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-fanctional 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. (C) 2002 Wiley-Liss, Inc.