Assessing Thorium-Peptide Interactions Using Hydrophilic Interaction Liquid Chromatography

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Scientists from the Paris-Saclay University in Gif-sur-Yvette, France analyzed how hydrophilic interaction liquid chromatography (HILIC) can assess thorium interaction with peptides in a new study published in the Journal of Chromatography A (1).

3D glass molecules or atoms on light blue background. Concept of biochemical, pharmaceutical, beauty, medical. Science or medical background. Vector 3d illustration | Image Credit: © Vector_Artist - stock.adobe.com

3D glass molecules or atoms on light blue background. Concept of biochemical, pharmaceutical, beauty, medical. Science or medical background. Vector 3d illustration | Image Credit: © Vector_Artist - stock.adobe.com

HILIC is a type of reversed-phase liquid chromatography that is performed using a polar stationary phase (2). The mobile phase employed is highly organic, typically containing a small percentage of aqueous solvent/buffer or other polar solvent. The water in the mobile phase forms an aqueous-rich layer adsorbed to the polar surface of the stationary phase into which analytes partition. The main advantage of using HILIC is the strong retention of polar, hydrophilic compounds that would normally be unretained under standard reversed-phase conditions without the need for an ion-pair additive in the mobile phase.

Thorium (Th) is a naturally occurring radioactive metal found at trace levels in soil, rocks, water, and more. It can be used to make ceramics, welding rods, camera and telescope lenses, and more. Under normal conditions, Th is solid, and all its forms, natural and man-made, are radioactive (3). Most people are not exposed to dangerous levels of Th, and but they are typically exposed to tiny amounts in air, food, and water. However, those who live near Th mining areas or near certain legacy industrial facilities may have increased exposure to it. This is concerning, as research shows that inhaling thorium dust can increase the risk of lung and pancreatic cancer; further, since thorium can be stored in bone, those exposed to thorium can increase their risk of bone cancer (4).

In this study, the scientists demonstrated HILIC’s potential in separating polar Th biomimetic peptide complexes. Th4+ was used as plutonium (Pu4+) analogue and pS16 and pS1368 as synthetic di- and tetra-phosphorylated peptides capable of mimicking the interaction sites of these actinides (An) in osteopontin (OPN), a hyperphosphorylated protein. The scientists determined the relative affinity of pS16 and pS1368 towards Th4+ and evaluated the pS1368 selectivity when Th4+ was in competition complexation reaction with UO22+ at physiological pH.

For this process, HILIC was coupled to electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS), allowing the scientists to simultaneously to identify online the molecular structure of the separated complexes and quantify them. Dedicated HILIC conditions were set up to separate the new dimeric Th2(peptide)2 complexes with good separation resolution. By adding pS16 and pS1368 in different proportions relatively to Th4+, the scientists found that lower or equal proportions of pS16 with respect to pS1368 were not sufficient to displace pS1368 from Th2pS13682 and pS16 proportion higher than pS1368 led to the formation of a predominant ternary complex Th2(pS16)(pS1368), showing how Th4+ demonstrated preferential binding to the tetra-phosphorylated peptide. Online identification and quantification of the formed complexes showed that in spite of pS1368 has been specifically designed to coordinate uranyl (UO22+), pS1368 is also Th4+-selective and exhibits stronger affinity for this latter than for UO22+.

By continuously developing HILIC stationary phases with decreased dimensions or core shell particles, sub-2 µm diameter particles and monolithic materials, scientists can gain further insight into a species separations based on this separation mode, all while decreasing sample consumption and waste production.

References

(1) Abou-Zeid, L.; Pell, A.; Saraiva, M. A.; Delangle, P.; Bresson, C. Hydrophilic Interaction Liquid Chromatography: An Efficient Tool for Assessing Thorium Interaction with Phosphorylated Biomimetic Peptides. J. Chromatogr. A 2024, 1735, 465341. DOI: 10.1016/j.chroma.2024.465341

(2) HILIC Overview. ThermoFisher Scientific 2025. https://www.thermofisher.com/us/en/home/industrial/chromatography/chromatography-learning-center/liquid-chromatography-information/hilic-hplc-uhplc-columns-information/hilic-overview.html (accessed 2025-2-3)

(3) Radionuclide Basics: Thorium. EPA 2025. https://www.epa.gov/radiation/radionuclide-basics-thorium (accessed 2025-2-4)

(4) Thorium. National Cancer Institute 2025. https://www.cancer.gov/about-cancer/causes-prevention/risk/substances/thorium (accessed 2025-2-4)

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