Oxidized fibrinogen HPLC-MS/MS

Fibrinogen is highly susceptible to oxidation compared to other plasma proteins. Fibrinogen oxidation damages its structure and affects the protein function. Fibrinogen was isolated from citrated human plasma by the modified cold ethanol precipitation technique. Oxidation of fibrinogen by ozone: a solution of 2.0 mg of fibrinogen in 1 ml of 0.05 M Tris/0.15 M NaCl buffer (pH 7.4) was introduced into a quartz reactor filled with ozone–oxygen mixture. The full exhaustion of ozone in each experiment was confirmed by spectrophotometry by the absorption band at 254 nm. The amount of ozone was varied in the range of 50–100 μM per 1 μM of fibrinogen. Ozone-induced oxidative modifications of the fibrinogen Aα, Bβ, and γ polypeptide chains upon addition of various amounts of the oxidizer have been studied by mass-spectrometry.