A novel method for identifying PEGylation sites of protein using biotinylated PEG derivatives.
The identification of PEGylation sites is essential in the characterization of PEGylated therapeutic proteins. This report describes a simple and novel method of finding poly(ethylene glycol) (PEG) conjugation sites in PEGylated proteins by using a hetero-functional biotin-PEG-N-hydroxyl succinimide derivative. PEGylated lysozyme species having a biotin moiety at each PEG chain end were separated and digested by trypsin. Among the digested lysozyme fragments, biotin-terminated PEGylated peptide fragments were purified by a monomeric avidin immobilized column. Their mass was analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry, directly indicating that PEG was conjugated to lysine 33, 97, 116 residues. Reversed-phase high-pressure liquid chromatography results for the PEGylated peptide fragments exhibited that PEGylation occurred preferentially at lysine 33> lysine 97> lysine 116.
Lee
Haeshin
H
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea (South).
Park
Tae Gwan
TG
D001665:Binding Sites / Q000502:physiology; D001710:Biotin / Q000032:analysis / Q000378:metabolism; D019921:Biotinylation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D009113:Muramidase / Q000032:analysis / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000032:analysis / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D011506:Proteins; D011092:Polyethylene Glycols; D001710:Biotin; D009113:Muramidase