Phosphoproteomics and Cell Signaling
Members: Maximiliane Hilger, Matthias Mann, Sara Zanivan
Signal transduction processes are of key importance for the biological function of eukaryotic cells. A major goal is to understand how external stimuli affect thousands of proteins to generate specific cellular responses. Recently, quantitative proteomics has emerged as a powerful tool to study cell signalling on a proteome-wide scale. We are using Stable-Isotope Labelling with Amino acids in Cell culture (SILAC) and mass spectrometry to investigate cellular signalling events. Our research interests include insulin and growth factor receptor signalling, signalling in the immune system (T-cells and dendritic cells), protein ubiquitination and host-pathogen interaction.
Protein phosphorylation is a ubiquitous and very important post translational modification (PTM), which affects an estimated one third of all proteins. Many critical events involved in cellular response are mediated by phosphorylation and dephosphorylation, such as regulation of enzymatic activity, protein conformational change, protein-protein interaction, and cellular localization. Abnormal phosphorylation often leads to severe diseases.
Phosphoproteomics in our department covers: (1) qualitative phosphoproteome mapping in prokaryotic and eukaryotic cells, as well as body fluids (e.g. human milk) and tissues (e.g. chicken egg shell) ;(2) quantitative analysis of phosphoproteome dynamics in signal transduction.
Our interest group focuses on development and applications of technologies used in phosphoproteomics. The adaptation of stable isotope labeling by amino acids in cell culture (SILAC) enables quantitative phosphoproteomic studies in different cell conditions. Currently we combine strong-cation ion exchange chromatography (SCX) or 1D gel electrophoresis (SDS-PAGE) and titanium dioxide bead chromatography (TiO2) for phosphopeptide separation and enrichment. Specifically to define tyrosine phosphorylations, we employ protein immunoprecipitation approach using anti-phosphotyrosine antibodies (e.g. 4G10 and pY100) prior to TiO2-enrichment. High accuracy identification of phosphopeptides with pinpointed phosphorylation sites is routinely performed in the LTQ-Orbitrap mass spectrometer using multi-stage activation in the ion-trap and/or higher energy CID (HCD) in the c-trap.
To determine sites of modification on phosphopeptides, we developed a statistical algorithm named PTM score, which is automatically calculated in the MSQuant software and greatly facilitates analysis of large datasets.
Currently our approach is able to detect 6600 phosphorylation sites in HeLa cells in one proteomics experiment, of which 14% are responsive to EGF stimulation (see Global, in vivo and site-specific phosphorylation dynamics in signaling networks, Cell, in press).
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