Large-Scale Analysis of Protein Folding and Stability Changes Associated with Breast Cancer

Abstract

Proteomic methods for disease state characterization and biomarker discovery have traditionally utilized quantitative mass spectrometry methods to identify proteins with altered expression levels in disease states. Unfortunately, these studies have not been as useful as expected at identifying disease-related proteins that can be exploited for diagnostic and therapeutic purposes, presumably due to the indirect link between a protein’s expression level and its function. Investigated here is the use of thermodynamic stability measurements to probe a more biologically relevant dimension of the proteome. It has the potential to become a new strategy for disease state characterization and to help elucidate the molecular basis of the disease. This thesis outlines the use of two discovery based techniques and one validation based technique to study protein folding and stability changes associated with breast cancer.The first part of this dissertation describes the application of a mass spectrometry-based technique, stable isotope labeling with amino acids in cell culture and stability of proteins from rates of oxidation (SILAC-SPROX), in a comparison of the MCF-7 versus BT-474 breast cancer cell lines and a comparison of the MCF-7 versus MDA-MB-468 breast cancer cell lines. This work enabled ~1000 proteins to be assayed for breast cancer-related thermodynamic stability differences. The 242 and 445 protein hits identified with altered stabilities in these comparative analyses created distinct molecular markers to differentiate the three cell lines.The second part of this dissertation describes the development of a SILAC-based limited proteolysis (SILAC-LiP) strategy. The applicability of the protocol was demonstrated in a proof-of-principle study using proteins from a yeast cell lysate and a ubiquitous ligand. The SILAC-LiP protocol was further applied in a comparison of the MCF-7 versus MCF-10A cell lines. This work 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 overlap between the SILAC-LiP hits reported here and the SILAC-SPROX hits previously identified in these same cell lines was relatively small (~20%). Thus, this work indicates that the SILAC-SPROX and SILAC-LiP techniques can be used together to provide complementary information on the disease states.Furthermore, the protein hits identified in both the SILAC-SPROX and SILAC- LiP experiments included a large fraction (∼70%) with no significant expression level changes. This suggests protein folding and stability measurements can provide information about disease states that is orthogonal to that obtained in protein expression level analyses.The last part of this dissertation focuses on the establishment of targeted mass spectrometry-based validation assays for the protein biomarker candidates with altered thermodynamic stabilities identified in the SILAC-SPROX experiments. Application of the PAB-SPROX protocol on the MCF-7 cell lysate enabled reproducible identification and quantitation of a subset of prioritized target peptides.