Gas-Phase Oxidation of Neutral Basic Residues in Polypeptide Cations by Periodate

Abstract

The gas-phase oxidation of doubly protonated peptides containing neutral basic residues to various products, including [M+H+O]+, [M-H]+, and [M-H-NH3]+, is demonstrated here via ion/ion reactions with periodate. It was previously demonstrated that periodate anions are capable of oxidizing disulfide bonds and methionine, tryptophan, and S-alkyl cysteine residues. However, in the absence of these easily oxidized sites, we show here that systems containing neutral basic residues can undergo oxidation. Furthermore, we show that these neutral basic residues primarily undergo different types of oxidation (e.g., hydrogen abstraction) reactions than those observed previously (i.e., oxygen transfer to yield the [M+H+O]+ species) upon gas-phase ion/ion reactions with periodate anions. This chemistry is illustrated with a variety of systems including a series of model peptides, a cell-penetrating peptide containing a large number of unprotonated basic sites, and ubiquitin, a roughly 8.6 kDa protein.

Gas-Phase Oxidation via Ion/Ion Reactions: Pathways and Applications

“…While peaks corresponding to this mass are also observed for arginine-containing species they are derived from a sequential ammonia loss from the [M-H] + species instead of directly from the complex, in contrast with the case for lysine-containing peptides. (Comparison of the spectra derived from MS/MS of the [M-H-NH 3 ] + species directly from the complex and MS 3 of the ammonia loss from the [M-H] + species are identical for arginine-containing species but different for lysine-containing species, as discussed in detail previously [48]. ) Furthermore, while the oxidation of lysine residues to the [M-H-NH 3 ] + derivative was first noticed in ion/ion reactions with periodate anion, these species are also observed in reactions with persulfate anion where they are likely derived from a similar mechanism.…”

“…Activation of the periodate-peptide complex exclusively results in loss of HIO 4 to generate the charge-reduced [M+H] + species. No oxidation is observed for this species, though previous reactions between periodate anion and other histidine-containing peptides have shown evidence for a very minor extent of oxygen transfer to the His side chain [48]. Activation of the HAHAHAA-persulfate complex, however, results in dominant formation of the oxygen transfer [M+H+O] + and hydrogen deficient [M-H] + species.…”

“…Potential mechanisms and structures for these pathways have been discussed in detail previously [46]. Recently, we showed that periodate is also capable of oxidizing neutral basic sites in certain systems [48]. Peptides lacking easily oxidized residues (i.e., Met, Trp, disulfide bonds, etc.)…”

“…Furthermore, additional steps of MS n on the various oxidized species, with the exception of [KAKAKAA+H+O] + , generates different spectra for peptides oxidized with periodate and persulfate (Supplemental Figures S-5 through S-8), indicating that the structures resulting from oxidation with the two reagents may be different. The oxidation of neutral basic sites is important in certain systems like proteins and cell-penetrating peptides, both of which contain a high density of unprotonated basic residues under traditional nESI conditions [48]. …”

“…Methionine-containing peptides then undergo efficient loss of 64 Da, corresponding to ejection of methanesulfenic acid, upon collision-induced dissociation (CID) of the [M+H+O] + species, in agreement with activation of methionine sulfoxide-containing peptides generated in solution [37–41,45]. Periodate anion is also capable of oxidizing neutral lysine and arginine residues, predominantly to the hydrogen deficient species [M-H] + , in systems lacking other easily oxidized residues [48]. A suite of reagents generated upon negative nanoelectrospray ionization (nESI) of an aqueous solution of sodium persulfate, i.e., persulfate anion (HS 2 O 8 − ), peroxymonosulfate anion (HSO 5 − ), and sulfate radical anion (SO 4 -• ), have also been shown to be oxidizing reagent anions for ion/ion reactions [46].…”