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

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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.
Publisher
JOHN WILEY & SONS INC
Issue Date
2003-01
Language
English
Article Type
Article
Keywords

POLYETHYLENE-GLYCOL; SUPEROXIDE-DISMUTASE; COVALENT ATTACHMENT; POSITIONAL ISOMERS; IMMUNOLOGICAL PROPERTIES; POLY(ETHYLENE GLYCOL); CHEMICAL MODIFICATION; STABILITY; LYSOZYME; PEPTIDE

Citation

JOURNAL OF PHARMACEUTICAL SCIENCES, v.92, no.11, pp.97 - 103

ISSN
0022-3549
DOI
10.1002/jps.10270
URI
http://hdl.handle.net/10203/13303
Appears in Collection
CH-Journal Papers(저널논문)BS-Journal Papers(저널논문)
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