A novel method for identifying PEGylation sites of protein using biotinylated PEG derivatives

  • Lee H
  • Park T
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Abstract

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.

Author-supplied keywords

  • Avidin-biotin
  • Identification of PEGylation sites
  • MALDI-TOF MS
  • Polyethylene glycol (PEG)

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Authors

  • Haeshin Lee

  • Tae Gwan Park

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