Journal: PLoS ONE

Article Title: Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

doi: 10.1371/journal.pone.0075518

Figure Lengend Snippet: Hepcidin-25 +40 was dissolved in 20% acetonitril (acn), 5% phosphoric acid or water and used as internal standards in blank serum samples containing end concentrations of 0, 0.5,1, 2, 3, 5, 7.5, 10, 15, 20 and 40 nM synthetic hepcidin-25 purchased from Peptides International. Linearity of measured hepcidin-25 was determined by the formulas: Y = 1.822X+0.097 (5% phosphoric acid); Y = 1.119X+0.494 (water); Y = 0.964X+0.069 (20% acn).

Article Snippet: To better understand the cause of different absolute hepcidin concentrations that are measured by different hepcidin assays throughout the world , we used the hepcidin-25 +40 standard to quantify synthetic hepcidin peptides from either Bachem or Peptides International that were spiked in blank serum with 10 nM of synthetic hepcidin-25 (according to the package inserts of the vendors).

Techniques:

Journal: PLoS ONE

Article Title: Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

doi: 10.1371/journal.pone.0075518

Figure Lengend Snippet: A. 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 (R 2 = 0.9950); hepcidin-24, Y = 1.145X−0.767 (R 2 = 0.9975); hepcidin-22, Y = 1.100X−0.197 (R 2 = 0.9998); hepcidin-20, Y = 0.867X+0.055 (R 2 = 0.9998). 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.

Article Snippet: To better understand the cause of different absolute hepcidin concentrations that are measured by different hepcidin assays throughout the world , we used the hepcidin-25 +40 standard to quantify synthetic hepcidin peptides from either Bachem or Peptides International that were spiked in blank serum with 10 nM of synthetic hepcidin-25 (according to the package inserts of the vendors).

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

doi: 10.1371/journal.pone.0075518

Figure Lengend Snippet: Panel A , peptide profile of a heparin plasma pool of IC patients; Panel B/C , peptide profile of heparin plasma pool of nephrology patients that were untreated ( B ) or pre-incubated with 1 molar excess of the anti-hepcidin molecule PRS-080 prior to WCX-TOF MS analysis ( C ); Panels D/E , 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.

Article Snippet: To better understand the cause of different absolute hepcidin concentrations that are measured by different hepcidin assays throughout the world , we used the hepcidin-25 +40 standard to quantify synthetic hepcidin peptides from either Bachem or Peptides International that were spiked in blank serum with 10 nM of synthetic hepcidin-25 (according to the package inserts of the vendors).

Techniques: Incubation, Derivative Assay

Journal: PLoS ONE

Article Title: Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

doi: 10.1371/journal.pone.0075518

Figure Lengend Snippet: 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.

Article Snippet: To better understand the cause of different absolute hepcidin concentrations that are measured by different hepcidin assays throughout the world , we used the hepcidin-25 +40 standard to quantify synthetic hepcidin peptides from either Bachem or Peptides International that were spiked in blank serum with 10 nM of synthetic hepcidin-25 (according to the package inserts of the vendors).

Techniques: Stable Transfection, Fluorescence

Journal: PLoS ONE

Article Title: Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

doi: 10.1371/journal.pone.0075518

Figure Lengend Snippet: 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 (R 2 = 0.9959); hepcidin-25 (IS HEP-24), with hep-24 correction, Y = 1.041X−0.425 (R 2 = 0.9960).

Article Snippet: To better understand the cause of different absolute hepcidin concentrations that are measured by different hepcidin assays throughout the world , we used the hepcidin-25 +40 standard to quantify synthetic hepcidin peptides from either Bachem or Peptides International that were spiked in blank serum with 10 nM of synthetic hepcidin-25 (according to the package inserts of the vendors).

Techniques:

Journal: PLoS ONE

Article Title: Improved Mass Spectrometry Assay For Plasma Hepcidin: Detection and Characterization of a Novel Hepcidin Isoform

doi: 10.1371/journal.pone.0075518

Figure Lengend Snippet: The theoretical concentrations of hepcidin-25 used in these experiments were adjusted towards 100% peptide content, based on the information provide in the package inserts of the respective Vendors (see ). Hep-25, m/z 2789.4; Hep-25 +40 (internal standard), m/z 2829.4.

Article Snippet: To better understand the cause of different absolute hepcidin concentrations that are measured by different hepcidin assays throughout the world , we used the hepcidin-25 +40 standard to quantify synthetic hepcidin peptides from either Bachem or Peptides International that were spiked in blank serum with 10 nM of synthetic hepcidin-25 (according to the package inserts of the vendors).

Techniques: