title: Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.
abstract: In order to identify new molecular markers for pancreatic intra-epithelial neoplasias (PanINs), the precursor lesions of pancreatic ductal adenocarcinoma, we established a proteomics approach analysing microdissected PanIN cells. Due to the limited amount of proteins available from microdissection, we developed a procedure including fluorescence dye saturation labelling in combination with high resolution two-dimensional gel electrophoresis. With this procedure we were able to analyse proteins extracted from 1000 microdissected cells with a high resolution of up to 2500 protein spots. Using protein lysates from the pancreatic carcinoma tissue as a reference proteome, we were able to successfully identify the proteins. Thus, we could match approximately 2200 protein spots (92%) of the microdissected sample proteome to the reference proteome for protein identification using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and nanoliquid chromatography-electrospray ionisation tandem mass spectrometry after in-gel digestion. The first proteome analysis of microdissected PanIN-2 grades revealed eight differentially expressed proteins. The differential expression of the three actin filament-associated proteins--transgelin, vimentin and MRCL3 as well as actin itself--indicates a relevant role of the actin cytoskeleton during pancreatic tumour progression. Additionally, two members of the annexin family (annexin A2 and A4) implicate a functional contribution of exocytotic and endocytotic pathways at that stage.
authors.affiliation: Medical Proteom-Center, Ruhr-University Bochum, Bochum, Germany.
authors.forename: Barabara
authors.initials: B
authors.lastname: Sitek
authors.forename: Jutta
authors.initials: J
authors.lastname: Lüttges
authors.forename: Katrin
authors.initials: K
authors.lastname: Marcus
authors.forename: Günter
authors.initials: G
authors.lastname: Klöppel
authors.forename: Wolff
authors.initials: W
authors.lastname: Schmiegel
authors.forename: Helmut E
authors.initials: HE
authors.lastname: Meyer
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Hahn
authors.forename: Kai
authors.initials: K
authors.lastname: Stühler
mesh_terms: D000230:Adenocarcinoma / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D044584:Carcinoma, Ductal / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D004210:Dissection; D015180:Electrophoresis, Gel, Two-Dimensional; D004798:Enzymes / Q000302:isolation & purification; D006801:Humans; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010190:Pancreatic Neoplasms / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D020543:Proteome; D012680:Sensitivity and Specificity
chemical_list: D004798:Enzymes; D009363:Neoplasm Proteins; D020543:Proteome