title: Disulfide bond characterization of human factor Xa by mass spectrometry through protein-level partial reduction.
abstract: Protein-level partial reduction was investigated as a novel sample preparation technique to characterize proteins with cystine knots or complex disulfide linkages. Human Factor Xa containing twelve disulfide bonds was selected as a model protein to demonstrate this methodology. Five in twelve disulfide linkages were characterized through conventional non-reduced samples while the other seven disulfide linkages containing cystine knots were successfully characterized though partially reduced samples. Each disulfide linkage was confirmed through product ions generated by an UPLC-ESI QTOF MS system equipped with data independent collision-induced dissociation (CID) acquisition. Free cysteines in the sample were also determined in this study.
authors: Mass Spectrometry Core Facility, KBI Biopharma, 1101 Hamlin Road, Durham, NC 27704, United States. Electronic address: Song.Klapoetke@gmail.com.
authors: Song
authors: S
authors: Klapoetke
authors: Suncadia Biopharmaceuticals, A Subsidiary of Hengrui Medicine Corporation, 218 Xinghu Street, B8-401, Suzhou, Jiangsu, 215000, China.
authors: Michael Hongwei
authors: MH
authors: Xie
mesh_terms: D000097:Acetonitriles; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D015951:Factor Xa / Q000032:analysis / Q000737:chemistry*; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000097:Acetonitriles; D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015951:Factor Xa; D003545:Cysteine; C032159:acetonitrile
keywords: Cystine knots; Disulfide bond characterization; Human factor Xa; LC–TOF MS; Mass spectrometry; Partial protein reduction