Identification of the unpaired cysteine residues in the VWF C1-CK fragment. (A) The C1-CK fragment contains unpaired cysteine thiols. The fragment was expressed in mammalian HEK cells, purified from the conditioned medium, labeled with a biotin-linked maleimide (MPB), and resolved on SDS-PAGE. The fragment was visualized by staining with colloidal Coomassie or blotted with either anti-VWF Abs or with streptavidin-peroxidase to detect the MPB label. (B) Amino acid sequence and peptide coverage (underlined) of the VWF C1-CK fragment by tandem MS analysis. Seventy percent of the residues and 68% of the cysteines were covered in the analysis. (C) Tandem mass spectrum of the GCDVCTCTDME peptide showing Cys2453 (underlined) labeled with carboxyamidomethyl (free cysteine) and Cys2448 and Cys2451 labeled with methyldisulfide (paired cysteines). The accurate mass spectrum of the peptide is also shown in the inset (observed [M + 2H]²⁺ = 663.1872 m/z; expected [M + 2H]²⁺ = 663.1859 m/z).