To gain deeper insights in the dynamics of metabolism we adapted available techniques for proteome and metabolome analyses, as well as stable isotope labeling. Therefore we installed three different mass spectrometers and established several metabolomics and proteomics techniques. Using these techniques we are able to measure the metabolic activity of cells and organs in a dynamic manner in vitro and in vivo. With these techniques we are able to quantify proteins and metabolites from cells, organs and organisms and to monitor their turnover rates in a genome-wide scale.
Overview about the different mass spectrometers installed in our group and the related applications: (1) two dimensional gas chromatography coupled mass spectrometer [GCxGC-TOF); (2) nano LC / UPLC liquid chromatography coupled triple quadrupole mass spectrometer [LC-QQQ]; (3) nano LC liquid chromatography coupled LTQ-Orbitrap mass spectrometer Software tools
Is a software tool that generates comprehensive quality reports from proteomics analyses. As input the analysis results of the MaxQuant software tool is used (Bielow et. al. under review).
Is a software tool developed to generate data matrices from GC-MS based metabolomics analyses. It extracts areas or mass intensities from exported mass spectra for stable isotope resolved metabolomics analyses as input a CSV export of the chromatof-software is used. MetMax is constantly maintained by our group.
Is a software tool that enables a fast and precise analysis of GC-EI-MS metabolomics data. The software-tool allows the automated analysis of our in house developed identification (IDENT) and quantification (QUANT) mixtures and includes additional measures for quality control. Early in 2015 we have organized the 1st GC-MS and MAUI-SILVIA user workshop incl. members of BIMSB and MDC groups. The software tool is declared as invention at the MDC.
Metabolomics and proteomics methods
The different mass spectrometric platforms that are established within our group allow the quantitative measurement of a broad spectrum of biomolecules - this setup offers a high flexibility for the establishment and the further development of metabolomics and proteomics techniques.
The analysis of metabolites and proteins is crucial to determine the metabolic network of cells and organs. Although transcript levels contain basic information about the metabolic settings, transcriptional studies will never be sufficient to describe the metabolic state. Because the regulation of metabolism appears at all biological layers: (post-) transcriptional, (post-) translational and on an allosteric level.
Our group established cutting edge mass spectrometry-based metabolomics and proteomics techniques to analyze cell cultures, organs and whole organisms. Proteomic techniques combined with stable isotope labelling as SILAC (stable isotope labelling of amino acids in cell culture) are used for quantitative proteome analyses. Gas chromatography coupled mass spectrometry (GC-MS) as well as liquid chromatography coupled mass spectrometry (LC-MS) based techniques are applied to monitor the metabolome. In addition, we have developed pulsed stable isotope resolved metabolomics (pSIRM) as a tool for a dynamic metabolic characterization of cellular metabolism. We use both strategies (I) direct infusion parent ion scan and neutral loss scan methods and (II) direct infusion high resolution applications for lipidome analyses.
Metabolic profiling / fatty acid profiling
Metabolic profiling (free fatty acid profiling) is an untargeted GC-MS based method that allows the quantitative analysis of a broad spectrum of small molecules. Using commercial and in house data analysis tools large data sets can be established as basis for downstream statistical and bioinformatics analyses.
pSIRM (pulsed stable isotope resolved metabolomics)
pSIRM is an in house established workflow that enables a quantitative and time resolved analysis of cellular metabolism. We have further developed the analysis of stable isotope incorporation in vivo (mouse models) and we have established methods that allow to decode the action of metabolic inhibitors (see below). In frame of a collaboration with Katharina Nöh from the Helmholtz center Jülich we are translating pSIRM data as input for metabolic modeling and calculation of metabolic fluxes. This work is performed in frame of the DynaMeTox project. Beside the publication of the workflow we have filed a patent to protect the intellectual properties of our inventions.
Lipid analyses (Lipidomics)
Lipids are an important class of metabolites that are crucial as structural components of biological membranes, signaling molecules and energy source. Lipids comprise the largest number among all classes of metabolites. In order to comprehensively analyze the lipidome of biological materials powerful methods are required. We use triple-quadrupol based parent ion and neutral loss scan methods as well as high resolution techniques to quantitatively measure the lipidome. A software tool for comprehensive data analysis is under development in context of the BIH metabolomics core.
Nucleotides and nucleosides are an important class of molecules that are building blocks of nucleic acids, signaling molecules and energy equivalents. Of special interest is the composition analysis of DNA and RNA and to quantify modified nucleotide species. We use triple-quadrupol SRM based methods to determine the composition of RNA; novel mass spectrometry based high resolution methods are in preparation. The new established methods will allow to measure nucelotides/nucleosides in an unbiased way.
Various proteomics methods were established in our lab. Using the established methods we have conducted a large number of projects and collaboration during the last years.