Phenylglyoxal-Based Visualization of Citrullinated Proteins on Western Blots
Sanne M. M. Hensen,
Wilbert C. Boelens,
Kimberly M. Bonger,
Remco T. P. van Cruchten,
Floris L. van Delft,
Ger J. M. Pruijn
Affiliations
Sanne M. M. Hensen
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
Wilbert C. Boelens
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
Kimberly M. Bonger
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
Remco T. P. van Cruchten
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
Floris L. van Delft
Department of Synthetic Organic Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands
Ger J. M. Pruijn
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
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.
