Plasma proteomics of pancreatic cancer by multi-dimensional liquid chromatography and two-dimensional gel electrophoresis

The mortality rate of pancreatic adenocarcinoma is the highest among all cancer types, because of a lack of specific symptoms in the early stages, limitations of diagnostic methods, and the lack of response to all present treatments. We conducted plasma proteomics to identify the plasma proteins, the expression of which is aberrantly regulated in pancreatic cancer, in order to develop the specific and sensitive tumor markers for early diagnosis. To reduce the complexity and dynamic range of plasma proteome, we combined multi-dimensional liquid chromatography with 2D-PAGE. In our system, highly abundant proteins were depleted by Multiple Affinity Removal Column (Agilent) and the flow through fraction was separated by anion exchange column. For accurate quantitative comparison of protein expression profiles, the fractioned proteins were labeled with fluorescent dyes, which were developed for two-dimensional difference gel electrophoresis (2D-DIGE). The number of protein spots was increased from 290 to 403 spots by depleting the abundant proteins, then further increased up to 1098 by fractionation with anion exchange chromatography, provably due to the reduced complexity and dynamic range of plasma proteome. We selected 39 protein spots, whose intensity was significantly different in amount by more than 2-fold between the patients with pancreatic cancer and the healthy volunteers. The proteins corresponding to the selected protein spots were determined by mass spectrometry, and the identification of the proteins was confirmed by specific antibodies. A combination of multi-dimensional liquid chromatography with 2D-PAGE is a powerful tool for plasma proteomics. The identified proteins will be a candidate for diagnostic tumor marker and provide a clue to understand the molecular background of pancreatic cancer.