Phenylglyoxal-Based Visualization of Citrullinated Proteins on Western Blots

Molecules

Sanne M. M. Hensen¹  Wilbert C. Boelens¹  Kimberly M. Bonger¹  Remco T. P. van Cruchten¹  Floris L. van Delft²  Ger J. M. Pruijn¹ 

[1] Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud Institute for Molecular Life Sciences and Netherlands Proteomics Centre, Radboud University Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands; E-Mails:;Department of Synthetic Organic Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands; E-Mail:

关键词: citrulline;    peptidylarginine deiminases;    anti-modified citrulline antibodies;    phenylglyoxal;    azide-alkyne cycloaddition;    rheumatoid arthritis;   

DOI  :  10.3390/molecules20046592

来源: mdpi

【 摘 要 】

Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.