Top-Down Electron Detachment Dissociation at ∼15–92 eV and 9.4 T with High Space Charge Capacity FT-ICR Cells

Abstract Top-down mass spectrometry (TD-MS) allows detailed characterization of biomolecular heterogeneity, e.g., sequence variants, chemical and biological modifications, and mutations. While relatively established in positive-ion mode, negative-ion-mode TD-MS of intact proteins has been less explored, in part due to a lack of suitable activation approaches for tandem mass spectrometry. Here, we show that electron detachment dissociation (EDD) can yield analytically useful sequence coverage up to ∼60% for β-casein (24 kDa), with relatively straightforward spectral interpretation due to favored formation of a• and x-type fragments with limited neutral loss. This leap in EDD performance from previously reported 5% sequence coverage was enabled by use of a custom wide-bore 9.4 T FT-ICR instrument with a solid dispenser cathode for optimal anion-electron overlap. In addition, a prototype─now commercially available─instrument with a dynamically harmonized ICR cell and a hollow cathode yielded 51% sequence coverage for β-casein, more than an order of magnitude higher than previous reports. EDD of phosphorylated α- and β-casein proteins showed phosphate retention in fragment ions and allowed phosphorylation site determination. High-energy, ∼72–92 eV, EDD is demonstrated to enable shorter irradiation times than previously reported, as low as 3 ms for an RNA 18-mer (100% sequence coverage) and 35 ms for carbonic anhydrase (47% sequence coverage), although higher electron energy results in lower average fragment ion charge. Numerous (y – 2H) fragments from backbone cleavage near proline were observed, indicative of electronic excitation under EDD conditions. Electron energy vs irradiation time heatmaps provide an in-depth understanding of how to tune EDD for desired fragmentation outcomes. Overall, the presented results show promise for incorporating EDD in online liquid chromatography-TD-MS workflows.