title: Quantification of pancreatic cancer proteome and phosphorylome: indicates molecular events likely contributing to cancer and activity of drug targets.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Britton
authors.affiliation: Institute of Liver Studies, King's College Hospital, London, United Kingdom.
authors.forename: Yoh
authors.initials: Y
authors.lastname: Zen
authors.affiliation: Institute of Liver Studies, King's College Hospital, London, United Kingdom.
authors.forename: Alberto
authors.initials: A
authors.lastname: Quaglia
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Stefan
authors.initials: S
authors.lastname: Selzer
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Vikram
authors.initials: V
authors.lastname: Mitra
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Christopher
authors.initials: C
authors.lastname: Löβner
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Stephan
authors.initials: S
authors.lastname: Jung
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Gitte
authors.initials: G
authors.lastname: Böhm
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Peter
authors.initials: P
authors.lastname: Schmid
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Petra
authors.initials: P
authors.lastname: Prefot
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Claudia
authors.initials: C
authors.lastname: Hoehle
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Sasa
authors.initials: S
authors.lastname: Koncarevic
authors.affiliation: Cardiff School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom.
authors.forename: Julia
authors.initials: J
authors.lastname: Gee
authors.affiliation: Cardiff School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom.
authors.forename: Robert
authors.initials: R
authors.lastname: Nicholson
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Malcolm
authors.initials: M
authors.lastname: Ward
authors.affiliation: Faculty of Medicine, Department of Surgery & Cancer, Imperial College, London, United Kingdom.
authors.forename: Leandro
authors.initials: L
authors.lastname: Castellano
authors.affiliation: Faculty of Medicine, Department of Surgery & Cancer, Imperial College, London, United Kingdom.
authors.forename: Justin
authors.initials: J
authors.lastname: Stebbing
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Hans Dieter
authors.initials: HD
authors.lastname: Zucht
authors.affiliation: Institute of Liver Studies, King's College Hospital, London, United Kingdom.
authors.forename: Debashis
authors.initials: D
authors.lastname: Sarker
authors.affiliation: Institute of Liver Studies, King's College Hospital, London, United Kingdom.
authors.forename: Nigel
authors.initials: N
authors.lastname: Heaton
authors.affiliation: Proteome Sciences plc, Cobham, United Kingdom.
authors.forename: Ian
authors.initials: I
authors.lastname: Pike
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D015415:Biomarkers / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D016002:Discriminant Analysis; D005109:Extracellular Matrix / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D063990:Gene Ontology; D006801:Humans; D016018:Least-Squares Analysis; D009363:Neoplasm Proteins / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism*; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D011554:Pseudopodia / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D000970:Antineoplastic Agents; D015415:Biomarkers; D009363:Neoplasm Proteins; D010748:Phosphopeptides; D020543:Proteome; D011494:Protein Kinases