Project summary: This revised proposal seeks funds to acquire a Thermo Orbitrap Eclipse mass spectrometer equipped with ETD and UVPD to address critical needs for HDX-MS and glycoproteomics studies at the University of Washington. This system would allow our groups to overcome the technical limitations and sample analysis bottlenecks that have hindered and stalled many NIH funded projects. The Obitrap Eclipse platform is the most suitable platform for expanding the projects scopes and throughput of our HDX-MS studies as it: 1) Offers state of the art sensitivity to lower limits of detection to enable HDX- MS studies of very material-limited samples, including intact virus particles and hard to purify proteins from native biological sources; 2) Achieves a mass resolution of 500,000 which is ten times higher than obtainable on our current instrumentation and is necessary for analyzing highly complex samples; 3) Is capable of routine and robust high-throughput electron transfer dissociation (ETD), which is vital for identification of complex protein samples and glycopeptides; 4) Incorporates ultra-violet photodissociation (UVPD) for expanding the resolution obtainable from HDX-MS studies; and 5) Enables top-down HDX-MS studies through both ETD and UVPD for proteins refractory to classical bottom-up approaches. The capacity to combine different fragmentation methods a rapid timescale will enable development of new tools for site-specific glycoproteomics studies and development of much needed tools for glycan structural elucidation for glycobiology. This instrument, with its broad capabilities, will also benefit general proteomics applications and identification of rare and novel post- translational modifications. While several mass spectrometers are available at UW, all the high-end systems are heavily utilized and devoted to other applications. The HDX-MS system that is required for the majority of our users is housed in the School of Pharmacy Mass Spectrometry Center and cannot be moved and interfaced with the instruments outside of the facility. Additionally, there is not a single UVPD enabled high resolution mass spectrometer at UW or anywhere in the greater Seattle area to support out current technological needs. This shared instrument will provide the capabilities and access needed to advance current NIH funded research and will allow us to push the boundaries of protein analysis by HDX-MS and site-specific glycoproteomics studies in biomedical research to support future NIH funded projects.