Sulfur Pentafluoride is a Preferred Reagent Cation for Negative Electron Transfer Dissociation

Negative mode proteome analysis offers access to unique portions of the proteome and several acidic post-translational modifications; however, traditional collision-based fragmentation methods fail to reliably provide sequence information for peptide anions. Negative electron transfer dissociation (NETD), on the other hand, can sequence precursor anions in a high-throughput manner. Similar to other ion-ion methods, NETD is most efficient with peptides of higher charge state because of the increased electrostatic interaction between reacting molecules. Here we demonstrate that NETD performance for lower charge state precursors can be improved by altering the reagent cation. Specifically, the recombination energy of the NETD reaction-largely dictated by the ionization energy (IE) of the reagent cation-can affect the extent of fragmentation. We compare the NETD reagent cations of C₁₆H₁₀^●+ (IE = 7.9 eV) and SF₅^●+ (IE = 9.6 eV) on a set of standard peptides, concluding that SF₅^●+ yields greater sequence ion generation. Subsequent proteome-scale nLC-MS/MS experiments comparing C₁₆H₁₀^●+ and SF₅^●+ further supported this outcome: analyses using SF₅^●+ yielded 4637 peptide spectral matches (PSMs) and 2900 unique peptides, whereas C₁₆H₁₀^●+ produced 3563 PSMs and 2231 peptides. The substantive gain in identification power with SF₅^●+ was largely driven by improved identification of doubly deprotonated precursors, indicating that increased NETD recombination energy can increase product ion yield for low charge density precursors. This work demonstrates that SF₅^●+ is a viable, if not favorable, reagent cation for NETD, and provides improved fragmentation over the commonly used fluoranthene reagent. Graphical Abstract ᅟ.