Reproducibility of the measurement of hepcidin-25 and its isoforms of the improved hepcidin assay with the novel hepcidin-25⁺⁴⁰ as internal standard.^*

*<p>Heparin samples used for intra-run experiments are the high and low QC samples and samples from nephrology patients (n = 2). Heparin samples used for inter-run experiments are the high and low QC samples.</p

Characteristics of commercial hepcidin peptides used.

*<p>catalog product of Peptide International but manufactured by Peptide Institute (Osaka, Japan);</p>∧<p>vialed at 100% peptide content;</p>†<p>due to an isotope content of 98% the actual mass is 1 Da less than the theoretical mass of this peptide.</p>#<p>delivered as 0.10 mg per vial; personal communication revealed that ≈ 0.11 mg was pipetted in each vial. PI, Peptide International (Louisville, KY, USA); B, Bachem LTD (St. Helens, UK); n.a., not applicable.</p

Hepcidin-mediated ferroportin internalization.

<p>Different concentrations of synthetic hepcidin-25, -24, -22 and -20 (indicated in nM on the horizontal axis) were added to the growth medium of a stable cell line that expresses green fluorescent protein-fused ferroportin (GFP-FPN). Hepcidin-mediated GFP-FPN internalization and degradation was quantified by measuring cellular fluorescence levels in arbitrary units.</p

Relative change of hepcidin at RT in heparin plasma^*.

*<p>Relative change in hepcidin-25, -24, -22 and -20 concentrations in heparin plasma samples expressed as percentage of the respective concentrations in fresh samples. Samples were from 19 intensive care (IC) patients and 5 controls and kept for 1 day and 7 days (1 week) at RT. Results <1.0 nM were removed from the calculations, among which are the results of the isoforms of 5 controls. Only results from samples with complete serial measurements are included.</p

Comparison of hepcidin concentrations obtained by the respective internal standards hepcidin-24 and hepcidin-25⁺⁴⁰, with and without correction for native hepcidin-24 concentrations.

<p>Samples (n = 14) consisted of serum samples from healthy controls (n = 3), heparin plasma from nephrology patients (n = 7), heparin plasma high and low QC pools, serum high and low QC pools. Description of the lines: hepcidin-25 (IS HEP-24), Y = 0.878X+0.059 (R² = 0.9959); hepcidin-25 (IS HEP-24), with hep-24 correction, Y = 1.041X−0.425 (R² = 0.9960).</p

Changes in time at RT of hepcidin-25, 24, 22, and 20 and their sum (total hepcidin) in a representative heparin sample of an IC patient to illustrate that decrease in hepcidin-25 levels is accompanied by an increase in hepcidin-24, -22, and -20 levels.

<p>Similar observations for an extended set of samples are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075518#pone.0075518.s001" target="_blank">Figure S1</a>. Note that the total amount of hepcidin decreases despite the increase in hepcidin isoform levels.</p

Quantification of hepcidin isoforms using hepcidin-25⁺⁴⁰ as internal standard.

<p><b>A.</b> Linearity (range 0–40 nM) for hepcidin-25, hepcidin-24, hepcidin-22, and hepcidin-20 as determined by hepcidin-25⁺⁴⁰ as internal standard. Linearity curves are assessed in different runs. Blank serum (hep-24, hep-20 and hep-25) or heparin plasma (used for hep-22 as serum yields an interfering peak near the position of this isoform) was used as matrix for the addition of the synthetic hepcidin isoforms (PI) to end concentrations of 0, 0.5,1, 2, 3, 5, 7.5, 10, 15, 20 and 40 nM. Since there is a small interfering peak at 2191.8 Da in blank serum, the linearity curve of hepcidin-20 was corrected for the base line hepcidin-20 peak (data not shown). Description of the lines: hepcidin-25, Y = 0.964X+0.064 (R² = 0.9950); hepcidin-24, Y = 1.145X−0.767 (R² = 0.9975); hepcidin-22, Y = 1.100X−0.197 (R² = 0.9998); hepcidin-20, Y = 0.867X+0.055 (R² = 0.9998). <b>B.</b> WCX-TOF MS profile of blank plasma that was spiked with 10 nM of each of the synthetic hepcidin-20, -22, -24, -25, and -25⁺⁴⁰ peptides, which illustrates that all these hepcidin analogues have the same WCX-binding characteristics and flight behavior during TOF MS.</p

WCX-TOF MS profiles of sample pools of patients with presumed hepcidin isoforms.

<p>Panel <b>A</b>, peptide profile of a heparin plasma pool of IC patients; Panel <b>B/C</b>, peptide profile of heparin plasma pool of nephrology patients that were untreated (<b>B</b>) or pre-incubated with 1 molar excess of the anti-hepcidin molecule PRS-080 prior to WCX-TOF MS analysis (<b>C</b>); Panels <b>D/E</b>, control peptide profile of plasma from patients with juvenile hemochromatosis and iron deficiency anemia, respectively, that lack hepcidin-25. Positions in the spectrum: hepcidin-25⁺⁴⁰ (internal standard), m/z 2829.4; hepcidin-25, m/z 2789.4; hepcidin-24, m/z 2673.9; hepcidin-22, m/z 2436.1; and hepcidin-20, m/z 2191.8. It should be noted that: i) profiles from IC and nephrology patients both clearly contain the m/z 2673.9 peak at the presumed position of hepcidin-24; ii) this peak disappears completely from the profile after incubation with PRS-080, similar to hepcidin-25/-22; iii) hepcidin-25⁺⁴⁰ does not disappear from the profile as it was added after the PRS-080 incubation period, which limits complex formation; iv) the intensity of the presumed peak of hepcidin-20 at m/z 2191.8 after PRS-080 incubation decreases but does not disappear completely suggesting that another hepcidin-unrelated peptide is also present at this position; v) the peptide spectra of patient that lack hepcidin-25 also contain a peak at m/z 2191.8 (calculated between 1–2 nM), providing further evidence for the unlikeliness that this peak is solely derived from hepcidin-20.</p

Comparison of hepcidin-25 concentrations measured in EDTA plasma and heparin plasma samples from healthy controls (n = 5), hemodialysis (HD) patients (n = 10) and intensive care (IC) patients (n = 10).

<p>Description of the line: Y = 1.083X+0.274 (R² = 0.9770).</p

Hepcidin quantification by WCX-TOF MS in human samples that were pre-incubated with 0, 5 or 10 nM of PRS-080 to block binding of hepcidin to the WCX beads.

<p>Decrease in hepcidin concentration was determined by measurement of 6 different samples, with total hepcidin concentrations between 10 and 40-080. Dotted line indicates the theoretically expected 1∶1 ratio. Description of the line: Y = 0.655X+0.013 (R² = 0.9373).</p