RUB »Aktuelle Seite


The Biomolecular Mass Spectrometry group elucidates biological pathways covering biotechnological questions as well as medicinal aspects. Clearly, the future of proteomics technologies will rely on robust quantification technologies for differential protein studies. Still, to a large extent differential analysis is carried out with 2D-PAGE. In this case intensities of distinct protein spots after Coomassie or silver staining are semi-quantified by image analysis software. Although these approaches are extremely powerful some intrinsic problems associated with 2D gels, such as detection of high or low molecular weight proteins, proteins with extreme pI values and hydrophobicity usually escape identification. Moreover, additional manual steps via a gel based approach comprising staining, gel washing, destaining, in gel digestion results in protein sample loss on multiple surfaces, which is especially critical if low abundant proteins or membrane proteins are targeted. In addition to traditional 2D-PAGE, our group has developed a non-gel-based approach called MudPIT (Multidimensional Protein Identification Technology). Prior to separation of all compounds, proteins are digested to peptides. The core of the MudPIT technology is a fused silica capillary (50-100 µm ID) consisting of a strong cation exchange (SCX) resin back-to-back with a reversed phase (RP) material. Depending on the complexity of the mixture, 10-15 salt steps are used to deplete all peptides from the two-dimensional column. Peptides are subjected to the mass spectrometer where collision induced dissociation fragments ions in a tandem mass spectrum. More than 1,500 unique proteins can be identified within 24 h. The MudPIT technology has been proven to be a powerful high-throughput tool to shotgun entire proteomes, addressing proteins which are difficult to access by 2D-PAGE, like low abundance and hydrophobic proteins, such as protein kinases, transcription factors and integral membrane protein. Recently, we developed an almost close-to-complete integral membrane protein enrichment and cleavage procedure and used it for the analysis with MudPIT. Non gel based labelling strategies are favourable for differential proteomics. Incorporation of stable isotopes, especially 13C, 15N in stable cell lines is relatively robust and well manageable. Relative ratios are calculated through mass spectrometric peak heights between labelled and unlabeled sample. In different projects related to aging, biotechnology, wound healing and cancer. Our group is interested in membrane processes and associated posttranslation modifications of these complex signal transduction pathways.