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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

FTICR mass spectra of folded and reduced synthetic hepcidin-25. We show the 3+ion of unlabelled folded hepcidin 25 (a), reduced unlabelled hepcidin (b), folded labelled hepcidin (c) and reduced labelled hepcidin (d). Spectra were acquired by direct infusion in 0.1% formic acid/50% acetonitrile into a ThermoFinnigan LTQ-FT. Reduced samples were treated with DTT, zip-tipped and kept at low pH prior to analysis.
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Figure 1: FTICR mass spectra of folded and reduced synthetic hepcidin-25. We show the 3+ion of unlabelled folded hepcidin 25 (a), reduced unlabelled hepcidin (b), folded labelled hepcidin (c) and reduced labelled hepcidin (d). Spectra were acquired by direct infusion in 0.1% formic acid/50% acetonitrile into a ThermoFinnigan LTQ-FT. Reduced samples were treated with DTT, zip-tipped and kept at low pH prior to analysis.

Mentions: During folding 4 disulphide bridges form with the loss of 8 hydrogen atoms resulting in an 8 Da decrease in molecular weight. We used ESI-FTICR mass spectrometry to detect this mass change. The 3+ ions of the folded and reduced forms of unlabelled and labelled hepcidin are shown in Figure 1. The measured masses of the folded and reduced unlabelled hepcidin, 2787.033 and 2795.106 Da, agree with predicted values (2787.025 and 2795.088 respectively). The measured masses of the folded and reduced labelled hepcidin are 2797.053 and 2805.134 Da (predicted values 2797.053 and 2805.116).


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)

FTICR mass spectra of folded and reduced synthetic hepcidin-25. We show the 3+ion of unlabelled folded hepcidin 25 (a), reduced unlabelled hepcidin (b), folded labelled hepcidin (c) and reduced labelled hepcidin (d). Spectra were acquired by direct infusion in 0.1% formic acid/50% acetonitrile into a ThermoFinnigan LTQ-FT. Reduced samples were treated with DTT, zip-tipped and kept at low pH prior to analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: FTICR mass spectra of folded and reduced synthetic hepcidin-25. We show the 3+ion of unlabelled folded hepcidin 25 (a), reduced unlabelled hepcidin (b), folded labelled hepcidin (c) and reduced labelled hepcidin (d). Spectra were acquired by direct infusion in 0.1% formic acid/50% acetonitrile into a ThermoFinnigan LTQ-FT. Reduced samples were treated with DTT, zip-tipped and kept at low pH prior to analysis.
Mentions: During folding 4 disulphide bridges form with the loss of 8 hydrogen atoms resulting in an 8 Da decrease in molecular weight. We used ESI-FTICR mass spectrometry to detect this mass change. The 3+ ions of the folded and reduced forms of unlabelled and labelled hepcidin are shown in Figure 1. The measured masses of the folded and reduced unlabelled hepcidin, 2787.033 and 2795.106 Da, agree with predicted values (2787.025 and 2795.088 respectively). The measured masses of the folded and reduced labelled hepcidin are 2797.053 and 2805.134 Da (predicted values 2797.053 and 2805.116).

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