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Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry
Authors:Friedman David B  Hill Salisha  Keller Jeffrey W  Merchant Nipun B  Levy Shawn E  Coffey Robert J  Caprioli Richard M
Institution:Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-8575, USA. david.friedman@vanderbilt.edu
Abstract:Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.
Keywords:Colorectal cancer  Difference gel electrophoresis  Internal standard  Matrix-assisted laser desorption/ionization-time of flight
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