Characterization of poly(ethylene glycol)-modified superoxide dismutase: Comparison of capillary electrophoresis and matrix-assisted laser desorption ionization mass spectrometry

The poly(ethylene glycol) (PEG)-modified enzyme superoxide dismutase (PEG-SOD) was characterized using capillary electrophoresis (CE), and the results were compared to those obtained using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, A free-solution CE method was developed which separated PEG-SOD species based on the number of attached PEG chains. The identity of individual CE peaks was established by isolation and subsequent molecular weight analysis by MALDI. The accuracy of the CE method in determining the average total number of PEG chains attached to the enzyme was established by comparing it with an HPLC method that quantitates the amount of PEG after hydrolytic cleavage from the modified enzyme. In addition to determining the average number of PEG chains attached to the protein, the CE method provides information on the distribution of PEG conjugation to the SOD. The total amount and distribution of coupling of PEG to the enzyme was also directly measured using MALDI. The distribution profile of PEG modification for a typical sample of PEG-SOD as determined by CE was found to be consistent with that obtained by MALDI. A quantitative comparison of the results obtained by the two techniques on PEG-SOD samples representing a range of values of PEG modification of the enzyme demonstrated good correlation, although differences were noted in some cases. Both techniques achieved a high degree of reproducibility when properly optimized. Data were also obtained on stressed samples of PEG-SOD demonstrating the ability of the CE method to detect changes in the degree and distribution of PEG conjugation for samples on stability, These two techniques provide a convenient means of characterizing the distribution of coupling of PEG to this enzyme which may have applications to other PEG modified proteins.