Abstract

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.