title: Characterization of a site-specific PEGylated analog of exendin-4 and determination of the PEGylation site.
abstract: PEGylation has been a successful strategy for improving the pharmacokinetic and pharmaceutical properties of proteins and peptides. However, PEGylated products also create significant challenges for detailed structural characterization. In this work, a site-specific PEGylation strategy was successfully performed on an exendin-4 analog (Ex4C) through a maleimide method. Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), analytical reversed phase HPLC (RP-HPLC) and MALDI-TOF were applied to verify the accomplishment of the PEGylation. Peptide mapping was investigated after tryptic digestion, and the PEGylaton site was successfully located on the C-terminal fragment of Ex4C. Amino acid analysis (AAA) of cysteine was then applied to verify the block in the thiol group caused by PEGylation. We believe that the combination of proper enzymatic digestion and amino acid analysis of cysteine provided an easy and convincing way to identify the PEGylation site in this maleimide method.
authors.affiliation: Department of Biochemistry, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Qian
authors.forename: Hongxia
authors.initials: H
authors.lastname: Dong
authors.forename: Hong
authors.initials: H
authors.lastname: Tian
authors.forename: Yue
authors.initials: Y
authors.lastname: Tong
authors.forename: Linfeng
authors.initials: L
authors.lastname: Guo
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Hu
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Gao
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D000077270:Exenatide; D008301:Maleimides / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013678:Technology, Pharmaceutical / Q000379:methods; D014688:Venoms / Q000737:chemistry*
chemical_list: D008301:Maleimides; D010455:Peptides; D014688:Venoms; C043592:maleimide; D011092:Polyethylene Glycols; D000077270:Exenatide; D003545:Cysteine
keywords: Amino acid analysis (AAA); Cysteine; Exendin-4; PEGylation site; Tryptic digestion