A STRATEGY FOR CHARACTERIZATION OF POLYETHYLENE GLYCOL-DERIVATIZED PROTEINS - A MASS-SPECTROMETRIC ANALYSIS OF THE ATTACHMENT SITES IN POLYETHYLENE GLYCOL-DERIVATIZED SUPEROXIDE-DISMUTASE

Base treatment of polyethylene glycol-derivatized superoxide dismutase in which the polyethylene glycol is linked to the protein via a succi nyl bridge, removes the polyethylene glycol leaving a succinyl marker. Exhaustive succinylation with d4-succinic anhydride completes the der ivatization in order to minimize fractionation in proteolysis, chromat ography, and desorption in the mass spectrometer. Production of peptid es from the derivatized protein for high resolution and high-resolutio n tandem MS allows identification of the site that had been derivatize d by polyethylene glycol and the determination of the amount of polyet hylene glycol originally at each site. The mass spectrometric strategy outlined herein can be applied to other proteins derivatized for ther apeutic administration.