Project Summary/Abstract Formaldehyde (FA) is a ubiquitous small-molecule that plays a diverse array of important roles in human health and disease. As the simplest aldehyde and reactive carbonyl species, FA is a major environmental toxin that is classified as a carcinogen and exposure is also connected to a variety of other serious diseases ranging from neurodegenerative diseases, diabetes, and chronic liver and heart disorders. At the same time, the body produces this reactive carbonyl species during normal physiology, primarily through enzymatic demethylation events as well as through the one-carbon cycle. We are developing and applying new chemical reagents for selective imaging and proteomics of FA in living systems to identify its molecular sources and targets, with the long-term goal of understanding how and in what context this reactive small molecule contributes to normal physiology and disease. This application will focus on creating new technologies that will enable highly selective molecular imaging of FA in cellular models to detect sources of FA generation, with accompanying chemoproteomics methods to identify targets of FA in genetic and cancer models where FA metabolism is compromised. Specific aims include developing new fluorescent FA probes with a variety of available colors and ratiometric detection, unbiased reactivity-based protein profiling (RBPP) methods to identify cysteine- and lysine-derived targets of FA in whole proteomes, and combining these complementary methods for studying sources and targets of FA biology.