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SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard.

Ward DG, Roberts K, Stonelake P, Goon P, Zampronio CG, Martin A, Johnson PJ, Iqbal T, Tselepis C - Proteome Sci (2008)

Bottom Line: However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency.We synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments.This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Birmingham, UK. d.g.ward@bham.ac.uk

ABSTRACT

Background: Hepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS.

Results: We synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin.

Conclusion: We demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.

No MeSH data available.


Related in: MedlinePlus

Hepcidin analysis in urine under different conditions. A urine sample was diluted to 20 μg protein/ml and spiked with 200 ng/ml labelled hepcidin and 0–32% (v/v) human serum devoid of hepcidin (as estimated from SELDI spectra – see the top spectrum in Figure 4). Panel a: endogenous hepcidin peak intensity with/without TIC-normalisation plotted against laser power. Filled symbols denote mean (± SEM) non-normalised intensity of all 8 samples at each laser setting (not [serum] dependent). Open symbols denote mean (± SEM) TIC-normalised intensity of the 4 samples with 0–32% serum added (the TIC normalised is [serum] dependent). Panel b plots the TIC-normalised hepcidin intensity and the concentration calculated from the peak ratio over a range of serum concentrations (constant laser power).
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Figure 7: Hepcidin analysis in urine under different conditions. A urine sample was diluted to 20 μg protein/ml and spiked with 200 ng/ml labelled hepcidin and 0–32% (v/v) human serum devoid of hepcidin (as estimated from SELDI spectra – see the top spectrum in Figure 4). Panel a: endogenous hepcidin peak intensity with/without TIC-normalisation plotted against laser power. Filled symbols denote mean (± SEM) non-normalised intensity of all 8 samples at each laser setting (not [serum] dependent). Open symbols denote mean (± SEM) TIC-normalised intensity of the 4 samples with 0–32% serum added (the TIC normalised is [serum] dependent). Panel b plots the TIC-normalised hepcidin intensity and the concentration calculated from the peak ratio over a range of serum concentrations (constant laser power).

Mentions: Serum was collected from women attending routine breast clinics at Russell's Hall Hospital, Dudley, UK between 2005 and 2007 (LREC Ref 05/Q2709/48). All subjects gave informed consent prior to venipuncture. Venous blood was taken into serum collection tubes and allowed to clot at room temperature for 1–2 hours. Samples were then centrifuged for 10 min at 3000 g and the supernatant stored in aliquots at -80°C. The urine sample used in the experiment of Figure 7 was selected on the basis of hepcidin peak height from a previously analysed set of urine samples [20].


SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard.

Ward DG, Roberts K, Stonelake P, Goon P, Zampronio CG, Martin A, Johnson PJ, Iqbal T, Tselepis C - Proteome Sci (2008)

Hepcidin analysis in urine under different conditions. A urine sample was diluted to 20 μg protein/ml and spiked with 200 ng/ml labelled hepcidin and 0–32% (v/v) human serum devoid of hepcidin (as estimated from SELDI spectra – see the top spectrum in Figure 4). Panel a: endogenous hepcidin peak intensity with/without TIC-normalisation plotted against laser power. Filled symbols denote mean (± SEM) non-normalised intensity of all 8 samples at each laser setting (not [serum] dependent). Open symbols denote mean (± SEM) TIC-normalised intensity of the 4 samples with 0–32% serum added (the TIC normalised is [serum] dependent). Panel b plots the TIC-normalised hepcidin intensity and the concentration calculated from the peak ratio over a range of serum concentrations (constant laser power).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571088&req=5

Figure 7: Hepcidin analysis in urine under different conditions. A urine sample was diluted to 20 μg protein/ml and spiked with 200 ng/ml labelled hepcidin and 0–32% (v/v) human serum devoid of hepcidin (as estimated from SELDI spectra – see the top spectrum in Figure 4). Panel a: endogenous hepcidin peak intensity with/without TIC-normalisation plotted against laser power. Filled symbols denote mean (± SEM) non-normalised intensity of all 8 samples at each laser setting (not [serum] dependent). Open symbols denote mean (± SEM) TIC-normalised intensity of the 4 samples with 0–32% serum added (the TIC normalised is [serum] dependent). Panel b plots the TIC-normalised hepcidin intensity and the concentration calculated from the peak ratio over a range of serum concentrations (constant laser power).
Mentions: Serum was collected from women attending routine breast clinics at Russell's Hall Hospital, Dudley, UK between 2005 and 2007 (LREC Ref 05/Q2709/48). All subjects gave informed consent prior to venipuncture. Venous blood was taken into serum collection tubes and allowed to clot at room temperature for 1–2 hours. Samples were then centrifuged for 10 min at 3000 g and the supernatant stored in aliquots at -80°C. The urine sample used in the experiment of Figure 7 was selected on the basis of hepcidin peak height from a previously analysed set of urine samples [20].

Bottom Line: However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency.We synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments.This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Birmingham, UK. d.g.ward@bham.ac.uk

ABSTRACT

Background: Hepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS.

Results: We synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin.

Conclusion: We demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.

No MeSH data available.


Related in: MedlinePlus