Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy |
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Authors: | Klaas W Decoene Kamil Unal An Staes Olivier Zwaenepoel Jan Gettemans Kris Gevaert Johan M Winne Annemieke Madder |
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Institution: | Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4, 9000 Ghent Belgium.; Department of Biomolecular Medicine, Ghent University, Ghent Belgium ; VIB Center for Medical Biotechnology, Technologiepark-Zwijnaarde 75, 9052 Ghent Belgium ; VIB Core Facility, VIB Center for Medical Biotechnology, Technologiepark-Zwijnaarde 75, 9052 Ghent Belgium |
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Abstract: | Labelling of tyrosine residues in peptides and proteins has been reported to selectively occur via a ‘tyrosine-click’ reaction with triazolinedione reagents (TAD). However, we here demonstrate that TAD reagents are actually not selective for tyrosine and that tryptophan residues are in fact also labelled with these reagents. This off-target labelling remained under the radar as it is challenging to detect these physiologically stable but thermally labile modifications with the commonly used HCD and CID MS/MS techniques. We show that selectivity of tryptophan over tyrosine can be achieved by lowering the pH of the aqueous buffer to effect selective Trp-labelling. Given the low relative abundance of tryptophan compared to tyrosine in natural proteins, this results in a new site-selective bioconjugation method that does not rely on enzymes nor unnatural amino acids and is demonstrated for peptides and recombinant proteins.A new strategy for selective tryptophan modification using triazolinedione (TAD) chemistry at pH 4 is shown on peptides and proteins. Additionally, off-target modification of tryptophan residues during the classical TAD-Y click reaction is uncovered. |
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