Investigations of the Copper Peptide Hepcidin-25 by LC-MS/MS and NMR

• Published in: International journal of molecular sciences Vol. 19; no. 8; p. 2271
• Main Authors: Abbas, Ioana M, Vranic, Marija, Hoffmann, Holger, El-Khatib, Ahmed H, Montes-Bayón, María, Möller, Heiko M, Weller, Michael G
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 02-08-2018; MDPI
• Subjects: Amino Acid Sequence; Ammonia; ATCUN motif; Binding Sites; Biological activity; Chromatography, Liquid; copper; Copper - chemistry; copper complex; Copper compounds; Emission spectroscopy; Hepcidin; hepcidin-25; Hepcidins - chemistry; Humans; Inductively coupled plasma mass spectrometry; Ionic mobility; Ions; Iron; Isoforms; Isomerism; isomerization; Isomers; Mass spectrometry; Mass spectroscopy; metal complex; metal peptide; metallopeptide; metalloprotein; Models, Molecular; N-Terminus; nickel; NMR; NMR structure; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Peptides; Protein Conformation; purity; racemization; reference material; Scientific imaging; Tandem Mass Spectrometry; NMR structure; metalloprotein; nickel; isomerization; purity; MS; copper complex; ATCUN motif; hepcidin-25; metal peptide; metal complex; copper; racemization; reference material; metallopeptide
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms19082271

Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.