Time-resolved protein phosphorylation analysis during epithelial mesenchymal transition of pancreatic adenocarcinoma

Pancreatic cancer (PanCa) is among the most aggressive adenocarcinomas and difficult to diagnose. Since it develops silently and exhibits high genetic complexity, a reliable diagnosis is only possible when metastasis have already spread to secondary sites. Unfortunately, the two biomarkers for PanCa detection (carbohydrate antigen 19-9 and carcinoembryonic antigen) do not allow a reliable detection during early states due to their high false positive rate in acute pancreatitis or other gastrointestinal problems. Consequently, mechanisms that trigger metastasis have to be unveiled to improve diagnostic tools and therapies. An underlying mechanism for migration and invasiveness of PanCa is epithelial to mesenchymal transition (EMT), promoting tumor progression and metastasis in adenocarcinomas. Fundamental changes in cell differentiation such as EMT are widely triggered by differential protein phosphorylation, known to be the mediator in regulation of cellular and metabolic processes (1) and aberrant protein phosphorylation has been found in various cancer forms rendering phosphorylation a key-mediator in cancerogenesis and progression. However, despite the importance of protein phosphorylation changes in cancer signaling, information gathered up until now is still limited since sample processing and data analysis are quite challenging for those not skilled in this art. Thus, changes in protein phosphorylation during EMT have never been investigated in detail. Considering the preceding and the expertise of the Leibniz-Institute for Analytical Sciences - ISAS - e.V. in Dortmund, Germany at dealing with complex samples and developing techniques more appropriate for phosphoproteome analysis, I propose a time-resolved study to be realized at the ISAS with the objective of monitoring protein phosphorylation changes during EMT induced in a pancreatic cancer cell line (PANC-1) employing 3-plex SILAC for a time-resolved screening 0, 15 and 60 minutes after EMT induction. For protein phosphorylation analysis, I will use a large-scale phosphoproteomics strategy, termed ERLIC-SCX/RP-LC-MS developed at the host institute that enables access to the entire dynamic range of phosphoproteins in a cell rendering it a perfect strategy to gain a comprehensive picture of protein phosphorylation events in pancreatic cancer cells. The long-standing experience in phosphoproteomics, the state-of-the-art instrumentation and the developed data analysis tools (Search GUI, Peptide Shaker) at ISAS will allow a comprehensive quantitative analysis during EMT in PanCA. In summary, a large-scale phosphoproteomics strategy combined with state-of-the-art LC-MS technology will be used to unveil key mechanisms during metastasis development of PanCa, a prerequisite to identify potential target proteins for diagnosis and/or therapy. (AU)