Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry.
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.
Friedman
David B
DB
Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University, Nashville, TN 37232-8575, USA. david.friedman@vanderbilt.edu
Hill
Salisha
S
Keller
Jeffrey W
JW
Merchant
Nipun B
NB
Levy
Shawn E
SE
Coffey
Robert J
RJ
Caprioli
Richard M
RM
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D003110:Colonic Neoplasms / Q000378:metabolism*; D019992:Databases as Topic; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005260:Female; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007413:Intestinal Mucosa / Q000473:pathology; D053558:Keratin-3; D007633:Keratins / Q000096:biosynthesis; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D020033:Protein Isoforms; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
C479360:KRT3 protein, human; D053558:Keratin-3; D020033:Protein Isoforms; D020543:Proteome; D007633:Keratins