Programmbereich Infektionen

Bioanalytische Chemie

Mission    Projekte    Drittmittel    Methoden  Publikationen    Mitarbeiter/innen

Methoden

Analytical and semi-preparative liquid chromatography (LC, HPLC)
We utilize liquid chromatography to fractionate and analyse components of complex bacterial extracts and isolation of fractions for structural analysis using NMR and MS (Gisch et al., Bioorg. Med. Chem. Lett. 2011; Zähringer et al., J. Biol. Chem. 2014; Ranf et al., Nat. Immunol. 2015; Gisch et al., J. Biol. Chem. 2018).

Bioinformatics
For the identification and quantitation of lipids we utilize LipidXplorer software platform (Herzog et al., Genome Biol. 2011; Herzog et al., PLOS ONE 2012; Herzog et al., Current Protocols in Bioinformatics 2013). Further information can be found on the Lipidomics Informatics for Life Scinces (LIFS) web portal (https://lifs.isas.de/lipidxplorer.html). We utilize a number of statistical methods and multivariate data analysis approaches to interpret lipidomics data (Eggers et al., Scientific Reports 2017).

Lipidome homology
We developed a new metrics to determine the similarity between lipidomes of different organisms, tissues and cell types. The homology concept was designed to enable functional associations of lipidome compositions and foster utilization of model organisms for studying the impact of lipid metabolic alterations in human diseases (Marella et al., PLOS Comp. Biol. 2015; Eggers et al., Scientific Reports 2017).
Details can be found on the LIFS web portal (https://lifs.isas.de/lux-score.html).

Gas chromatography-mass spectrometry (GC-MS)
We perform quantitative fatty acid analysis and perform component analysis of carbohydrates, amino acids and glycoconjugates using specific chemical derivatization reactions.
Instruments:
Agilent Technologies 5975 inert XL Mass Selective Detector;
Agilent Technologies 5975C inert XL Mass Selective Detector

Mass spectrometry
Omics experiments as well as tandem mass spectrometric experiments for structural characterization are performed in our group (Turska-Szewcuk etal., Mar Drugs. 2014; Abdullah et al, Mol Microbiol. 2014; Hogendorf et al., Chemistry 2014; Gisch et al., J. Biol. Chem. 2018).
Instrument platforms:
I) High resolution 7 Tesla hybrid Qq / FT-ICR MS (Apex Qe, Bruker Daltonics) equipped with a Dual MALDI / ESI ion source and capability to perform MS/MS with CID, IRMPD, and ECD;
TriVersa Nanomate (Advion Biosciences) a chip-based nanoESI-system and autosampler;
II) Quadrupole Time of Flight MS (Q-ToF Ultima, Micromass now Waters);
Microflow HPLC-system (1100 Series, Agilent);
III) Hybrid quadrupole-orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific);
Microflow HPLC-system (1100 Series, Agilent);
Nanoflow LC-system (EASY-nLC, Proxeon now part of Thermo Scientific);
IV) Triple quadrupole mass spectrometer (Quattro Premier XE, Micromass now Waters) equipped with 1100 LC-systems (Agilent).

Nuclear magnetic resonance (NMR)
For structural characterization we utilize 1D- and 2D (COSY, NOESY, HSQC; HMBC etc.) experiments for 1H, 13C und 31P (Gisch et al., J. Biol. Chem. 2013; Heß, Waldow, Kohler et al., Nat. Commun. 2017; Waldow et al., J. Biol. Chem. 2018; Gisch et al., J. Biol. Chem. 2018).
Instruments:
Bruker Avance lll 700 MHz; equipped with an inverse 5-mm quadruple-resonance (1H,13C,15N,31P) CryoProbe or an inverse 1.7-mm triple-resonance (1H,13C,15N) micro-CryoProbe;
Bruker Avancell 360 MHz; equipped with an inverse 5-mm double-resonance Broad Band Probe (among others)

Shotgun lipidomics
Complex biological lipid extracts are directly analysed using tandem mass spectrometric approaches (Schwudke et al., Cold Spring Harb Perspect Biol. 2011; Eggers and Schwudke, Clinical Metabolomics, Methods in Molecular Biology 2018). We further utilize lipidomics screens to analyse sample of large scales studies (Schwudke et al., Anal Chem. 2007; Subramanian et al., Dis Model Mech. 2013; Müller et al., J. Virol. 2018; Hofmann et al., BBA - Molecular and Cell Biology of Lipids 2018).