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

      Analytical Biochemistry
      Alkylation, Amino Acid Sequence, Animals, Chromatography, Liquid, Cricetinae, Disulfides, chemistry, Humans, Mice, Molecular Sequence Data, Nanotechnology, Oxidation-Reduction, Peptide Mapping, methods, Protein Structure, Tertiary, Proteins, isolation & purification, metabolism, Receptors, Tumor Necrosis Factor, Recombinant Proteins, Sensitivity and Specificity, Tandem Mass Spectrometry

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          Abstract

          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.

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