Characterization of disulfide linkages in proteins by 193 nm ultraviolet photodissociation (UVPD)

Inter-linked disulfide bonds connecting peptide chains are homolytically cleaved with 193 nm ultraviolet photodissociation (UVPD). Analysis of insulin demonstrates the ability for UVPD to cleave multiple disulfide bonds and provide sequence coverage of multiple peptide chains in the same MS/MS event. The information provided by UVPD of disulfide-bound peptides is comparable to information garnered from other high energy dissociation methods but requires an activation period an order of magnitude faster than these methods. This phenomenon was investigated, along with a partial reduction and alkylation sample preparation to determine disulfide connectivities of enzymatically digested proteins. The 4 disulfide bonds of lysozyme and the 19 disulfide bonds of serotransferrin were unambiguously characterized through non-reduced and partially reduced protein digestions.