title: Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using Limited Proteolysis.
abstract: Conformational changes in proteins can lead to disease. Thus, methods for identifying conformational changes in proteins can further improve our understanding and facilitate detection of disease states. Here we combine limited proteolysis (LiP) with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to characterize breast cancer-related conformational changes in proteins on the proteomic scale. Studied here are the conformational properties of proteins in two cell culture models of breast cancer, including the MCF-10A and MCF-7 cell lines. The SILAC-LiP approach described here identified ∼200 proteins with cell-line-dependent conformational changes, as determined by their differential susceptibility to proteolytic digestion using the nonspecific protease, proteinase K. The protease susceptibility profiles of the proteins in these cell lines were compared to thermodynamic stability and expression level profiles previously generated for proteins in these same breast cancer cell lines. The comparisons revealed that there was little overlap between the proteins with protease susceptibility changes and the proteins with thermodynamic stability and/or expression level changes. Thus, the large-scale conformational analysis described here provides unique insight into the molecular basis of the breast cancer phenotypes in this study.
authors: Department of Chemistry, Duke University , Durham, North Carolina 27708, United States.
authors: Fang
authors: F
authors: Liu
authors: Department of Chemistry, Duke University , Durham, North Carolina 27708, United States.
authors: Michael C
authors: MC
authors: Fitzgerald
mesh_terms: D001943:Breast Neoplasms / Q000737:chemistry*; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D055550:Protein Stability; D059748:Proteolysis
chemical_list: D009363:Neoplasm Proteins; D010447:Peptide Hydrolases
keywords: MCF-10A; MCF-7; SILAC; breast cancer; limited proteolysis; mass spectrometry; protein folding; proteomics