New PEG's for peptides and protein modification, suitable for the identification of the peghilation site

New PEG derivatives were studied for peptide and protein modification, based upon an amino acid arm, Met-Nle or Met-βAla, activated as succinimidyl ester. PEG-Met-Nle-OSu or PEG-Met-βAla-OSu react with amino groups in protein-yielding conjugates with stable amide bond. From these conjugates PEG may be removed by BrCN treatment, leaving Nle or βAla as reporter amino acid, at the site where PEG was bound. The conjugation of PEG and its removal by BrCN treatment was assessed on a partial sequence of glucagone and on lysozyme as model peptide or protein. Furthermore, insulin, a protein with three potential sites of PEGylation, was modified by PEG-Met-Nle, and the PEG isomers were separated by HPLC. After removal of PEG, as reported above, the sites of PEGylation were identified by characterization of the two insulin chains obtained after reduction and carboxymethylation. Mass spectrometry, amino acid analysis and Edman sequence, could reveal the position of the reporter norleucine that corresponds to the position of PEG binding.