Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry

Proteomics. 2004 Mar;4(3):793-811. doi: 10.1002/pmic.200300635.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Colonic Neoplasms / metabolism*
  • Databases as Topic
  • Electrophoresis, Gel, Two-Dimensional / methods*
  • Female
  • Humans
  • Image Processing, Computer-Assisted
  • Intestinal Mucosa / pathology
  • Keratin-3
  • Keratins / biosynthesis
  • Male
  • Mass Spectrometry / methods*
  • Middle Aged
  • Protein Isoforms
  • Proteome*
  • Proteomics / methods*
  • Sensitivity and Specificity
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods


  • KRT3 protein, human
  • Keratin-3
  • Protein Isoforms
  • Proteome
  • Keratins