Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform

Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role...

Full description

Saved in:

Bibliographic Details
Published in:	PloS one 2013-10, Vol.8 (10), p.e75518-e75518
Main Authors:	Laarakkers, Coby M M, Wiegerinck, Erwin T, Klaver, Siem, Kolodziejczyk, Maria, Gille, Hendrik, Hohlbaum, Andreas M, Tjalsma, Harold, Swinkels, Dorine W
Format:	Article
Language:	eng
Subjects:	Amino acids Anticoagulants Assaying Bioassays Cation exchange Cation exchanging Coefficient of variation Hepcidin Hepcidins - blood Homeostasis Humans Ions Iron Isoforms Isotopes Kidney diseases Laboratories Mass spectrometry Mass Spectrometry - methods Mass spectroscopy Medicine Metabolic disorders Metabolism Methods Optimization Patients Peptides Physiological aspects Protein Isoforms - blood Renal function Reproducibility of Results Scientific imaging Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Spectroscopy Standardization Urine
Online Access:	Get full text
Tags:	Add Tag No Tags, Be the first to tag this record!

Description
Summary:	Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25(+40) isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25(+40) as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.
ISSN:	1932-6203 1932-6203