Objective: LC-MS/MS phospho-proteomics is an essential technology to help unravel the complex molecular events that
lead to and propagate cancer. We have developed a global phospho-proteomic workflow to determine activity of signaling
pathways and drug targets in pancreatic cancer tissue for clinical application.
Methods: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal
adenocarcinoma and background pancreas (n = 12), were labelled with tandem mass tags (TMT 8-plex), separated by strong
cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using
IMAC and TiO2, prior to analysis. In-house, commercial and freeware bioinformatic platforms were used to identify relevant
biological events from the complex dataset.
Results: Of 2,101 proteins identified, 152 demonstrated significant difference in abundance between tumor and non-tumor
tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were
new candidate markers such as HIPK1 & MLCK. Of the 6,543 unique phosphopeptides identified (6,284 unique
phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho
guanine nucleotide exchange factors & MRCKa) and formation of focal adhesions. Activator phosphorylation sites on FYN,
AKT1, ERK2, HDAC1 and other drug targets were found to be highly modulated ($2 fold) in different cases highlighting
their predictive power.
Conclusion: Here we provided critical information enabling us to identify the common and unique molecular events likely
contributing to cancer in each case. Such information may be used to help predict more bespoke therapy suitable for an
individual case.