Cleavage of Multiple Disulfide Bonds in Insulin via Gold Cationization and Collision-induced Dissociation

Abstract

Intact bovine insulin, with its two chains linked via two disulfide linkages, has been used as a model system to study the incorporation of one or more gold cations as means for facilitating the cleavage of multiple disulfide bonds in a tandem mass spectrometry experiment. Gas-phase ion/ion reactions involving Au(I)Cl(2) (-) or Au(III)Cl(4) (-) were used to incorporate either one or two gold cations into multiply-protonated insulin cations, followed by ion trap collision-induced dissociation (CID) of the products. The incorporation of a single gold cation followed by CID showed little evidence for disulfide bond cleavage. Rather, the CID spectra were similar to those acquired for the same charge state with only excess protons present. However, the incorporation of two gold cations, regardless of oxidation state, resulted in efficient cleavage of the disulfide bonds connecting the two chains of insulin. Furthermore, ion trap CID of the insulin complexes containing two gold cations showed more sequence information compared to the complexes containing only one gold cation or no gold cations. The partitioning of the gold cations between the two chains upon CID proved to be largely asymmetric, as both gold cations tended to stay together. There appeared to be a slight preference for both gold cations to partition into the B-chain. However, the relatively low contribution from single chain ions with only one gold ion suggests a degree of cooperativity in the overall mechanism for separation of the two chains.

Figure 1

Product ion spectrum derived from ion trap CID of [Insulin+Au(I)+4H]⁵⁺ : (a) mass-to-charge range from 300–1700; (b) mass-to-charge range from 1700–2900 . * denotes ions with a gold adduct; Ion nomenclature: for example, By₄ is a y₄ ion from B-chain; ABb₂₁ is an ion that contains entire A-chain and b₂₁ ion from B-chain.

Figure 2

Product ion spectrum derived from ion trap CID of [Insulin+2Au(I)+2H]⁴⁺ : (a) mass-to-charge range from 300–1700; (b) mass-to-charge range from 1700–2900. * denotes ions with a gold adduct; ** denotes ions with two gold adducts. Ion nomenclature: for example, By₄ is a y₄ ion from B-chain; ABb₂₁ is an ion that contains entire A-chain and b₂₁ ion from B-chain.

Figure 3

Product ion spectra derived from MS³ of: (a) [B-Chain+3H]³⁺ and (b) [B-Chain+2Au(I)+H]³⁺. * denotes ions with one Au(I) adduct; ** denotes ions with two Au(I) ions.