Quantification of Pancreatic Cancer Proteome and Phosphorylome: Indicates Molecular Events Likely Contributing to Cancer and Activity of Drug Targets

Abstract

ObjectiveLC-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.MethodsPeptides 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.ResultsOf 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.ConclusionHere 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.

Decreased plasma C‐reactive protein levels in APOE ε4 allele carriers

“…In case of multiple probes mapping to a single transcript, the probe with the most interquartile‐range variation across the samples, was retained in the analysis. Protein and phosphopeptide levels were assessed from 36 protein samples from the temporal cortex using SysQuant™ liquid chromatography tandem mass spectrometry (LC‐MS/MS) global proteomics (Proteome Sciences) as described 26, 27. LC‐MS/MS ‐based global proteomics identified 146,396 peptides mapping to 7516 unique proteins.…”

A global proteomic study identifies distinct pathological features of IgG4‐related and primary sclerosing cholangitis

“…This novel approach has been used for in‐vitro studies, and was rarely applied to tissue samples. We recently developed a workflow called SysQuant that enables examination of the phosphorylation status of proteins extracted from frozen human tissue samples, by means of several phosphopeptide enrichment steps with immobilized metal affinity columns (IMAC) or titanium dioxide (TiO 2 ) . Crucially, SysQuant also measures protein expression by means of a non‐enrichment arm focusing on quantification of non‐phosphorylated peptides.…”

“…Crucially, SysQuant also measures protein expression by means of a non‐enrichment arm focusing on quantification of non‐phosphorylated peptides. Our previous study on human pancreatic cancers revealed that 635 of 6543 phosphopeptides identified were modulated significantly between cancerous and background pancreatic tissue . The combined conventional proteomic and phosphoproteomic approach helped us to determine activated signalling pathways in pancreatic cancer tissue on a case‐by‐case basis …”

Network‐based integration of mRNA and miRNA profiles reveals new target genes involved in pancreatic cancer

“…Numerous studies have been carried out to explore molecular mechanisms underlying the development of PC, including genetic variations and environmental exposures . In recent years, with the wide applications of next‐generation sequencing and other high‐throughput technologies, genetic alternations in the genome, methylome, transcriptome, and proteome of PC cells have been intensively studied, which provide a systematic and unbiased view of variations at different levels . Genome‐wide sequencing approaches have identified a set of somatic mutations and genomic rearrangements that are associated with tumorigenesis of PC .…”