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Antelmann, Haike Greifswald

Expertise: Proteomics in bacteria, response and post-translational thiol-modifications caused by ROS and reactive electrophilic species (RES) in B. subtilis, regulatory redox-switch mechanisms of MarR/DUF24-family regulators in B. subtilis, the physiological role of the bacillithiol redox buffer in Firmicutes bacteria

Methodology provided: To elucidate the mode of action of ROS and RES, the group of Haike Antelmann provides transcriptomics, proteomics and metabolomics approaches. To study reversible disulfide bond formation in the proteome, the 2D-gel-based thiol-redox proteomics approach that relies on the reduction of disulfides and the labelling of newly released thiols with fluorescent dyes (e.g. Bodipy-IAM) is applied. Oxidized fluorescently-labelled proteins are quantified using the Decodon-Delta-2D software and identified using MALDI-TOF mass spectrometry. In addition, the group uses other thiol-trapping methods (e.g. AMS-labelling and non-reducing/reducing diagonal SDS-PAGE) to study Cys oxidations. For direct identification of specific Cys residues that form intramolecular, intermolecular disulfides or S-thiolations Orbitrap LC-MS/MS analysis is applied. Using DNA-binding assays, in vitro transcription assays, transcriptional analysis, phenotype or enzyme activity assays the effect of specific Cys oxidations or Cys mutations on protein functions is studied. Methods provided to the group include: MS-based relative quantitative proteomics using stable isotope metabolic labelling (14N/15N-isotope-labelling) (e.g. for NEM-Biotin-switch assay); MS-based identification of S-thiolations, inter- or intramolecular disulfides using Orbitrap Velos LC-MS/MS analysis; 2D gel-based redox proteomics using BODIPY-FL-IAM for quantification of reversibly oxidized proteins (e.g. bacilliredoxin‑, glutaredoxin- und thioredoxin substrates); high-throughput Ettan-spot-handling proteomics platform for spot digestion and MALDI-TOF/TOF MS.