Quantification of VWF sialic acid expression with HPLC. (A) Reverse-phase HPLC analysis was used to quantify sialic acid expression on purified pd-VWF. A sample chromatograph of liberated sialic acid from bovine fetuin is represented. Hydrolyzed sialic acid (Neu5Ac) was labeled with OPD, and a baseline-resolved peak representing Neu5Ac, with a retention time of 36.3 minutes, was observed. A structural representation of Neu5Ac derivatized with OPD is shown. (B) To determine relative quantitative sialic acid expression on the N- and O-linked carbohydrate structures of VWF, HPLC analysis of residual VWF-bound sialic acid and free (flow-through, FT) was performed on digestion with either PNGase F or O-glycosidase, respectively. Bound sialic acid refers to sialic acid still intact on the glycans of VWF after digestion with a specific glycosidase; FT refers to liberated sialic acid that has been removed from VWF after glycosidase treatment. (C) Purified human VWF was incubated overnight with various concentrations of α2-3,6,8,9 neuraminidase as before, and residual α2-3,6–linked sialic acid expression on VWF was analyzed by HPLC.