Lectures/Exercise:
Preparation of samples: general properties, sampling, methods of cell disruption, handling of protein samples, protein digestion.
Separation of methods for proteome analysis: principles of the 1D and 2D SDS-PAGE, preparative IEF, capillary electrophoresis and capillary isoelectric focusing, HPLC (reversed phase, ion-exchange, size exclusion chromatography), multidimensional LC, LC-MALDI.
Methods in proteome analytics based on mass spectrometry: basics, MALDI-TOF-MS, MALDI-TOF/TOF-MS, ESI-MS and ESI-MS/MS, analyzers (ion trap, quadrupole, TOF), sequencing of peptides, SELDI, protein databases and search algorithms
Applications: mining, peptide mass fingerprinting, 2D SDS PAGE linked with MALDI-TOF, LC-ESI-MS/MS, MALDI-TOF/TOF-MS; expression profiling, comparative 2D SDS PAGE analysis, isotope markers, protein-protein interactions, immune precipitation, Yeast-Two-Hybrid System, Shot Gun approach, Bait/Reverse Bait, posttranslational modifications
Protein separation: affinity, hydrophobicity, gel filtration and ion exchange chromatography, ultrafiltration, protein precipitation, determination of purity, protein characterization
Laboratory module:
Purification and determination of the specific activity of a protein, which is, expressed in E. Coli cells. 2D-gel analysis of a protein in induced and uninduced E. coli cells. Peptide cleavage of lysozyme with trypsin and analysis of the digested peptides by reverse phase HPLC.