Pinpointing disulfide linkage pattern is critical in the characterization of proteins and peptides consisting of multiple disulfide bonds. Herein, we report a method based on coupling online disulfide modification and tandem mass spectrometry (MS/MS) to distinguish peptide disulfide regio-isomers. Such a method relies on a new disulfide bond cleavage reaction in solution, involving methanol as a reactant and 254 nm ultraviolet (UV) irradiation. This reaction leads to selective cleavage of a disulfide bond and formation of sulfenic methyl ester (-SOCH) at one cysteine residue and a thiol (-SH) at the other. Under low energy collision-induced dissociation (CID), cysteine sulfenic methyl ester motif produces a signature methanol loss (-32 Da), allowing its identification from other possible isomeric structures such as S-hydroxylmethyl (-SCHOH) and methyl sulfoxide (-S(O)-CH). Since disulfide bond can be selectively cleaved and modified upon methoxy addition, subsequent MS CID of the methoxy addition product provides enhanced sequence coverage as demonstrated by the analysis of bovine insulin. More importantly, this reaction does not induce disulfide scrambling, likely due to the fact that radical intermediates are not involved in the process. An approach based on methoxy addition followed by MS CID has been developed for assigning disulfide linkage patterns in peptide disulfide regio-isomers. This methodology was successfully applied to characterizing peptide systems having two disulfide bonds and three disulfide linkage isomers: side-by-side, overlapped, and looped-within-a-loop configurations. Graphical Abstract ᅟ.

Homocysteine sulfinyl radical ((SO⋅) Hcy) is a reactive intermediate involved during oxidative damage of DNA in the presence of high concentrations of homocysteine (Hcy). The short lifetime of (SO⋅) Hcy makes its preparation, isolation, and characterization challenging using traditional chemical measurement tools. Herein, we demonstrate the first study on mass-selected protonated (SO⋅) Hcy ions in the gas phase by investigating its unimolecular dissociation pathways from low energy collision-induced dissociation (CID). Tandem mass spectrometry (MS/MS), stable-isotope labeling, and theoretical calculations were employed to rationalize the observed fragmentation pathways. The dominant dissociation channel of protonated (SO⋅) Hcy was a charge-directed H2 O loss from the protonated sulfinyl radical (-SO⋅) moiety, forming a thiyl radical (-S⋅), which further triggered sequential radical-directed ⋅SH loss through multiple pathways. Compared to cysteine sulfinyl radical ((SO⋅) Cys), the small structural change induced by one additional methylene group in the side chain of (SO⋅) Hcy significantly promotes its base property while reducing the radical reactivity of sulfinyl radical. This observation provides new insight into studying reactions of (SO⋅) Hcy with biomolecules, which are critical in understanding toxicity induced by high levels of Hcy in biological conditions.

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