Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in XXXX .

@entity1 @entity11746 @entity788 @entity1124 @entity575 @entity5 @entity2975 @entity460 @entity214
@entity1124 ( @entity1124 ) is the most lethal of all the common @entity5 and markers for early detection or targets for treatment of this disease are urgently required. The disease is characterised by a strong stromal response, with @entity5 cells usually representing a relatively small proportion of the cells in the @entity5 mass. We therefore performed laser capture microdissection (LCM) to enrich for both normal and malignant pancreatic ductal epithelial cells. Proteins extracted from these cells were then separated by two-dimensional gel electrophoresis (2-DE). The limited amounts of protein in the LCM procured samples necessitated the detection of 2-DE resolved proteins by @entity575 staining. Consequently, loading equivalent amounts of protein onto gels was essential. However, we found that conventional means of measuring total protein in the samples were not sufficiently accurate. We therefore adopted a strategy in which the samples were first separated by one-dimensional @entity214 - @entity2975 gel electrophoresis, stained with @entity575 stain and subjected to densitometry. Evaluation of the staining intensity was then used to normalise the samples. We found that the protein profiles from undissected normal pancreas and LCM-acquired non-malignant ductal epithelial cells from the same tissue block were different, underpinning the value of LCM in our analysis. The comparisons of protein profiles from nonmalignant and malignant ductal epithelial cells revealed nine protein spots that were consistently differentially regulated. Five of these proteins showed increased expression in @entity5 cells while four showed diminished expression in these cells. One of the proteins displaying enhanced expression in @entity5 cells was identified as the @entity460 -binding protein, @entity11746 . To determine the incidence of @entity11746 overexpression in @entity788 , we carried out immunohistochemical analysis on sections from a @entity788 tissue array containing 174 duplicate normal and malignant pancreatic tissue samples, from 46 @entity788 @entity1 . Normal pancreatic ductal epithelia were either devoid of detectable @entity11746 or showed weak expression only. Moderately or poorly differentiated @entity5 , by contrast, showed a higher incidence and a higher level of @entity11746 expression. These observations indicate that the combination of LCM with 2-DE provides an effective strategy to discover proteins that are differentially expressed in @entity1124 .
