Intrigued by these findings, we decided to further investigate the bioconjugation site in a more rational manner. As bTG has been described to crosslink BSA , we reproduced and optimized the bioreaction between BSA and dansylcadaverine, catalyzed by the bTG enzyme (Figure ). The reaction mixture was digested for peptide mapping experiments and a control digestion was performed on BSA alone (Figure ). The digested tryptic peptides were analyzed by mass spectrometry and the dansylcadaverine modification could be detected on two asparagine residues, N290 and N324. The extraction of the dansylcadaverine signature fragment confirmed the presence of the modification on these two amino acids. Surprisingly, no modification was detected on glutamine residues. By comparing the decrease in the intensity of the unmodified peptides containing N324 and N290 respectively, we propose the main conjugation site might be on N324 (Figure ). Therefore, we ordered the two identified peptide sequences ("YICDNQDTISSK" and "DAIPENLPPLTADFAEDK", Figures and) and two control peptide sequences containing asparagine residues that are present in the BSA sequence but where no modification could be identified (Figure and Figure ). Bioconjugation reactions with the dansylcadaverine amine were performed on the peptide sequences (Figures S23 to S26 and Table ) where product formation could only be observed with the peptide sequence identified as the main binding site (N324, 96% conversion). Interestingly, this sequence possesses the same "ENL" motif as found in the TEV recognition sequence for which conversion could also be detected.