LAUSR

HighlightsProtein‐level partial reduction is proposed first time in complex disulfide linkage study.Disulfide bonds in Human Factor Xa are fully characterized first time by MS‐based method.Partial reduction can be used for disulfide bond mapping in recombinant protein by tandem MS. 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.