Proteomics analysis of serum protein profiling in pancreatic cancer patients by DIGE: up-regulation of mannose-binding lectin 2 and myosin light chain kinase 2

Abstract

Background: Pancreatic cancer has significant morbidity and mortality worldwide. Good prognosis relies on an early diagnosis. The purpose of this study was to develop techniques for identifying cancer biomarkers in the serum of patients with pancreatic cancer.

Methods: Serum samples from five individuals with pancreatic cancer and five individuals without cancer were compared. Highly abundant serum proteins were depleted by immuno-affinity column. Differential protein analysis was performed using 2-dimensional differential in-gel electrophoresis (2D-DIGE).

Results: Among these protein spots, we found that 16 protein spots were differently expressed between the two mixtures; 8 of these were up-regulated and 8 were down-regulated in cancer. Mass spectrometry and database searching allowed the identification of the proteins corresponding to the gel spots. Up-regulation of mannose-binding lectin 2 and myosin light chain kinase 2, which have not previously been implicated in pancreatic cancer, were observed. In an independent series of serum samples from 16 patients with pancreatic cancer and 16 non-cancer-bearing controls, increased levels of mannose-binding lectin 2 and myosin light chain kinase 2 were confirmed by western blot.

Conclusions: These results suggest that affinity column enrichment and DIGE can be used to identify proteins differentially expressed in serum from pancreatic cancer patients. These two proteins 'mannose-binding lectin 2 and myosin light chain kinase 2' might be potential biomarkers for the diagnosis of the pancreatic cancer.

Figure 1

Differentially expressed protein spots identified by DIGE analysis. Eight protein spot-features were found to be significantly up-regulated (red) and eight were significantly down-regulated in the sera of patients with pancreatic cancer (blue).

Figure 2

3-D view of differentially expressed proteins. 3-D view of differentially expressed proteins in the serum of pancreatic cancer patients and the cancer-free controls. The computational analysis of the images with the DyCyder software allowed for the detection of significant abundance changes based on the variance of the mean change within the cohort. MBL2 (A) and MLCK2 (B) were over-expressed in the pancreatic cancer patients' serum. T: the pancreatic patients; N: the controls.

Figure 3

Western blot analysis. Western blot analysis confirms the DIGE findings of increased protein levels of MBL2 and MLCK2 in serum from pancreatic cancer patients (n = 16).

Figure 4

The expression level of MBL2 and MLCK2. The expression level of MBL2 and MLCK2 was assessed by analyzing the intensity of its bands normalized with β-actin. Comparison of serum protein levels between patients with pancreatic cancer and the cancer-free controls (n = 16) showed increased protein levels of MBL2 (A) and MLCK2 (B) (**p < 0.05).