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 FA exposure is also connected to a variety of other serious diseases ranging from chronic liver disorders to cancer, cardiovascular and neurodegenerative diseases, and diabetes. 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 seek to develop and apply 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 both physiology and disease. This application will focus on new technologies to enable selective molecular imaging of FA in biological models to study sources of FA generation and metabolism, with accompanying chemoproteomics methods to identify targets of FA in genetic models where FA metabolism is compromised. Specific aims include developing new fluorescent probes for subcellular imaging of FA and enzymatic oxidation to formate, applying unbiased activity-based protein profiling (ABPP) methods to identify cysteine-derived targets of FA in whole proteomes, and performing biochemical and cellular studies to decipher roles of FA targets in regulating one-carbon metabolism.