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Disulfide bond characterization of human factor Xa by mass spectrometry through protein-level partial reduction.
Journal of Pharmaceutical and Biomedical Analysis 2017 January 6
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.
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