Project Z2
Differential proteomics for the characterisation of proteolysis-dependent proteome-wide changes in cells and tissues
Summary of the project
The Z2 proteomic platform in Kiel is distributed over three different institutes, i. e. the Institute for Immunology, the Zoological Institute, and the Institute for Experimental Medicine covering the 2DDIGE unit, the MALDI-TOF/TOF MSMS unit, and the Systematic Proteome Research Division, respectively. The expertise of the platform can be summarized as follows: i) sensitive gel-based assays for fluorescently-labelled soluble and membrane-bound proteins including e. g. 1-D PAGE, various 2- D PAGE technologies, (Co)-IPs, and pull-down assays, ii) quantitative analyses of protein regulation by 2-D DIGE technology using covalent minimal Cy-Dye labelling of protein samples, iii) the qualitative and quantitative analysis of proteomes by non-gel-based assays, iv) multiplexing of protein samples prior to quantification and identification, v) two-dimensional LC, vi) automated mediumthroughput sample preparation for MALDI-TOF/TOF MSMS analyses, vii) protein identifications by MALDI-TOF/TOF MSMS and LC-ESI-MSMS, viii) development of methods for quantitative MALDI MS, ix) the determination of enzyme activities, in particular of proteases, x) the analysis of posttranslational modifications, e.g. phosphorylation, glycosylation, acylation, and oxidative modifications, xi) the analysis of non-covalent interactions between proteins and metal ions. Previous work includes method development/validation as well as the application of these methods on biomedical and biotechnological problems. A major focus lies on the development of separation techniques based on multidimensional chromatography for the separation of proteolytic peptides or of intact proteins combined with either ESI or MALDI MS. Within the SFB, existing methods will be adapted and applied on particular problems of cooperation partners. Additionally, methods will be developed for the proteome-wide determination of proteolytic processes, mainly focussing on (i) the quantitative analysis of the products of proteolysis, e.g. by analysis of cleavage sites via products Nand C-termini; (ii) the role of posttranslational modifications (phosphorylation, oxidative processes; others when occurring in partner projects) for the regulation of proteolysis; (iii) methods for the analysis of proteolytic processes involving membrane proteins; (iv) activity-based proteomics: chemical derivatization will be used for the proteome-wide identification and quantification of classes of proteases; the basic principle is the targeted chemical modification of defined active-site residues. The research will cover all major steps of protein and proteome analysis: sample preparation, identification, characterisation, quantification and investigation of interactions/functional aspects. The major goals will be the reduction of the necessary number of experiments and technical replicates at simultaneously increased confidence of identification and quantification and a deeper coverage of the proteomes/subproteomes (e.g. posttranslational modifications). We will pay special attention to the development of multiplexed methods and parallelization, miniaturisation and improved sensitivities, both reached by improved sample preparation (e.g. enrichment) and separation methods, and by instrumental developments. A major point will be the parallel application of different complementary techniques together with the integration of nowadays still widely unused - but inherent - analytical information, e.g. retention times and isoelectric points by combination of analytical and bioinformatics approaches. Novel methods will first be developed and validated on model systems, e.g. bacterial proteomes and human cell cultures; thereafter, they will be directly applied on projects of the partners in the SFB.
Training and Events:
1st Baltic MS Meeting - Kiel 2011 - From Bacteria to Human Diseases(PDF file)
Date: 5 May 2011
Time: 09:00 am - 04:15 pm
Proteomics 2.0 Initiative
Neue Entwicklungen und methodische Möglichkeiten in der Proteom-Analyse(PDF file)
Date: 18 May, 2011
Time: 09:00 am - 01:00 pm
