Project Summary/Abstract This project will develop an automated capillary electrophoresis-tandem mass spectrometry platform for the structural analysis of mixtures of glycosaminoglycan oligomers that are isolated in protein binding assays. This class of carbohydrates can be found on the surface of all animal cells, and play a key role in many important biological processes. Glycosaminoglycans are linear, polysulfated copolymers, typically with a repeating disaccharide unit composed of an N-acetyl-hexosamine and glucuronic acid, and can be more than 200 repeat units long. Modifications can occur anywhere along the chain, and these include O-sulfation, N- deacetylation/N-sulfation, and epimerization of the uronic acid. These are a particularly challenging class of compounds to characterize due to their heterogeneous structures, an outcome of their non-template biosynthesis. Nevertheless, within the long glycan chains are short regions of defined modification that target specific proteins with remarkable specificity. These glycan-protein interactions are the basis for the biological activity of the glycosaminoglycan. There is a critical need for tools to find these organized regions with the long glycan chain, and to determine their pattern of modification. A few specialized laboratories have developed mass spectrometry methods for the structural analysis of glycosaminoglycans, but these have not found their way into biological laboratories at large. The goal of this project is to produce a platform with a high degree of automation so that it can be deployed in the laboratories of biology researchers. The accessibility of these advanced mass spectrometry methods will result from innovations in hardware, software, and methods development.