Abstract
Several mass spectrometry techniques were combined to characterize polyethylene glycol (PEG)– superoxide dismutase (SOD). This chapter describes a determination of the locations at which PEGs are attached to SOD as well as a strategy for determining the number of PEGs attached to SOD. Fast atom bombardment mass spectrometry and tandem mass spectrometry of proteolytic digestion products were used to locate the attachment sites, and electrospray ionization mass spectrometry to estimate the numbers of PEGs attached. This study indicates that mass spectrometry can be used to characterize PEG–SOD and provides a general strategy for analyzing other PEG derivatized proteins. Release of PEG by base treatment of PEG–SOD leaves a modified lysine group. Location of the modified lysines can be accomplished by a combination of proteolytic digestion, high performance liquid chromatography, and mass spectrometry. The number of modifications can be seen in the electrospray ionization mass spectrum of the dePEGated protein. Work in progress includes an investigation of the electrospray ionization behavior of intact PEG–SOD and site specific quantitative analysis of partial derivatization. The chapter presents reports that indicates that the heterogeneity in PEG–SOD is due to the partial derivatization of the ten lysines in SOD and to the numbers of PEGs attached to each molecule of SOD.
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