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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

Assessment of reproducibility. Two serum samples (A: grey bars, B: black bars) were assayed in quadruplicate each day for 5 days. The bars represent the mean concentration calculated each day for each sample by the peak height ratio approach. Error bars represent +/- 1 standard deviation.
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Figure 9: Assessment of reproducibility. Two serum samples (A: grey bars, B: black bars) were assayed in quadruplicate each day for 5 days. The bars represent the mean concentration calculated each day for each sample by the peak height ratio approach. Error bars represent +/- 1 standard deviation.

Mentions: This was estimated by analysing 2 serum samples (A: ~50 ng endogenous hepcidin/ml, B: ~120 ng endogenous hepcidin/ml), spiked with 200 ng labelled hepcidin/ml in quadruplicate over 5 successive days (Figure 9). The mean intra-assay CVs of the endogenous hepcidin concentrations estimated by the peak height ratio approach were 9% and 8% for samples A and B respectively. The mean inter-assay CVs across the 5 days were 15% and 7%. The CVs calculated using peak heights alone were, for sample A, 17% and 16% (intra and inter) and for sample B 28% and 26%. After TIC normalisation the CVs were 11% and 7% for sample A and 17% and 15% for sample B.


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)

Assessment of reproducibility. Two serum samples (A: grey bars, B: black bars) were assayed in quadruplicate each day for 5 days. The bars represent the mean concentration calculated each day for each sample by the peak height ratio approach. Error bars represent +/- 1 standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Assessment of reproducibility. Two serum samples (A: grey bars, B: black bars) were assayed in quadruplicate each day for 5 days. The bars represent the mean concentration calculated each day for each sample by the peak height ratio approach. Error bars represent +/- 1 standard deviation.
Mentions: This was estimated by analysing 2 serum samples (A: ~50 ng endogenous hepcidin/ml, B: ~120 ng endogenous hepcidin/ml), spiked with 200 ng labelled hepcidin/ml in quadruplicate over 5 successive days (Figure 9). The mean intra-assay CVs of the endogenous hepcidin concentrations estimated by the peak height ratio approach were 9% and 8% for samples A and B respectively. The mean inter-assay CVs across the 5 days were 15% and 7%. The CVs calculated using peak heights alone were, for sample A, 17% and 16% (intra and inter) and for sample B 28% and 26%. After TIC normalisation the CVs were 11% and 7% for sample A and 17% and 15% for sample B.

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