ABSTRACT Genome- and proteome-based gene expression profiling studies have been widely used over the past decade to characterize biological states including those associated with normal biological process (e.g., aging) and with disease (e.g., cancer). While such differential gene expression profiling studies can help identify the cellular pathways and physiological processes associated with a biological state they often fail to produce a complete understanding of the biological state because gene expression levels are not directly tied to protein function. This has limited the number of useful protein biomarkers and therapeutic targets discovered from such studies, and left gaps in our understanding of disease states. Proposed here is the development and application of thermodynamic measurements of protein stability to characterize different biological states including those associated with aging and cancer. Such thermodynamic measurements of protein stability are expected to be more closely related to protein function than are protein expression levels, and thus produce more useful protein biomarkers and therapeutic targets of disease and generate a better understanding of the molecular basis of disease. The proposed work will further develop and utilize three mass spectrometry-based proteomics methods, termed SPROX, LiP and PP, to characterize the protein folding and stability changes associated with a fundamental biological process, aging, and a disease, colorectal cancer. The specific aims of this work are: (1) to interface SPROX and LiP with middle- down proteomics methods to enable proteoform specific folding and stability measurments; (2) to utilize the middle down SPROX and LiP workflows developed in (1) to make proteoform specific thermodynamic stability measurements on mouse brain proteins derived from a mouse model of aging; (3) to utilize the SPROX, LiP, and PP techniques to identify protein biomarker with altered folding and stability in patient-derived colorectal cancer cell lines with different sensitivities to oxaliplatin treatment; (4) to utilize the protein biomarkers discovered in (3) to develop a clinically relevant assay for predicting oxaliplatin resistance/sensitivity in colorectal cancer.