Optimised methods (SDS/PAGE and LC-MS) reveal deamidation in all examined transglutaminase-mediated reactions

Éva Sivadó, Meddy El Alaoui, Robert Kiraly, László Fesüs, Frédéric Delolme, Adeline Page, Saïd El Alaoui

Research output: Contribution to journalArticle


Transglutaminases (TGs) are a family of structurally and functionally related enzymes that catalyse calcium-dependent post-translational modifications of proteins through protein–protein crosslinking, amine incorporation, or deamidation. For many years deamidation mediated by TGs was considered to be a side reaction, but recently substrate-specific deamidations have been reported. Here we describe an optimised SDS/PAGE assay for the easy and rapid monitoring of the TG reaction with small peptides. The relative proportion of deamidation to transamidation was evaluated by densitometric analysis and confirmed by nano-liquid chromatography–nano-electrospray ionisation MS. We further investigated the effect of reaction conditions on transamidation and deamidation of TG1, TG2 and blood coagulation factor XIII A-subunit (FXIII-A) enzymes using a panel of glutamine-containing peptide substrates. The ratio of transamidation to deamidation was enhanced at high excess of the acyl-acceptor substrate and increasing pH. In addition, it was influenced by peptide substrates as well. Whereas deamidation was favoured at low cadaverine concentrations and acidic pH, no significant effect of calcium was observed on the ratio of transamidation/deamidation. Under our experimental conditions, deamidation always occurred in vitro even at high excess of the acyl-acceptor substrate, and the reaction outcome was shifted to deamidation at neutral pH. Our results provide clear evidence of the deamidation in the TG reaction, and may serve as an important approach for in vivo analysis of deamidation to better understand the role of TGs in biological events.

Original languageEnglish
Pages (from-to)396-404
Number of pages9
JournalFEBS Open Bio
Issue number2
Publication statusPublished - Feb 2019



  • deamidation
  • fluorescence detection
  • transamidation
  • transglutaminases

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this