Negative Electron Transfer Dissociation of Deprotonated Phosphopeptide Anions: Choice of Radical Cation Reagent and Competition between Electron and Proton Transfer

Despite significant developments in mass spectrometry technology in recent years, no routine proteomics sequencing tool is currently available for peptide anions. The use of a molecular open-shell cation is presented here as a possible reaction partner to induce electron transfer dissociation with deprotonated peptide anions. In this negative electron transfer dissociation (NEW) scheme, an electron is abstracted from the peptide anion and transferred to the radical cation. This is demonstrated for the example of the fluoranthene cation, C16H10+center dot, which is reacted with deprotonated phosphorylated peptides in a 3-D ion trap mass spectrometer. Selective backbone cleavage at the C-alpha-C bond is observed to yield a and x fragments, similarly to electron detachment dissociation (EDD) of peptide anions. Crucially, the phosphorylation site is left intact in the dissociation process, allowing an identification and localization of the post-translational modification (PTM) site. In contrast, NETD using Xe+center dot as the reagent cation results in sequential neutral losses (CO2 and H2PO4) from a/x fragments, which complicate the interpretation of the mass spectra. This difference in dissociation behavior can be understood in the framework of the reduced recombination energy of the electron transfer process for fluoranthene, which is estimated at 2.5-4.5 eV, compared to 6.7-8.7 eV for xenon. Similarly to ETD, proton transfer is found to compete with electron transfer processes in NETD. Isotope fitting of the charge-reduced species shows that in the case of fluoranthene-mediated NETD, proton transfer only accounts for <20%, whereas this process highly abundant for Xe+center dot (43 and 82%). Since proton abstraction from Xe+center dot is not possible, this suggests that Xe+center dot ionizes other transient species in the ion trap, which then engage in proton transfer reactions with the peptide anions.