title: Differential secreted proteome approach in murine model for candidate biomarker discovery in colon cancer.
abstract: The complexity and heterogeneity of the plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. We used cell culture as a model system and identified differentially expressed, secreted proteins which may constitute serological biomarkers. A stable isotope labeling by amino acids in cell culture (SILAC) approach was used to label the entire secreted proteomes of the CT26 murine colon cancer cell line and normal young adult mouse colon (YAMC) cell line, thereby creating a stable isotope labeled proteome (SILAP) standard. This SILAP standard was added to unlabeled murine CT26 colon cancer cell or normal murine YAMC colon epithelial cell secreted proteome samples. A multidimensional approach combining isoelectric focusing (IEF), strong cation exchange (SCX) followed by reversed phase liquid chromatography was used for extensive protein and peptide separation. A total of 614 and 929 proteins were identified from the YAMC and CT26 cell lines, with 418 proteins common to both cell lines. Twenty highly abundant differentially expressed proteins from these groups were selected for liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) analysis in sera. Differential secretion into the serum was observed for several proteins when Apc(min) mice were compared with control mice. These findings were then confirmed by Western blot analysis.
authors.affiliation: Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160, USA.
authors.forename: Kannan
authors.initials: K
authors.lastname: Rangiah
authors.forename: Montri
authors.initials: M
authors.lastname: Tippornwong
authors.forename: Vineet
authors.initials: V
authors.lastname: Sangar
authors.forename: David
authors.initials: D
authors.lastname: Austin
authors.forename: Marie-Pier
authors.initials: MP
authors.lastname: Tétreault
authors.forename: Anil K
authors.initials: AK
authors.lastname: Rustgi
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Yu
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D015415:Biomarkers / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D003110:Colonic Neoplasms / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000737:chemistry; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009363:Neoplasm Proteins / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D015415:Biomarkers; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D020543:Proteome