Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform
| Autor: | Harold Tjalsma, Dorine W. Swinkels, Andreas Hohlbaum, Maria Kolodziejczyk, Erwin T. Wiegerinck, Hendrik Gille, Coby M. Laarakkers, Siem M. Klaver |
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| Rok vydání: | 2013 |
| Předmět: |
Gene isoform
inorganic chemicals congenital hereditary and neonatal diseases and abnormalities Ferroportin Iron metabolism Pathogenesis and modulation of inflammation [IGMD 7] lcsh:Medicine Mass spectrometry Mass Spectrometry 03 medical and health sciences 0302 clinical medicine Hepcidins Hepcidin hemic and lymphatic diseases Blood plasma Humans Protein Isoforms Iron metabolism [IGMD 7] lcsh:Science 030304 developmental biology 0303 health sciences Multidisciplinary biology Chemistry lcsh:R N4i 1 - pathogenesis and modulation of inflammation Oncol 5 - Aetiology screening and detection Reproducibility of Results nutritional and metabolic diseases Transporter Iron metabolism Aetiology screening and detection [IGMD 7] Matrix-assisted laser desorption/ionization Biochemistry 030220 oncology & carcinogenesis Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein lcsh:Q Time-of-flight mass spectrometry Research Article |
| Zdroj: | PLoS One, 8, 10 PLoS One, 8 PLoS ONE PLoS ONE; Vol 8 PLoS ONE, Vol 8, Iss 10, p e75518 (2013) |
| ISSN: | 1932-6203 |
| Popis: | Contains fulltext : 125929.pdf (Publisher’s version ) (Open Access) 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. |
| Databáze: | OpenAIRE |
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