Analysis of oncogenic KRAS impact on the phospho-proteome of PDAC cells.

To reveal the impact of mutant KRAS on the phospho-proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a quantitative phospho-proteomic analysis of tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). Tandem Mass Tagging (TMT) was employed for quantitative analysis of Dox induced (24hrs) vs. non-induced iKras-PDAC cells. To assess the impact of ERK1/2 signalling, we also analysed cells induced with Dox in presence of Trametinib (10nM), a potent and specific inhibitor of ERK1/2 signalling. A total of 3 biological replicates per condition were assessed, with all samples barcoded and pooled together using TMT 10plex labelling kit (Thermo). To enrich for phospho-peptides, TiO phospho-peptide enrichment was performed. Both the phospho-enriched as well as the total peptide pools were analysed by mass spectrometry and the phopho to total ratio was calculated as a specific measure of phosphorylation change.