Selective Gas-Phase Ion/Ion Reactions: Enabling Disulfide Mapping via Oxidation and Cleavage of Disulfide Bonds in Intermolecularly-Linked Polypeptide Ions

Abstract

The selective gas-phase oxidation of disulfide bonds to their thiosulfinate form using ion/ion reactions and subsequent cleavage is demonstrated here. Oxidizing reagent anions are observed to attach to all polypeptides, regardless of amino acid composition. Direct proton transfer yielding a charge-reduced peptide is also frequently observed. Activation of the ion/ion complex between an oxidizing reagent anion and a disulfide-containing peptide cation results in oxygen transfer from the reagent anion to the peptide cation to form the [M+H+O](+) species. This thiosulfinate derivative can undergo one of several rearrangements that result in cleavage of the disulfide bond. Species containing an intermolecular disulfide bond undergo separation of the two chains upon activation. Further activation can be used to generate more sequence information from each chain. These oxidation ion/ion reactions have been used to illustrate the identification of S-glutathionylated and S-cysteinylated peptides, in which low molecular weight thiols are attached to cysteine residues in peptides via disulfide bonds. The oxidation chemistry effectively labels peptide ions with readily oxidized groups, such as disulfide bonds. This enables a screening approach for the identification of disulfide-linked peptides in a disulfide mapping application involving enzymatic digestion. The mixtures of ions generated by tryptic and peptic digestions of lysozyme and insulin, respectively, without prior separation or isolation were subjected both to oxidation and proton transfer ion/ion chemistry to illustrate the identification of peptides in the mixtures with readily oxidized groups.

Figure 1

Oxidation of two disulfide-linked peptides derived from a tryptic digest of somatostatin. (a) Ion/ion reaction between AGCKNFFWK/TFTSC [M+2H]²⁺ and IO₄⁻. Activation of the (b) [M+2H+IO₄]⁺ and (c) [M+H+O]⁺ from the ion/ion reaction between doubly protonated AGCKNFFWK/TFTSC and IO₄⁻. (d) Activation of the [M+H+O]⁺ derived from the reaction between doubly protonated AGCK/TFTSC and HS₂O₈⁻. Asterisks indicate ammonia losses, degree signs indicate water losses, and lightning bolts indicate species subjected to CID.

Figure 2

CID of ion/ion complex between periodate anion and doubly protonated (a) S-glutathionylated ARACAKA and (d) S-cysteinylated ARACAKA. (b) CID of [M+H+O]⁺ species of S-glutathionylated ARACAKA. (c) Control CID of the [M+H]⁺ species of S-glutathionylated ARACAKA. Structures of S-glutathionylated and S-cysteinylated ARACAKA inset in (b)/(c) and (d), respectively. ‘A’ refers to the peptide ARACAKA; “Glu”, and “Cys” refer to glutathione and cysteine, respectively. Lightning bolts indicate the species subjected to CID, while degree signs indicate water losses. Pyroglutamic acid loss indicated by -pyr.

Figure 3

Activation of the ion/ion complexes between periodate anion and (a) doubly protonated GYSLGNWVCAAK/CK, (b) triply protonated CELAAAMK/GCR, and (c) triply protonated WWCNDGR/NLCNIPCSALLSSDITASVNCAK. Degree signs indicate water losses, and lightning bolts indicate species subjected to CID.

Figure 4

(a) Positive nESI spectrum of the tryptic digest of lysozyme, (b) ion/ion reaction between the entirety of (a) and IO₄⁻, and (c) DDC CID of the entirety of (b). The peptide WWCNDGR/NLCNIPCSALLSDITASVNCAK is indicated in the figure by the shortened form, WWCNDGR/NLC-C-CAK.

Figure 5

Difference spectrum where the DDC CID spectrum resulting from the ion/ion reaction with IO₃⁻ is subtracted from that with IO₄⁻. The peptide WWCNDGR/NLCNIPCSALLSDITASVNCAK is indicated in the figure by the shortened form, WWCNDGR/NLC-C-CAK.

Scheme 1

Proposed Mechanisms for the Oxidation of Disulfide Bonds and Cleavage of the Oxidized Disulfide Bonds