Selective Gas-Phase Oxidation and Localization of Alkylated Cysteine Residues in Polypeptide Ions via Ion/Ion Chemistry

Abstract

The thiol group in cysteine residues is susceptible to several post-translational modifications (PTMs), including prenylation, nitrosylation, palmitoylation, and the formation of disulfide bonds. Additionally, cysteine residues involved in disulfide bonds are commonly reduced and alkylated prior to mass spectrometric analysis. Several of these cysteine modifications, specifically S-alkyl modifications, are susceptible to gas-phase oxidation via selective ion/ion reactions with periodate anions. Multiply protonated peptides containing modified cysteine residues undergo complex formation upon ion/ion reaction with periodate anions. Activation of the ion/ion complexes results in oxygen transfer from the reagent to the modified sulfur residue to create a sulfoxide functionality. Further activation of the sulfoxide derivative yields abundant losses of the modification with the oxidized sulfur as a sulfenic acid (namely, XSOH) to generate a dehydroalanine residue. This loss immediately indicates the presence of an S-alkyl cysteine residue, and the mass of the loss can be used to easily deduce the type of modification. An additional step of activation can be used to localize the modification to a specific residue within the peptide. Selective cleavage to create c- and z-ions N-terminal to the dehydroalanine residue is often noted. As these types of ions are not typically observed upon collision-induced dissociation (CID), they can be used to immediately indicate where in the peptide the PTM was originally located.

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

“…Activation of the [M+H+O] + species generated in the gas-phase was compared to the [M+H+O] + species generated via solution-phase oxidation and the resulting spectra were identical suggesting that these two species were of the same primary structure. It was then found that the gas-phase ion/ion oxidation of thioethers was a general phenomenon as S-alkyl cysteine residues were also shown to undergo this oxygen transfer chemistry [53]. This was demonstrated with S-ethyl, S-farnesyl, and S-carbamidomethyl cysteine residues upon ion/ion reactions between doubly protonated peptides and both periodate and peroxymonosulfate anions.…”

“…For methionine residues this results in a loss of 64 Da corresponding to the ejection of methanesulfenic acid, a pathway that is well documented by groups investigating the solution-phase oxidation of methionine residues [37–41]. Interestingly, the loss of alkyl sulfenic acids from oxidized S-alkyl cysteine residues results in the generation of a dehydroalanine residue (Dha) at the site of the original modified residue [53,54,55,56]. This is especially useful as Dha residues open a low energy pathway that leads to cleavage of the N-C α bond of the Dha residue to generate c- and z-ions upon CID [56,57,58].…”

“…While we have reported on the oxidation of cysteine residues alkylated with common protecting agents (e.g., iodoacetamide and alkyl halides) [53], maleimide chemistry is another common method of protecting cysteine residues, the oxidation behavior of which is related here. The peptide ARACAKA was reacted with N-hydroxymaleimide to generate ARAC m AKA (superscript m indicates that the cysteine has been protected with N-hydroxymaleimide) and the doubly protonated species was subjected to ion/ion reactions with an oxidizing reagent anion (Supplemental Figure S-1).…”

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

“…As demonstrated previously, peptides containing easily oxidized moieties (i.e., methionine residues [25], S-alkylated cysteine residues [24], and disulfide bonds [23]) yield dominant [M+H+O] + species due to oxygen transfer from the reagent anion to the reactive side-chain upon activation of peptide/periodate ion/ion complexes. Here, we show that peptides lacking easily oxidized residues can also undergo oxidation upon ion/ion reaction with IO 4 − .…”

“…The gas-phase oxidation of disulfide bonds [23], S-alkyl cysteine residues [24], and methionine and tryptophan residues [25] via ion/ion reactions with periodate anion has recently been demonstrated. Furthermore, the oxidation of protonated polypeptides to [M+H+O] + , [M-H] + , and M +• species upon ion/ion reactions with a suite of reagents derived from persulfate has been demonstrated [26].…”