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Development of Disulfide Peptide Mapping and Determination of Disulfide Structure of Recombinant Human Osteoprotegerin Chimera Produced in Escherichia Coli

Development of Disulfide Peptide Mapping and Determination of Disulfide Structure of Recombinant Human Osteoprotegerin Chimera Produced in Escherichia Coli

L A Merewether, J Le, M D Jones, R Lee, G Shimamoto, H S Lu

Arch Biochem Biophys. 2000 Mar 1;375(1):101-10.

PMID: 10683254

Abstract:

Recombinant human osteoprotegerin chimera is a 90-kDa protein containing a human IgG Fc domain fused to human osteoprotegerin. The molecule is a dimer linked by two intermolecular disulfide bonds and contains eleven intramolecular disulfide bonds per monomer. A cysteine-rich region in osteoprotegerin contains nine disulfide bridges homologous to the cysteine-rich signature structure of the tumor necrosis factor receptor/nerve growth factor receptor superfamily. In this report, we have developed peptide mapping procedures suitable to generate disulfide-containing peptides for disulfide structure assignment of the fusion molecule. The methods employed included proteolytic digestion using endoproteinases Glu-C and Lys-C in combination followed by LC-MS analyses. Disulfide linkages of peptide fragments containing a single disulfide bond were assigned by sequence analysis via detection of (phenylthiohydantoinyl) cystine and/or by MS analysis. Disulfide bonds of a large, core fragment containing three peptide sequences linked by four disulfides were assigned after generation of smaller disulfide-linked peptides by a secondary thermolysin digestion. Disulfide structures of peptide fragments containing two disulfide bonds were assigned using matrix-assisted laser desorption ionization mass spectrometry with postsource decay. Both the inter- and intramolecular disulfide linkages of the chimeric dimer were confirmed.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4248628	Nerve Growth Factor Receptor/Fc Chimera human			Price

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