Gold(I) Cationization Promotes Ring Opening in Lysine-Containing Cyclic Peptides

Abstract

A strategy to sequence lysine-containing cyclic peptides by MSⁿ is presented. Doubly protonated cyclic peptides ions are transformed into gold (I) cationized peptide ions via cation switching ion/ion reaction. Gold(I) cationization facilitates the oxidation of neutral lysine residues in the gas phase, weakening the adjacent amide bond. Upon activation, facile cleavage N-terminal to the oxidized lysine residue provides a site-specific ring opening pathway that converts cyclic peptides into acyclic analogs. The ensuing ion contains a cyclic imine as the new N-terminus and an oxazolone, or structural equivalent, as the new C-terminus. Product ions are formed from subsequent fragmentation events of the linearized peptide ion. Such an approach simplifies MS/MS data interpretation as a series of fragment ions with common N- and C-termini are generated. Results are presented for two cyclic peptides, sunflower trypsin inhibitor and the model cyclic peptide, β-Loop. The power of this strategy lies in the ability to generate the oxidized peptide, which is easily identified via the loss of HAuNH₃ from [M + Au]⁺. While some competitive processes are observed, the site of ring opening can be pinpointed to the lysine residue upon MS⁴ enabling the unambiguous sequencing of cyclic peptides.

Keywords: Cyclic peptides; Gold cationization; Ion/ion reactions; Sunflower trypsin inhibitor.

Figure 1.

Structures of a) sunflower trypsin inhibitor and b) β-Loop. Stereochemistry not shown.

Figure 2.

Activation of a) [M + Au]⁺ and b) [M − H − NH₃]⁺ where M = reduced and alkylated sunflower trypsin inhibitor. Open circles (°) indicate water loss and shaded circles (•) indicate ammonia loss. The lightning bolt () indicates the species subjected to CID.

Figure 3.

MS product ion spectrum of [M − H − NH₃ − 91 − 91]⁺ formed via collisional activation as shown in Supplemental Figure S1. M = reduced and alkylated sunflower trypsin inhibitor. Open circles (°) indicate water loss and shaded circles (•) indicate ammonia loss. The lightning bolt () indicates the species subjected to CID. Product ions corresponding to opening at lysine are highlighted in red. Fragment ions corresponding to opening at Dha are highlighted in blue and green.

Figure 4.

Activation of a) [M + Au]⁺ and b) [M − H − NH₃]⁺ where M = β-Loop. c) expanded view of b) between m/z 500 and m/z 1500. Open circles (°) indicate water loss and shaded circles (•) indicate ammonia loss. The star superscript (*) indicates an aurated ion. The lightning bolt () indicates the species subjected to CID.

Figure 5.

Activation of a) [M + Au]⁺, b) [M − H − NH₃]⁺, and c) y₁₀ where M = KGAILPGAILR. Activation of d) [GAILPGAILR + H]⁺. Open circles (°) indicate water loss and shaded circles (•) indicate ammonia loss. The lightning bolt () indicates the species subjected to CID. Lysine residue loss (i.e. 147 Da lower in mass) is represented with a triangle superscript (^Δ).

Figure 6.

Activation of the y_13GK fragment ion of Figure 4b. Open circles (°) indicate water loss and shaded circles (•) indicate ammonia loss.

Scheme 1.

General strategy for cyclic peptide analysis via gold (I) cationization utilizing gas-phase ion/ion chemistry.