Development of disulfide peptide mapping and determination of disulfide structure of recombinant human osteoprotegerin chimera produced in Escherichia coli.
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.
Merewether
L A
LA
Department of Protein Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California, 91320, USA.
Le
J
J
Jones
M D
MD
Lee
R
R
Shimamoto
G
G
Lu
H S
HS
D000595:Amino Acid Sequence; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D053244:Osteoprotegerin; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics*; D012680:Sensitivity and Specificity; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism
D004220:Disulfides; D006023:Glycoproteins; D053244:Osteoprotegerin; D010446:Peptide Fragments; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; C506030:TNFRSF11B protein, human; D004228:Dithionitrobenzoic Acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D013820:Thermolysin