SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard
© Ward et al; licensee BioMed Central Ltd. 2008
Received: 26 July 2008
Accepted: 14 October 2008
Published: 14 October 2008
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.
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.
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.
Hepcidin, a 25-residue peptide hormone, is a key regulator of iron homeostasis [1–3]. It is produced by hepatocytes and to a lesser extent by macrophages, bacteria-activated neutrophils and colorectal cancer cells [2–5]. The major stimuli for hepcidin expression include iron excess, inflammation and infection. Hepcidin exerts its biological effect at the level of cellular iron export by binding to and causing the internalisation and degradation of ferroportin . Thus in macrophages; the major cell type responsible for iron recycling, the iron becomes trapped resulting in an anaemia which in the context of inflammation and infection is characterised as the anaemia of chronic disease .
There has been intense research into how hepcidin is regulated and its role in pathologies including haematological disorders, liver disease and carcinogenesis [1, 5, 8]. The method most commonly employed for measuring hepcidin in serum and urine is surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI) [5, 9–12]. SELDI offers facile high-throughput sample preparation via on-chip retentate chromatography with hepcidin binding to NP20, CM10 and IMAC surfaces (normal-phase silica, cation exchange or immobilised metal ion chromatography respectively). It is assumed the height of the SELDI peak at m/z 2791 is related to hepcidin concentration. However, although Tomosugi et al report a linear relationship between SELDI peak height and hepcidin concentration under ideal conditions  and Bozzini et al demonstrate a correlation between SELDI peak height and a dot-blot immunoassay , this may not be a valid assumption when comparing samples with variable proteomic backgrounds or using different instruments. Doubts have been raised about the reproducibility of SELDI data [13–15]. Recently Swinkels and coworkers [16, 17] have used a truncated version of hepcidin (hepcidin-24) as an internal standard. Most recently, Ganz et al and Kobold et al have reported ELISA and LC-ESI-MS with a stable isotope labelled standard to quantitate hepcidin [18, 19]. We now report the development of a simple alternative method to assay hepcidin in human serum combining the use of stable isotope labelled hepcidin and SELDI-TOF-MS.
Hepcidin synthesis and folding
Human hepcidin was synthesised with or without 13C/15N phenylalanine at position 9 (AltaBioscience, University of Birmingham). This was dissolved at 0.1 mg/ml in 6 M urea, 30 mM MOPS (pH 7.0) and incubated overnight at room temperature with stirring. The folded hepcidin was purified by C18 RP-HPLC in 0.1% TFA/acetonitrile. Hepcidin concentrations were determined by BCA assay calibrated with bovine serum albumin (Pierce).
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 .
Sera were analysed on Cu2+-loaded IMAC30 ProteinChips using a PBS IIc time-of-flight mass spectrometer (BioRad). Sera were diluted 5-fold in 8 M urea, 1% CHAPS in binding buffer (0.5 M NaCl, 0.1 M sodium phosphate, pH 7.0) followed by a further 10-fold dilution in binding buffer and 100 μl applied to the chips. Following 30 minutes binding the chips were washed with binding buffer, rinsed with water, dried and 2 × 1 μl of 50% saturated sinapinic acid in 50% acetonitrile/0.5% TFA added. Spectra were collected over m/z 0–20,000 focussed at m/z 2800 using a laser power of 165. Following mass calibration, total ion current normalisation and baseline subtraction the hepcidin peaks were manually picked and intensities (peak heights) extracted using ProteinChip software.
THP-1 cells were cultured in RPMI 1640 media containing 10% FCS, 2 mM L-glutamine, 1% penicillin/streptomycin. To promote differentiation/activation cells were incubated with 16 nM phorbol-12-myristate-13-acetate for 48 hours. Cells were then treated for 24 hrs with/without 0.5 μM folded or reduced labelled hepcidin (reduced with 2 mM tris(2-carboxyethyl)phosphinehydrochloride). Cells were then fixed, blocked and incubated for 1 hr with an anti-ferroportin rabbit polyclonal (1:100, clone 3566, this antibody was raised against the oligopeptide CGKQLTSPKDTEPKPLEGTH corresponding to amino acids 247–264 of murine ferroportin as previously described ). Cells were then labelled with FITC goat anti-rabbit (Jackson Immunoresearch, 1:500), washed and visualised.
Spectra were acquired at 25°C on a Varian INOVA-800 spectrometer equipped with a cryoprobe as previously reported . Briefly, hepcidin (0.5 mg/ml) was prepared in 50 mM phosphate buffer (pH 4.0) containing 120 mM KCl in 90% H2O/10% D2O and 1H-1H through space correlations obtained using standard homonuclear 2D NOESY experiments with a 150-ms mixing time.
Statistics and analysis of reproducibility
Statistical significance was calculated using an unpaired two tailed Student's t test. Trend lines were fitted using standard least squares linear regression. Reproducibility was estimated by analysing 2 serum samples spiked with 200 ng hepcidin/ml in quadruplicate on ProteinChip arrays on 5 successive days. The intra-assay coefficients of variation (CV (mean/standard deviation)) were calculated from the 4 replicates of each sample on each ProteinChip array and the inter-assay CVs by comparing across days.
Hepcidin Measurement by SELDI
Hepcidin Assay Validation
To assess the effect of background proteome and instrument variability we used the peak height ratio method to determine hepcidin in urine with varying amounts of irrelevant proteins added and at various laser settings (Figure 7). Urine with ~200 ng/ml endogenous hepcidin was spiked with 200 ng/ml labelled hepcidin and increasing amounts of a serum sample not containing hepcidin. The absolute height of the endogenous hepcidin peak was strongly dependent on laser power but not affected by the addition of serum (Figure 7a). Total ion current (TIC) normalisation overcame the dependence on laser power but was influenced by addition of serum (Figure 7b). The concentration of endogenous hepcidin calculated from the peak height ratio was not influenced by sample composition or instrument performance as shown in Figure 7b.
Comparison with non-spiked SELDI data
Hepcidin Levels in Breast Cancer Patients
We had previously used IMAC chips to analyse serum from 140 patients with breast cancer and 53 non-cancer control subjects (mean ages 61.9 and 60.1 years respectively). On average, the hepcidin peak height was significantly higher in the cancer group than the control group (5.46 ± 2.93 versus 4.54 ± 1.86, mean ± SD, p = 0.0126). The current re-analysis of 24 of these serum samples (with endogenous hepcidin concentrations spanning the observed range) spiked with stable isotope labelled hepcidin indicates that this increase in peak height likely represents a genuine ~20% increase in systemic hepcidin concentration in this patient group. The mean peak height in the non-cancer controls corresponds to ~50 ng/ml hepcidin with a range from greater than 200 ng/ml to below the limit of detection (~10 ng/ml in this experiment, although this may be improved by spiking at a lower concentration and using a higher laser power).
Analysis of reproducibility
Since the discovery of hepcidin there has been great interest in this master regulator of iron metabolism . For the last seven years the scientific community has awaited a robust hepcidin assay. To date the two main approaches for measuring hepcidin in biological samples have been ELISA and SELDI. Two ELISA approaches exist, one measuring pro-hepcidin (which has proved controversial [11, 23–25]) and a recently described ELISA for hepcidin measurement in serum . This latter assay is based upon competition for antibody binding between endogenous hepcidin and added biotinylated hepcidin. Although further validation is required to ensure that this assay is solely specific for bioactive hepcidin 25, it likely represents a promising high throughput approach. In contrast SELDI has been widely used to measure urinary and serum hepcidin [5, 9–12]. The advantages of SELDI include the ability to measure different forms of hepcidin, e.g. hepcidin 20 and 25, and furthermore the assay can be conducted under denaturing conditions so that protein-protein interactions should not interfere. Unfortunately, due to the lack of good internal standards SELDI based hepcidin measurements are at best only semi-quantitative. This issue has now been partially resolved by Swinkels and co-workers utilising hepcidin 24 as an internal standard . The use of a stable isotope labelled hepcidin as an internal has so far been limited to LC-ESI-MS experiments . We have now combined the use of stable isotope labelled hepcidin and SELDI-TOF-MS to generate a technically simple high-throughput quantitative hepcidin assay.
The use of stable isotope labelled peptides to make mass spectrometry based proteomic experiments quantitative is well established and widely used as exemplified by the SILAC and ICAT methods [26, 27]. Stable isotope labelling introduces additional neutrons altering the mass of a peptide but does not alter the electronic structure and hence the binding and ionisation/detection during SELDI should be unaffected. In this study we apply this approach to the measurement of hepcidin. We synthesise a stable isotope labelled hepcidin that is 10 Da heavier than endogenous hepcidin. After checking that the synthetic hepcidin adopts the correct folded structure using FTICR-MS, NMR and a bio-assay we validate its use as an internal standard in SELDI experiments and demonstrate the merits of this approach.
The isotopic envelope of the labelled hepcidin does not overlap with the isotopic envelope of endogenous hepcidin and even when using the PBSIIc instrument (a sensitive but low resolution mass spectrometer) there is minimal overlap between the labelled and endogenous hepcidin peaks. A small amount of overlap with oxidised endogenous hepcidin does occur and it is therefore important to minimise sample (and standard) oxidation. Oxidation of hepcidin has previously been reported as an ex vivo artefact  and is usually minimal in serum but does occur in urine over time. Hepcidin oxidation can largely be prevented by rapid sample processing and avoiding long periods at room temperature. It should be noted that, regardless of internal standards, total hepcidin levels cannot be determined in oxidised samples from the sum of the non-oxidised and oxidised peak heights as the relative ionisation efficiencies are unknown. Additional precautions when using SELDI with or without spiking are to collect and average a large number of laser shots and to use appropriate laser power to ensure substantial, but not saturating peak intensities. The experiments presented here show that, when these conditions are met, the peak height ratio approach offers a quantitative assay for hepcidin.
We find a good correlation between SELDI peak heights and the peak height ratio method adding weight to previous reports inferring changes in hepcidin concentration from SELDI peak heights [5, 9–12]. In addition we now report a small but significant increase in systemic hepcidin in breast cancer patients. We find the average hepcidin concentration in healthy females to be ~50 ng/ml similar to the 65 ng/ml that Ganz et al determined by ELISA .
The use of the stable isotope labelled hepcidin alone does not increase the sensitivity or precision of SELDI measurements as demonstrated by intra- and inter-assay CVs similar to those using TIC normalised peak height alone. The major advance is that the labelled hepcidin approach should allow hepcidin levels to be measured in absolute concentration units independent of instrument/operator or background proteome variations. In the absence of labelled hepcidin spiking, conversion of peak heights into concentrations requires external calibration using a series of dilutions of hepcidin. This is not ideal as at high dilutions hepcidin either adheres to plasticware, precipitates or aggregates and mass spectrometry detection becomes variable (Ward et al, unpublished observations). This problem can be largely overcome by storing a concentrated labelled hepcidin solution in aliquots at -80°C, doing a single dilution in 8 M urea/1% CHAPS and spiking directly into samples. Addition of stable isotope labelled hepcidin to serum prior to sample work-up for mass spectrometry will enable multi-step hepcidin enrichment (followed by SELDI or MALDI) which will ultimately improve the sensitivity of the assay over the single step retentate chromatography of SELDI.
We conclude that by spiking stable isotope labelled hepcidin into clinical samples it is possible to turn high-throughput SELDI analyses into robust hepcidin assays. SELDI measurements without spiking reflect hepcidin concentrations but addition of stable isotope labelled hepcidin improves confidence in the data and provides absolute concentrations facilitating inter-study and inter-laboratory hepcidin comparisons.
coefficient of variation
liquid chromatography-electrospray ionisation-mass spectometry
matrix assisted laser desorption/ionisation
Nuclear Overhauser Enhancement Spectroscopy
reverse-phase high-performance liquid chromatography
surface enhanced laser desorption/ionisation time-of-flight mass spectrometry
total ion current
Fourier transform ion cyclotron resonance
nuclear magnetic resonance.
This work was funded by Cancer Research UK. NMR was carried out by Barry Levine, Wayne Mitchell, Christian Ludwig and Mark Jeeves (HWB.NMR, University of Birmingham).
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