Quantification of pancreatic cancer proteome and phosphorylome: indicates molecular events likely contributing to cancer and activity of drug targets

David Britton, Yoh Zen, Alberto Quaglia, Stefan Selzer, Vikram Mitra, Christopher Löβner, Stephan Jung, Gitte Böhm, Peter Schmid, Petra Prefot, Claudia Hoehle, Sasa Koncarevic, Julia Gee, Robert Nicholson, Malcolm Ward, Leandro Castellano, Justin Stebbing, Hans Dieter Zucht, Debashis Sarker, Nigel HeatonIan Pike

Research output: Contribution to journalArticlepeer-review

48 Citations (Scopus)

Abstract

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 & MRCKα) 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.
Original languageEnglish
Article numbere90948
Number of pages20
JournalPLoS ONE
Volume9
Issue number3
DOIs
Publication statusPublished - 26 Mar 2014

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