1. Validation of hepcidin quantification in plasma using LC-HRMS and discovery of a new hepcidin isoform
Bertrand Rochat, et al. Bioanalysis. 2013 Oct;5(20):2509-20. doi: 10.4155/bio.13.225.
Background: Hepcidin, a 25 amino acid peptide, plays an important role in iron homeostasis. Some hepcidin truncated peptides have antibiotic effects. Results: A new analytical method for hepcidin determination in human plasma using LC-HRMS operating in full-scan acquisition mode has been validated. The extraction consists of protein precipitation and a drying reconstitution step; a 2.1 x 50 mm (idxL) C18 analytical column was used. Detection specificity, stability, accuracy, precision and recoveries were determined. The LOQ/LOD were 0.25/0.1 nM, respectively. More than 600 injections of plasma extracts were performed, allowing evaluation of the assay robustness. Hepcidin-20, hepcidin-22 and a new isoform, hepcidin-24, were detected in patients. Conclusion: The data underscore the usefulness of LC-HRMS for in-depth investigations related to hepcidin levels and pathways.
2. Improved mass spectrometry assay for plasma hepcidin: detection and characterization of a novel hepcidin isoform
Coby M M Laarakkers, Erwin T Wiegerinck, Siem Klaver, Maria Kolodziejczyk, Hendrik Gille, Andreas M Hohlbaum, Harold Tjalsma, Dorine W Swinkels PLoS One. 2013 Oct 4;8(10):e75518. doi: 10.1371/journal.pone.0075518. eCollection 2013.
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.
