Picomole-level mapping of protein disulfides by mass spectrometry following partial reduction and alkylation

We have deduced the disulfide bond linkage patterns, at very low protein levels (<0.5 nmol), in two cysteine-rich polypeptide domains using a new strategy involving partial reduction/alkylation of the protein, followed by peptide mapping and tanden mass spectrometry (MS/MS) sequencing on a nanoflow liquid chromatography-MS/MS system. The substrates for our work were the cysteine-rich ectodomain of human Fn14, a member of the tumor necrosis factor receptor family, and the IgV domain of murine TIM-1 (T-cell, Ig domain, and mucin domain-1). We have successfully determined the disulfide linkages for Fn14 and independently confirmed those of the IgV domain of TIM-1, whose crystal structure was published recently. The procedures that we describe here can be used to determine the disulfide structures for proteins with complex characteristics. They will also provide a means to obtain important information for structure-function studies and to ensure correct protein folding and batch-to-batch consistency in commercially produced recombinant proteins.