Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.
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.
Sitek
Barabara
B
Medical Proteom-Center, Ruhr-University Bochum, Bochum, Germany.
Lüttges
Jutta
J
Marcus
Katrin
K
Klöppel
Günter
G
Schmiegel
Wolff
W
Meyer
Helmut E
HE
Hahn
Stephan A
SA
Stühler
Kai
K
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
D004798:Enzymes; D009363:Neoplasm Proteins; D020543:Proteome