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.

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

“…The fragmentation of the triply charged quasi-molecular ion as the most intense precursor ion resulted in the same fragment y22 2+ (1218.2 Da) compared to the fragmentation pattern of hepcidin-25, and a fragment of 415 Da which corresponds to a copper-adduct of the N-terminal fragment containing the three amino acids of the ATCUN motif (Asp-Thr-His). The difference of 61 Da corresponds indeed to the addition of one copper (62.9 Da) and the loss of 2 Da and 2 positive charges (2 H + ), in accordance with previous findings [28,33]. Analyzing the solution of hepcidin-Cu 2+ (molar ratio 1:10) revealed a hepcidin-25 species complexed with two Cu 2+ -ions.…”

“…Some authors applied FTICR-MS measurements at pH of 6.8 and found total complex formation at molar ratio hepcidin-25:copper of 1:1 [28], while other authors reported the presence of unreacted peptide at equimolar concentrations, in a q-ToF-MS (Quadrupol-Time-of-flight mass spectrometry) experiment at pH of 8.2. [33]. Fig.…”

“…This indicates the stability of the copper-binding motif that is preserved even during MS fragmentation. Furthermore, the affinity of hepcidin-25 for copper allowed the quantification of the peptide in serum samples using inductively coupled plasma mass spectrometry (ICP-MS) by employing direct biomolecule labeling [33]. Publications regarding N-terminus of Hep-25 reported this motif to be unstructured in contrast to the rest of the molecule which is highly rigid due to four intramolecular disulfide bridges [11,12].…”

“…The LC separation improves detection and reduces ion suppression effect in LC-MS analysis. Anion exchange chromatography was previously employed for the separation of the copper-labeled peptide from the excess of copper in an LC-ICP-MS study for the quantification of Hep-25 by measuring the amount of copper bound to the peptide [33]. Unfortunately, this method provides no molecular information on the peptide species present in solution.…”

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

“…This indicates the stability of the copper-binding motif that is preserved even during MS fragmentation. Furthermore, the affinity of hepcidin-25 for copper allowed the quantification of the peptide in serum samples using inductively coupled plasma mass spectrometry (ICP-MS) by employing direct biomolecule labeling [29]. Publications regarding N-terminus of Hep-25 reported this motif to be unstructured in contrast to the rest of the molecule which is highly rigid due to four intramolecular disulfide bridges [5,6].…”