This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Knowledge of protein structures and protein-protein interactions is essential for our understanding of biological processes. Obtaining such knowledge often presents a severe challenge. Recent advances in protein crosslinking and mass spectrometry (MS) have shown significant potential to contribute to this area. Here we are developing a novel method to rapidly and accurately identify crosslinked peptides based on their unique isotope signature when digested in the presence of H218O. This method overcomes the need for specially synthesized crosslinkers and/or multiple MS runs required by other techniques. We will validate our method by analyzing proteins/complexes of known structure. The computational technique Rosetta will be modified to validate crosslinks observed against published atomic structures.
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