title: Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein.
abstract: SDS-PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein-containing conjugates, mostly pegylated proteins. PEG-maleimide (MAL) is frequently used to site-specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C-S linkage between protein and PEG-MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein was observed. It is a thiol-independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG-MAL modified conjugates although PEG-vinylsulfone conjugates to thiol-containing proteins also through a C-S linkage. Cleavage kinetics of PEG40k-MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide-thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG-MAL modified protein/peptide by SDS-PAGE analysis.
authors.affiliation: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China; Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, China.
authors.forename: Chun
authors.initials: C
authors.lastname: Zhang
authors.forename: Yongdong
authors.initials: Y
authors.lastname: Liu
authors.forename: Cui
authors.initials: C
authors.lastname: Feng
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.forename: Hong
authors.initials: H
authors.lastname: Shi
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhao
authors.forename: Rong
authors.initials: R
authors.lastname: Yu
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Su
mesh_terms: D020934:Ciliary Neurotrophic Factor / Q000032:analysis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005030:Ethylenes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry; D013696:Temperature
chemical_list: D020934:Ciliary Neurotrophic Factor; D005030:Ethylenes; D008301:Maleimides; D011506:Proteins; D013451:Sulfonic Acids; C043592:maleimide; D011092:Polyethylene Glycols; C085726:ethylenesulfonic acid
keywords: Loss of PEG; PEG-maleimide; SDS-PAGE; Thioether cleavage; Thiosuccinimide