PROJECT SUMMARY – TR&D 1 We direct this TR&D toward improved biomolecule characterization. The rationale is simple: you cannot quantify what you cannot characterize! Tandem mass spectrometry (MS/MS), the central approach for characterizing biomolecules with MS, is typically achieved by collisional activation. This process often fails, however, when the peptide precursor contains amino acids that inhibit random backbone protonation and PTMs that dissociate by a lower energy pathway than that involved in the cleavage of the amide linkage. Beyond these limitations, several biomolecule classes – O-glycopeptides, glycosaminoglycans, and modified nucleic acids, to name a few – preferentially ionize in the negative mode and are therefore extremely difficult to sequence with current technologies. To access these challenging biomolecules, researchers across the globe have turned to electron transfer dissociation (ETD). Now a mainstay in proteomic technology, ETD reacts peptide cations with small molecule anions. That said, future breakthroughs in biomolecule characterization will require key advancements in ETD technology. First, ETD reaction rates are now an order of magnitude longer (~60–90 ms per scan) than collision-based methods, limiting the impact ETD can have in a modern era that demands high throughput and high sensitivity. Second, the success rate of assigning peptide sequence to ETD scans suffers for precursors with low charge density (>1,000 m/z). That impedes ETD’s use in a number of applications, especially those involving large biomolecules or PTMs that add mass without adding charge (e.g., glycosylation). And finally, a variant of ETD – negative electron transfer dissociation (NETD) – has tremendous potential to allow access to acidic biomolecules, but is not widely available because there is not yet an ion source that can produce a high flux of the reagent cations needed to conduct the reaction. Here we leverage our deep history and expertise in ETD to propose creative, exportable technologies that remedy these crucial bottlenecks.