We investigated the aberrant expression of plasma proteins in patients with pancreatic cancer. High-abundance plasma proteins (albumin, transferrin, haptoglobin, alpha-1-antitrypsin, IgG and IgA) were depleted by use of an immuno-affinity column, and low-abundance ones were separated into five fractions by anion-exchange chromatography. The fractionated plasma proteins were subjected to 2D-DIGE with highly sensitive fluorescent dyes. The quantitative protein expression profiles obtained by 2D-DIGE were compared between two plasma protein mixtures: one from five non-cancer bearing healthy donors and the other from five patients with pancreatic cancer. Among 1200 protein spots, we found that 33 protein spots were differently expressed between the two mixtures; 27 of these were up-regulated and six were down-regulated in cancer. Mass spectrometry and database searching allowed the identification of the proteins corresponding to the gel spots. Up-regulation of leucine-rich alpha-2-glycoprotein (LRG), which has not previously been implicated in pancreatic cancer, was observed. Western blotting with an anti-LRG antibody validated the up-regulation of LRG in an independent series of plasma samples from healthy controls, patients with chronic pancreatitis, and patients with pancreatic cancer. Our results demonstrate the application of a combination of multi-dimensional liquid chromatography with 2D-DIGE for plasma proteomics and suggest the clinical utility of LRG plasma level measurement.
Plasma proteomics of pancreatic cancer patients by multi-dimensional liquid chromatography and two-dimensional difference gel electrophoresis (2D-DIGE): Up-regulation of leucine-rich alpha-2-glycoprotein in pancreatic cancer