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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

NMR analysis of folded synthetic hepcidin. Section of the 1H-1H NOESY spectrum showing crosspeaks associated with the backbone -NH and aromatic groups of hepcidin. Through space correlations of the -NH of several of the Cys residues are identified. Some of the 1H proximities prescriptive of the published fold of the human hepcidin molecule are circled.
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Figure 2: NMR analysis of folded synthetic hepcidin. Section of the 1H-1H NOESY spectrum showing crosspeaks associated with the backbone -NH and aromatic groups of hepcidin. Through space correlations of the -NH of several of the Cys residues are identified. Some of the 1H proximities prescriptive of the published fold of the human hepcidin molecule are circled.

Mentions: We have used 2D proton NMR to characterize the folded structure of our synthetic hepcidin. Spatial proximities identified in the NOESY spectrum of hepcidin were used to deduce the correspondence between our folded hepcidin and the published structure (Figure 2 and reference [22]). Homology of folding can be recognised by spectral shift pattern matching in combination with observed long-range NOE and it is this dual approach, focussing on specific residues involved in critical contacts within the hepcidin molecule fold that we used to confirm the architecture of our synthetic hepcidin. Consistent with the homology of S-S linkages, the pattern and dispersion of chemical shifts of backbone -NH (and cysteine -CaH, 4.9–5.5 ppm – data not shown) obtained for the synthetic hepcidin matches the shift and intramolecular 1H-1H distance fingerprint of the published human hepcidin structure [22].


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)

NMR analysis of folded synthetic hepcidin. Section of the 1H-1H NOESY spectrum showing crosspeaks associated with the backbone -NH and aromatic groups of hepcidin. Through space correlations of the -NH of several of the Cys residues are identified. Some of the 1H proximities prescriptive of the published fold of the human hepcidin molecule are circled.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: NMR analysis of folded synthetic hepcidin. Section of the 1H-1H NOESY spectrum showing crosspeaks associated with the backbone -NH and aromatic groups of hepcidin. Through space correlations of the -NH of several of the Cys residues are identified. Some of the 1H proximities prescriptive of the published fold of the human hepcidin molecule are circled.
Mentions: We have used 2D proton NMR to characterize the folded structure of our synthetic hepcidin. Spatial proximities identified in the NOESY spectrum of hepcidin were used to deduce the correspondence between our folded hepcidin and the published structure (Figure 2 and reference [22]). Homology of folding can be recognised by spectral shift pattern matching in combination with observed long-range NOE and it is this dual approach, focussing on specific residues involved in critical contacts within the hepcidin molecule fold that we used to confirm the architecture of our synthetic hepcidin. Consistent with the homology of S-S linkages, the pattern and dispersion of chemical shifts of backbone -NH (and cysteine -CaH, 4.9–5.5 ppm – data not shown) obtained for the synthetic hepcidin matches the shift and intramolecular 1H-1H distance fingerprint of the published human hepcidin structure [22].

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