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.
Foley
Susan F
SF
Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA.
Sun
Yaping
Y
Zheng
Timothy S
TS
Wen
Dingyi
D
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D036103:Nanotechnology; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D000074050:TWEAK Receptor; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004220:Disulfides; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; C000613297:TNFRSF12A protein, human; D000074050:TWEAK Receptor; C000613299:Tnfrsf12a protein, mouse