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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

SELDI analysis of hepcidin mixes. a) spectra of serum with 200 ng/ml labelled hepcidin (L) added in addition to 67–400 ng/ml unlabelled hepcidin (U) as indicated in the grey boxes. b) spectra of labelled (dotted line) and unlabelled (solid line) hepcidin at 200 ng/ml in serum and the sum of the 2 spectra (bold line). c) shows the concentration of unlabelled hepcidin calculated from the peak height ratio plotted against the actual concentration in the experiment of Figure 6a and two independent replicates in different sera. The trend line represents a least-squares linear regression (r2 = 0.990).
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Figure 6: SELDI analysis of hepcidin mixes. a) spectra of serum with 200 ng/ml labelled hepcidin (L) added in addition to 67–400 ng/ml unlabelled hepcidin (U) as indicated in the grey boxes. b) spectra of labelled (dotted line) and unlabelled (solid line) hepcidin at 200 ng/ml in serum and the sum of the 2 spectra (bold line). c) shows the concentration of unlabelled hepcidin calculated from the peak height ratio plotted against the actual concentration in the experiment of Figure 6a and two independent replicates in different sera. The trend line represents a least-squares linear regression (r2 = 0.990).

Mentions: Labelled hepcidin was titrated into serum (0–400 ng hepcidin/ml) without influencing the intensity of the endogenous hepcidin SELDI peak (Figure 5). In experiments adding 0–50 ng hepcidin/ml to serum, the lowest concentration of labelled hepcidin generating a reliable peak was ~10 ng/ml indicating that this would also be the limit of detection of endogenous hepcidin in serum by this method (data not shown). A working concentration of 200 ng labelled hepcidin/ml was adopted as this produces a robust peak similar to the highest endogenous peak observed. We then added 0–400 ng/ml unlabelled and 200 ng/ml labelled hepcidin to a serum sample devoid of endogenous hepcidin to test whether the peak height ratio is representative of the concentration (Figure 6a). Although the edges of the peaks overlap, when the spectra of 200 ng/ml labelled and unlabelled hepcidin are superimposed neither peak contributes significantly to the peak height of the other (Figure 6b). Plotting unlabelled hepcidin concentrations calculated from peak height ratios against actual concentration (Figure 6c) reveals a strong correlation (the non-zero intercept is largely due to the endogenous hepcidin in the sera used and a slight downwards curvature is seen at very high hepcidin concentrations). This indicates that the peak height ratio produces a reliable estimate of hepcidin concentration.


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)

SELDI analysis of hepcidin mixes. a) spectra of serum with 200 ng/ml labelled hepcidin (L) added in addition to 67–400 ng/ml unlabelled hepcidin (U) as indicated in the grey boxes. b) spectra of labelled (dotted line) and unlabelled (solid line) hepcidin at 200 ng/ml in serum and the sum of the 2 spectra (bold line). c) shows the concentration of unlabelled hepcidin calculated from the peak height ratio plotted against the actual concentration in the experiment of Figure 6a and two independent replicates in different sera. The trend line represents a least-squares linear regression (r2 = 0.990).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: SELDI analysis of hepcidin mixes. a) spectra of serum with 200 ng/ml labelled hepcidin (L) added in addition to 67–400 ng/ml unlabelled hepcidin (U) as indicated in the grey boxes. b) spectra of labelled (dotted line) and unlabelled (solid line) hepcidin at 200 ng/ml in serum and the sum of the 2 spectra (bold line). c) shows the concentration of unlabelled hepcidin calculated from the peak height ratio plotted against the actual concentration in the experiment of Figure 6a and two independent replicates in different sera. The trend line represents a least-squares linear regression (r2 = 0.990).
Mentions: Labelled hepcidin was titrated into serum (0–400 ng hepcidin/ml) without influencing the intensity of the endogenous hepcidin SELDI peak (Figure 5). In experiments adding 0–50 ng hepcidin/ml to serum, the lowest concentration of labelled hepcidin generating a reliable peak was ~10 ng/ml indicating that this would also be the limit of detection of endogenous hepcidin in serum by this method (data not shown). A working concentration of 200 ng labelled hepcidin/ml was adopted as this produces a robust peak similar to the highest endogenous peak observed. We then added 0–400 ng/ml unlabelled and 200 ng/ml labelled hepcidin to a serum sample devoid of endogenous hepcidin to test whether the peak height ratio is representative of the concentration (Figure 6a). Although the edges of the peaks overlap, when the spectra of 200 ng/ml labelled and unlabelled hepcidin are superimposed neither peak contributes significantly to the peak height of the other (Figure 6b). Plotting unlabelled hepcidin concentrations calculated from peak height ratios against actual concentration (Figure 6c) reveals a strong correlation (the non-zero intercept is largely due to the endogenous hepcidin in the sera used and a slight downwards curvature is seen at very high hepcidin concentrations). This indicates that the peak height ratio produces a reliable estimate of hepcidin concentration.

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