The increasing number of people with type 2 diabetes is mainly due to insulin resistance as a dominant symptom. In addition, this resistance is not only the driving force behind type 2 diabetes, but is also associated with many other diseases such as high blood pressure, obesity, skeletal muscle remodeling, and neurodegeneration.
Enhanced insulin secretion induces the activation of the insulin signalling pathway, translocate GLUT4 vesicles to the plasma membrane, allowing them to transport glucose into the cell. The cascade starts with the activation of the insulin receptor and induces the phosphorylation of signalling molecules, including IRS1, PI-3K, and the AKT kinase as the key regulator for glucose transport. So far much effort has been invested in identifying proteins that impair intracellular insulin signalling, which can lead to the development of insulin resistance. Using state-of-the-art mass spectrometry in combination with transgenic diabetes mouse models, the Krüger laboratory is interested to decipher insulin-dependent cellular networks under regular and disease-related conditions. In particular, we ask which posttranslational modifications (PTMs) are associated with the insulin signaling and control glucose uptake, protein expression and metabolic activity. In addition, we are interested how insulin regulates the cross-talk to other signaling cascades.
Both projects focusing on high resolution mass spectrometry using quadrupole Orbitrap (QExactive HF-X) mass spectrometer. Sample preparation and PTM enrichment will be achieved with chromatographic separation techniques, including size exclusion and high pH reversed phase chromatography. In addition, kinase motif antibodies will be used to enrich specific phosphorylation sites to gain a more comprehensive view on activated signaling pathways. Bioinformatics analysis using different software tools will be an important part of the project to analyse the complex datasets. Protein quantification will be achieved by the stable isotope labeling of amino acids in cell culture (SILAC) approach, with in vivo SILAC, and with label free protein quantification.
Transgenic mouse models are established in the laboratory and further models based on the Crisper/Cas9 approach will be generated upon demand. Standard immunohistochemistry (IHC) methods and RNA hybridization will be used to visualize protein and gene expression changes. In collaboration with the laboratory of Jens Brüning from the MPI for Metabolomics, we will perform hyperinsulinemic-euglycemic clamp experiments to assess insulin sensitivity in vivo.
In the protein-protein interaction project we will also use an Orbitrap Fusion Lumos mass spectrometer which allows different peptide fragmentation modes such as Collision-induced Dissociation (CID) and Electron-transfer/higher-Energy Collision Dissociation (EThcD) to identify cross-linked proteins. In collaboration with the laboratory of Fan Liu (Potsdam), we have established the XL-MS approach, which used different fragmentation techniques for optimized identification of direct protein-protein interactions.
Basic knowledge in cell and molecular biology, quantitative proteomics and interest in bioinformatics analysis of large-scale datasets.