Determination of the affinity of biomimetic peptides for uranium through the simultaneous coupling of HILIC to ESI-MS and ICP-MS

Several proteins have been identified in the past decades as targets of uranyl (UO22+) in vivo. However, the molecular interactions responsible for this affinity are still poorly known which requires the identification of the UO22+ coordination sites in these proteins. Biomimetic peptides are efficient chemical tools to characterize these sites. In this work, we developed a dedicated analytical method to determine the affinity of biomimetic, syn-thetic, multi-phosphorylated peptides for UO22+ and evaluate the effect of several structural parameters of these peptides on this affinity at physiological pH. The analytical strategy was based on the implementation of the simultaneous coupling of hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). An essential step had been devoted to the definition of the best separation conditions of UO22+ complexes formed with di-phosphorylated peptide isomers and also with peptides of different structure and degrees of phosphorylation. We performed the first separations of several sets of UO22+ complexes by HILIC ever reported in the literature. A dedicated method had then been developed for identifying the separated peptide complexes online by ESI-MS and simultaneously quantifying them by ICP-MS, based on uranium quantification using external calibration. Thus, the affinity of the peptides for UO22+ was determined and made it possible to demonstrate that (i) the increasing number of phosphorylated residues (pSer) promotes the affinity of the peptides for UO22+, (ii) the position of the pSer in the peptide backbone has very low impact on this affinity (iii) and finally the cyclic structure of the peptide favors the UO22+ complexation in comparison with the linear structure. These results are in agreement with those previously obtained by spectroscopic techniques, which allowed to validate the method. Through this approach, we obtained essential information to better understand the mechanisms of toxicity of UO22+ at the molecular level and to further develop selective decorporating agents by chelation.

Automated summary

This study developed an analytical method to determine the affinity of biomimetic peptides for uranyl ions and evaluate the effect of structural parameters on this affinity. By combining hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS), the UO22+ complexes formed with different peptides were successfully separated and quantified. The results showed that an increasing number of phosphorylated residues promotes the affinity of the peptides for UO22+, while the position of the phosphorylated residues in the peptide backbone has minimal impact on this affinity, and the cyclic structure of the peptide favors the complexation with UO22+ compared to the linear structure.