Elemental Labeling and Isotope Dilution Analysis for the Quantification of the Peptide Hepcidin-25 in Serum Samples by HPLC-ICP-MS

• Published in: Analytical chemistry (Washington) Vol. 84; no. 19; pp. 8133 - 8139
• Main Authors: Konz, T, Montes-Bayón, M, Sanz-Medel, A
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 02-10-2012
• Subjects: Analytical chemistry; Antimicrobial Cationic Peptides - blood; Binding sites; Biomarkers; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, High Pressure Liquid - instrumentation; Exact sciences and technology; Hepcidins; Humans; Isotope Labeling; Mass spectrometry; Other chromatographic methods; Peptides; Reference Values; Sensitivity and Specificity; Spectrometric and optical methods; Spectrometry, Mass, Electrospray Ionization - instrumentation; Copper II; Biological fluid; Stoichiometry; Cysteine; Isotope labelling; Peptides; HPLC chromatography; Biological marker; Iron; Metal; Inductive coupling plasma spectrometry; Electrospray; Postcolumn; Serum; Diagnosis; Dissociation; Monitoring; Quantitative analysis; Hormone; Collisional activation; Stability; Coupled method; Sample; Use; Methionine; Isotope dilution; Liquid chromatography; Chemical ionization; Time of flight method; Strategy; Detection; Mass spectrometry; Tracers
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac300578n

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 65Cu spike and the second, by synthesizing the labeled 65Cu: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.