The focus of the research unit „Instrumental Bioanalysis“ is the identification of protein biomarkers, the characterization of these proteins (characterization, structure analysis, identification of posttranslational modifications) as well as the identification/quantification of low molecular weight molecules (e.g. protein ligands). Therefore, multi-Omics strategies (especially LC-MS/MS, GC-MS) as well as spectroscopic methods are established (UV-VIS, FT-IR).
The development of single- and multiplex assays as well as the validation of biomarkers is performed in the unit „protein biomarker“ (headed by Stefan Kalkhof) of the department therapy validation of the Fraunhofer Institute for Cell Therapy and Immunology – IZI (https://www.izi.fraunhofer.de/de/abteilungen/standort-leipzig/therapievalidierung.html). For validation ELISA, western blot, peptide- or bead array (Luminex) are applied using high affine monoclonal antibodies, which are often self-developed.
Structural Analysis of the TSH/TSH Receptor interaction
The Thyrotropin Receptor (TSH-Receptor) is a G-Protein-coupled Receptor, which is activated by the peptide hormone Thyrotropin. This activation stimulates the thyroid to secreted thyroid hormones. An abnormal reduced or increased activation is correlated to diseases such as morbus basedow.
Aim of this collaboration project with the research groups of Prof. Dr. Jens Meiler (Vanderbilt University, USA) and Prof. Dr. Ralf Paschke (University Calgary, Canada) is the elucidation of the full-length structure of this receptor in an active and inactive state. This would be a profund basic to guide the development of activating and inhibiting ligands.
Reference: Schaarschmidt und Nagel et al., J Biol Chem. 2016 Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation
Development of novel implant materials
Due to demographic changes and co-morbidities the number of patients with impaired tissue healing or chronic wounds is increasing. Thus, the development of functionalized biomaterials and implants as well as novel preparation technics are required to improve tissue regeneration. For an efficient guidance of this development process fast, robust, and reliable analytical techniques are required. Among the required information markers needs to be measured to provide information on the wound healing state as well as information on the status of implant material. To develop these kind of methods and validate them for clinical application is our goal.
Reference: Förster und Schmidt et al. PLoS One. 2016; 11(7) Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles
How do chemicals trigger cellular reactions?
According to REACH chemicals need to be tested according its potential environmental and health risk. Are there molecular effects at subtoxic concentrations? Which defense mechanism are activated? How do cell culture models reflect the complex situation in organism? These questions were addressed in my former group at the Helmholtz Centre for environmental research using toxicoproteomics, metabolomics, and functional assays and investigations will be continued in Coburg for selected chemicals.
Reference: Toxicol In Vitro. 2017 Apr;40:153-160 Hepatic co-cultures in vitro reveal suitable to detect Nrf2-mediated oxidative stress responses on the bladder carcinogen o-anisidine. Wewering F, Jouy F, Caliskan S, Kalkhof S, von Bergen M, Luch A, Zellmer S.
Proteins are key players in all kind of biological processes. Given our experience and state-of-the-art equipment, we are capable and interested to support projects by
Protein sequence analysis (also non-mammalian samples)
Detection of protein modification
Protein extraction, purification and quantification from body fluids or tissues
Protein interaction and structure analysis
Proteomics (only for small scale projects)
Development of high-affinity monoclonal antibodies (at the Fraunhofer Unit)
Wewering F, Jouy F, Caliskan S, Kalkhof S, von Bergen M, Luch A, Zellmer S. Hepatic co-cultures in vitro reveal suitable to detect Nrf2-mediated oxidative stress responses on the bladder carcinogen o-anisidine. Toxicol In Vitro. 2017 Jan 12;40:153-160
Rother S, Samsonov SA, Hofmann T, Blaszkiewicz J, Köhling S, Moeller S, Schnabelrauch M, Rademann J, Kalkhof S, von Bergen M, Pisabarro MT, Scharnweber D, Hintze V. Acta Biomater.Structural and functional insights into the interaction of sulfated glycosaminoglycans with tissue inhibitor of metalloproteinase-3 – A possible regulatory role on extracellular matrix homeostasis. 2016 Nov;45:143-154. doi: 10.1016/j.actbio.2016.08.030.
Förster Y, Schmidt JR, Wissenbach DK, Pfeiffer SE, Baumann S, Hofbauer LC, von Bergen M, Kalkhof S, Rammelt S. Microdialysis Sampling from Wound Fluids Enables Quantitative Assessment of Cytokines, Proteins, and Metabolites Reveals Bone Defect-Specific Molecular Profiles. PLoS One. 2016 Jul 21;11(7):e0159580. doi: 10.1371/journal.pone.0159580.
Schaarschmidt J, Nagel MB, Huth S, Jaeschke H, Moretti R, Hintze V, von Bergen M, Kalkhof S, Meiler J, Paschke R. Rearrangement of the Extracellular Domain/Extracellular Loop 1 Interface Is Critical for Thyrotropin Receptor Activation.J Biol Chem. 2016 Jul 1;291(27):14095-108. doi: 10.1074/jbc.M115.709659.
Kalkhof S, Schildbach S, Blumert C, Horn F, von Bergen M, Labudde D. PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data. Biomed Res Int. 2016;2016:2891918. doi: 10.1155/2016/2891918.
Franziska Wewering, Florent Jouy, Dirk K. Wissenbach, Scarlett Gebauer, Matthias Blüher, Rolf Gebhardt, Ralph Pirow, Martin von Bergen, Stefan Kalkhof, Andreas Luch, Sebastian Zellmer A human hepatic in vitro co-culture system for the analysis of DILI related signaling Zeitschrift für Gastroenterologie 53(12) 2016
Wewering F, Jouy F, Wissenbach DK, Gebauer S, Blüher M, Gebhardt R, Pirow R, von Bergen M, Kalkhof S, Luch A, Zellmer S. Characterization of chemical-induced sterile inflammation in vitro: application of the model compound ketoconazole in a human hepatic co-culture system. Arch Toxicol. 2016 Mar 10. [Epub ahead of print]
Schmidt JR, Kliemt S, Preissler C, Moeller S, von Bergen M, Hempel U, Kalkhof S Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans. Mol Cell Proteomics. 2016 Feb;15(2):558-72. doi: 10.1074/mcp.M115.049718.