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Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma
Author(s) -
Sitek Barabara,
Lüttges Jutta,
Marcus Katrin,
Klöppel Günter,
Schmiegel Wolff,
Meyer Helmut E.,
Hahn Stephan A.,
Stühler Kai
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401298
Subject(s) - labelling , pancreatic ductal adenocarcinoma , pathology , fluorescence , adenocarcinoma , chemistry , difference gel electrophoresis , gel electrophoresis , microbiology and biotechnology , biology , pancreatic cancer , proteomics , medicine , biochemistry , cancer , gene , physics , quantum mechanics
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.