Shear stress greatly enhances oxidation of M1606 at the ADAMTS13 cleavage site in plasma VWF. Plasma VWF (0.75 μg/mL) was incubated with buffer alone, 50μM H₂O₂, or 50μM H₂O₂ and 25nM MPO at 37°C for 1 hour under either shear or static conditions. Reactions were quenched by adding excess L-methionine. The treated proteins were digested with trypsin, and the resultant tryptic peptides were analyzed by LC-MS/MS. (A) Selected reaction monitoring of the M1606-containing peptide (EQAPNLVYM¹⁶⁰⁶VTGNPASDEIK), MS² of m/z 1088.5 → 1424.6 (left) and the corresponding oxidized peptide (EQAPNLVYM¹⁶⁰⁶(O)VTGNPASDEIK), MS² of m/z 1096.5 → 1440.6 (right). (B) Oxidation of M1606 after treatment under the indicated conditions. The percentage of methionine converted to the sulfoxide was determined by dividing the area of the Met(O) peak by the sum of the areas of the peaks for Met(O) and unoxidized Met. The results represent the means ± SD of 3 experiments.