Discovery and validation of colonic tumor-associated proteins via metabolic labeling and stable isotopic dilution

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-10, Vol.106 (40), p.17235-17240
• Main Authors: Huttlin, Edward L, Chen, Xiaodi, Barrett-Wilt, Gregory A, Hegeman, Adrian D, Halberg, Richard B, Harms, Amy C, Newton, Michael A, Dove, William F, Sussman, Michael R
• Format: Article
• Language: English
• Subjects: Adenomatous Polyposis Coli - genetics; Adenomatous Polyposis Coli - metabolism; Adenomatous Polyposis Coli - pathology; Adenomatous Polyposis Coli Protein - genetics; Algae; Animal models; Animals; Biological Sciences; Chromatography, Liquid; Colonic Neoplasms - genetics; Colonic Neoplasms - metabolism; Colonic Neoplasms - pathology; Colorectal cancer; Diet; Disease Models, Animal; Female; Gene expression; Gene Expression Profiling - methods; Humans; Isotope Labeling - methods; Linear Models; Metabolism; Mice; Mice, Inbred C57BL; Mutation; Neoplasm Proteins - analysis; Neoplasm Proteins - metabolism; Nitrogen Isotopes - metabolism; Oligonucleotide Array Sequence Analysis; Peptides; Proteins; proteomics; Proteomics - methods; Reproducibility of Results; RNA, Messenger - genetics; RNA, Messenger - metabolism; Rodents; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spirulina - chemistry; synthetic peptides; Tumors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0909282106

The unique biology of a neoplasm is reflected by its distinct molecular profile compared with normal tissue. To understand tumor development better, we have undertaken a quantitative proteomic search for abnormally expressed proteins in colonic tumors from ApcMin/⁺ (Min) mice. By raising pairs of Min and wild-type mice on diets derived from natural-abundance or ¹⁵N-labeled algae, we used metabolic labeling to compare protein levels in colonic tumor versus normal tissue. Because metabolic labeling allows internal control throughout sample preparation and analysis, technical error is minimized as compared with in vitro labeling. Several proteins displayed altered expression, and a subset was validated via stable isotopic dilution using synthetic peptide standards. We also compared gene and protein expression among tumor and nontumor tissue, revealing limited correlation. This divergence was especially pronounced for species showing biological change, highlighting the complementary perspectives provided by transcriptomics and proteomics. Our work demonstrates the power of metabolic labeling combined with stable isotopic dilution as an integrated strategy for the identification and validation of differentially expressed proteins using rodent models of human disease.