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Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein
Alternative TitleElectrophoresis
Zhang, Chun1,2; Liu, Yongdong1; Feng, Cui1,3; Wang, Qi1; Shi, Hong1; Zhao, Dawei1; Yu, Rong2; Su, Zhiguo1
2015
Source PublicationELECTROPHORESIS
ISSN0173-0835
Volume36Issue:2Pages:371-374
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.

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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.

KeywordLoss Of Peg Peg-maleimide Sds-page Thioether Cleavage Thiosuccinimide
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine ; Physical Sciences
DOI10.1002/elps.201400373
URL查看原文
Indexed BySCI
Language英语
WOS KeywordPEGYLATION ; STABILITY ; GLYCOL)
WOS Research AreaBiochemistry & Molecular Biology ; Chemistry
WOS SubjectBiochemical Research Methods ; Chemistry, Analytical
WOS IDWOS:000348739200017
Citation statistics
Cited Times:5[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/11885
Collection研究所(批量导入)
Affiliation1.Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China
2.Sichuan Univ, West China Sch Pharm, Minist Educ, Key Lab Drug Targeting & Drug Delivery Syst, Chengdu 610064, Peoples R China
3.Beijing Univ Chem Technol, Coll Life Sci & Technol, Beijing 100029, Peoples R China
Recommended Citation
GB/T 7714
Zhang, Chun,Liu, Yongdong,Feng, Cui,et al. Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein[J]. ELECTROPHORESIS,2015,36(2):371-374.
APA Zhang, Chun.,Liu, Yongdong.,Feng, Cui.,Wang, Qi.,Shi, Hong.,...&Su, Zhiguo.(2015).Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein.ELECTROPHORESIS,36(2),371-374.
MLA Zhang, Chun,et al."Loss of PEG chain in routine SDS-PAGE analysis of PEG-maleimide modified protein".ELECTROPHORESIS 36.2(2015):371-374.
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