title: Elemental labeling and isotope dilution analysis for the quantification of the peptide hepcidin-25 in serum samples by HPLC-ICP-MS.
abstract: Hepcidin-25 is a peptide-hormone that has been proposed as the key biomarker for the diagnosis and monitoring of iron disorders. Structurally, hepcidin-25 is a S-rich peptide (with 8 cysteines and 1 methionine) that contains a metal binding motif in the N-terminus. That domain binds preferably Cu(II) ion forming a stable complex. Such selective binding can be used as mean to determine hepcidin-25 in biological fluids by highly sensitive Cu measurement. Thus, we use liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS) to perform hepcidin-25 determination via Cu detection. For this purpose, the incubation conditions were optimized to address the complex formation and stability by electrospray-MS (ESI-q-TOF). It was found that Cu:hepcidin-25 complex is stable under physiological conditions and shows an equimolar stoichiometry (1:1). The collisional induced dissociation (CID) experiments confirmed the specific binding of Cu to the N-terminal motif. For Cu quantification, two isotope dilution strategies have been developed. The first one, including postcolumn addition of a (65)Cu spike and the second, by synthesizing the labeled (65)Cu:hepcidin-25 complex as tracer (species-specific). Both methods have been optimized and critically compared in real samples. The determination of hepcidin-25 in different serum samples from healthy individuals based on Cu monitoring showed a mean value of 21.6 ng mL(-1) which is in good agreement to previously published data.
authors.affiliation: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julián Clavería 8, 33006 Oviedo, Spain.
authors.forename: T
authors.initials: T
authors.lastname: Konz
authors.forename: M
authors.initials: M
authors.lastname: Montes-Bayón
authors.forename: A
authors.initials: A
authors.lastname: Sanz-Medel
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D064451:Hepcidins; D006801:Humans; D007553:Isotope Labeling; D012016:Reference Values; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation
chemical_list: D023181:Antimicrobial Cationic Peptides; D064451:Hepcidins; C545475:hepcidin 25, human