Loss of PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein. Issue 2 (10th November 2014)

• Record Type: Journal Article
• Authors: Zhang, Chun; Liu, Yongdong; Feng, Cui; Wang, Qi; Shi, Hong; Zhao, Dawei; Yu, Rong; Su, Zhiguo
• Is Part Of: Electrophoresis. Volume 36:Issue 2(2015)
• Page Start: 371
• Page End: 374
• Publication Date: 2014-11-10
• Subjects: Electrophoresis -- Periodicals; Electrophoresis -- Periodicals; 541.372
• DOI: 10.1002/elps.201400373
• Languages: English
• ISSNs: 0173-0835

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>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.</p> </abstract>