key to protein function and can provide insight into disease mechanisms. Neurodegenerative diseases like Alzheimer’s are frequently caused by accumulation of toxic protein species in neuronal cells. We are using quantitative mass spectrometry and network analysis to analyze protein-protein interactions (PPIs) involved in disease pathogenesis. A useful method to study PPIs is affinity purification using a bait molecule. However, such pull-down assays suffer from the trade-off between sensitivity and specificity: While more stringent purification conditions can remove some contaminating proteins they might also eliminate specific interaction partners with weak affinities and/or of low abundance. Quantitative proteomics solves this problem by comparing the abundance of proteins identified in a pull-down experiment with a suitable internal control. This allows the development of assays that can directly lead to understanding of biological systems. We are employing this strategy to identify interaction partners of proteins involved in neurodegeneration. Particularly, we are interested in identifying interactions affected by disease-associated mutations. (Fabian Hosp, Florian Paul)
Fig. 2: Screening for interaction partners of neurological disease proteins by quantitative mass spectrometry. Neuroblastoma cells are cultivated in the presence of essential amino acids containing light or heavy stable isotopes (SILAC). Lysate from differentially labelled cells is combined with immobilized protein bait or suitable control bait. Proteins binding to the baits are eluted, combined, digested and analyzed by high throughput mass spectrometry (LC-MS/MS). Specific interaction partners are identified by their abundance ratio (stars).
In vivo quantitative proteomics: The SILAC zoo
Cell culture-based experiments cannot recapitulate all of the complex interactions among different cell types and tissues that occur in vivo. Small animal models such as worms, fruit flies and zebrafish are attractive alternatives that are extensively used in many areas of biomedical research, especially in genetics and development. While SILAC has enormously improved quantitative proteomics in cultured cells, the method was not yet used in small animal models. We are currently extending this technology to three of the most important model organisms in biomedical research: Caenorhabditis elegans, Drosophila melanogaster and Danio rerio. (Jiaxuan Chen, Marieluise Kirchner, Matthias Sury)