Large-Scale Analysis of Breast Cancer-Related Conformational Changes in Proteins Using Limited Proteolysis

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.