ABSTRACT Cataract is the leading cause of blindness worldwide. Surgical removal of cataractous lenses places an enormous financial burden on our economy and is not without side effects. Moreover, the prevalence of cataract in the U.S. is expected to more than double by the year 2050. Although new therapies are being developed to prevent or reverse early cataract, our understanding of the underlying mechanisms of cataractogenesis remains unclear. The long- term goals of our research are to identify modifications to lens proteins during aging and cataractogenesis in order to define protein aging mechanisms and to develop ways to prevent, delay, or reverse opacification. Recently, we have identified multiple age-dependent, non- enzymatic biochemical processes that result in both age-related lens protein modification and cataract-related lens protein crosslinking. The lens proteins and amino acids involved have been identified; however, mechanistic details remain unresolved. In addition, we have observed a dramatic re-distribution of soluble lens proteins to the membrane fraction in aged lens tissue. Again, the operative mechanism(s) of this protein shift remains unknown. Our hypothesis is that specific lens protein-protein crosslinks, peptide/lipid alterations, and crystallin binding to lens membranes are cataractogenic. To test this hypothesis we will employ state-of-the-art imaging mass spectrometry and proteomics/lipidomics methodology to further define age-related and cataract-specific modifications and define conditions upon which the chemistry occurs to determine chemical mechanism. Specifically we propose to: 1) define the protein sites and chemistry of protein crosslinks in cataractous human lenses and, 2) define cataract-specific peptide and lipid changes in human lenses, and 3) elucidate the molecular events that lead to increased lens protein membrane binding with age and cataract formation. The proposed experiments are expected to provide new mechanistic details on protein aging that will not only inform the development of new cataract treatments, but also guide therapeutic development for other aging and protein aggregation diseases.