Article pubs.acs.org/ac
Elemental Labeling and Isotope Dilution Analysis for the Quantification of the Peptide Hepcidin-25 in Serum Samples by HPLC-ICP-MS T. Konz, M. Montes-Bayón,* and A. Sanz-Medel* Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julián Clavería 8, 33006 Oviedo, Spain S Supporting Information *
ABSTRACT: Hepcidin-25 is a peptide-hormone that has been proposed as the key biomarker for the diagnosis and monitoring of iron disorders. Structurally, hepcidin-25 is a S-rich peptide (with 8 cysteines and 1 methionine) that contains a metal binding motif in the N-terminus. That domain binds preferably Cu(II) ion forming a stable complex. Such selective binding can be used as mean to determine hepcidin-25 in biological fluids by highly sensitive Cu measurement. Thus, we use liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICPMS) to perform hepcidin-25 determination via Cu detection. For this purpose, the incubation conditions were optimized to address the complex formation and stability by electrospray-MS (ESI-q-TOF). It was found that Cu:hepcidin-25 complex is stable under physiological conditions and shows an equimolar stoichiometry (1:1). The collisional induced dissociation (CID) experiments confirmed the specific binding of Cu to the N-terminal motif. For Cu quantification, two isotope dilution strategies have been developed. The first one, including postcolumn addition of a 65Cu spike and the second, by synthesizing the labeled 65Cu:hepcidin-25 complex as tracer (species-specific). Both methods have been optimized and critically compared in real samples. The determination of hepcidin-25 in different serum samples from healthy individuals based on Cu monitoring showed a mean value of 21.6 ng mL−1 which is in good agreement to previously published data.
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Soon after the discovery of hepcidin-25 some attempts to develop reliable assays for its determination in biological samples (urine and plasma) revealed this as a very challenging task.11 However, ongoing improvements of analysis techniques for hepcidin have been essential to assess concentration levels of this peptide in the different biological fluids and cell cultures.12 Initial methods based on immunochemical reactions were hampered by several limitations mainly due to the few antigenic epitopes within the hepcidin molecule and the lack of specificity to distinguish between the different isoforms. These problems still remain13 although some recent publications have revealed the suitability of ELISA methods using more specific “custom-made” antibodies.14 Trying to overcome the limitations of immunoassays, Mass Spectrometry (MS) based methodologies have been also applied in hepcidin-25 determination. Particularly, surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF), which combines the features of surface chromatography with MALDI ionization, were able to distinguish between the different hepcidin forms and provided semiquantitative data to allow comparisons between pathological stages.15 However, absolute quantification could not be attained until the proper internal standard
he understanding of iron metabolism has increased enormously during recent years because of the discovery of the iron regulatory hormone peptide hepcidin.1,2 This sulfurrich peptide inhibits ferroportin which transports iron out of the cell and is strongly expressed by the enterocytes and liver macrophages.3 As body iron increases, hepcidin is induced in the liver which then inhibits the efflux of iron through ferroportin binding.4 Thus, by modulating hepcidin biosynthesis, an organism controls intestinal iron absorption, iron release from the macrophages, and mobilization from stores to meet the body iron need.5 The bioactive form of hepcidin is a peptide of 25 aminoacids that derives from a precursor (prohepcidin) that undergoes two enzymatic cleavages.6 Other isoforms containing 20 and 22 amino acids respectively are detectable in human serum and urine, although the biological significance of these latter forms is still unknown. Additionally, recent studies have reported that the five N-terminal amino acids of hepcidin (only present in hepcidin-25 and absent in the 20 and 22 isoforms) are essential to mediate its iron regulatory role.7 Furthermore, three of those terminal residues (aspartic acid, threonine and histidine) are responsible for the metal binding capabilities of hepcidin-25 for divalent ions, particularly Cu.8,9 This affinity for Cu ions has been found to be particularly advantageous for the purification of hepcidin-25 using IMAC chips that are previously loaded with this metal ion.10 © 2012 American Chemical Society
Received: February 27, 2012 Accepted: September 4, 2012 Published: September 4, 2012 8133
dx.doi.org/10.1021/ac300578n | Anal. Chem. 2012, 84, 8133−8139
Analytical Chemistry
Article
increase of the mobile phase B from 0% (5 min) to 100% within 30 min. The ESI-q-Tof mass spectrometer (QStar XL, Applied Biosystems, Darmstadt, Germany) used for the Cu:hepcidin25 complex structure studies was equipped with a quadrupole time-of-flight mass spectrometer and a nano ion spray source which was maintained at 2.8 kV. Mass calibration was achieved daily by analyzing a reserpine solution of 1 pmol μL−1. Cu:hepcidin-25 samples were prepared in 10 mM ammonium acetate pH = 6.2 and injected by a syringe pump maintaining a flow rate of 2 μL min−1. With the mass spectrometer a typical scanning range of m/z 400 to m/z 2000 was chosen. Chemicals and Materials. All solutions were prepared by using 18 MΩ cm−1 deionized water obtained from a Milli-Q system (Millipore, Bedford, MA, USA). Synthetic human hepcidin (MW = 2789.4 g mol−1) was purchased from Pepta Nova (Sandhausen, Germany) and dissolved in distilled water to give a 0.1 mM stock solution which was separated in aliquots and stored at −20 °C and used within a few weeks. Each aliquot was defrosted only once to be used. After thawing, the quantification of the standard was conducted by measuring the S-content via ICP-MS and compared with the concentration given by the producer. The different aliquots proved to be stable under these conditions. Serum samples from healthy volunteers were kindly provided by the Hospital Central of Asturias, Laboratory for Biochemical Analysis (Oviedo, Spain). Samples were anonymous and collected in accordance with protocols approved by the relevant institutional review boards and with the Declaration of Helsinki. All blood samples were collected using BD Vacutainer containing gel for serum separation. The blood samples were allowed to coagulate and centrifuged at 5000 rpm. The clear serum was aliquoted in 2 mL tubes and frozen at −20 °C. For sample cleaning 30 kDa Amicon ULTRA centrifugal filters (MilliPore, Tullagreen, Ireland), SpinTrap affinity columns for the depletion of albumin (HSA) and IgG (GE Healthcare, Buckinghamshire, U.K.) and protein precipitation using acetonitrile (99.9%; VWR International Eurolab, Barcelona, Spain) were compared to each other. Serum samples and hepcidin standard were incubated with CuCl2 anhydrous (Merck, Darmstadt, Germany) and CuSO 4 pentahydrate (Merck) to form the Cu:hepcidin-25 complex. Buffer solutions used for anion exchange chromatography were prepared from tris base (pro analysis, Merck) and ammonium acetate (≥99%, Fluka, Sigma-Aldrich, St. Louis, MO, USA). Typical compositions were 25 mM Tris base pH 9.5 for mobile phase A and 25 mM Tris base and 0.5 M ammonium acetate pH = 9.5 for mobile phase B. A solution of isotopically enriched 65 Cu (abundances: 65Cu 99.62%, 63Cu 0.38%), used for the quantification of the complex (postcolumn IDA) and the synthesis of the isotopically labeled 65Cu:hepcidin-25 complex (species specific IDA), as well as isotopically enriched 34S (abundances: 34S 99.91%, 32S 0.09%) were purchased from Euriso-top (Saint-Aubin Cedex, France). An aqueous copper standard solution in 2% nitric acid after appropriate dilution (1000 mg L−1 Cu; Merck) and a sulfur standard solution (1000 mg L−1 S) from the same purchaser were used for mass-bias correction of the Thermo Element 2. All gases used for ICP-MS and ESI applications (argon and nitrogen, each 99.999%) were purchased from Air Liquide (Barcelona, Spain). Procedures. All sample preparation steps were processed at room temperature. The freshly collected serum samples were
(hepcidin-24 lacking the terminal asparagine residue) was recently synthesized.16 Similarly, liquid chromatography with Electrospray-MS (LC-ESI-MS/MS) has proved to be successful for hepcidin-25 determination using isotopically labeled internal standards of hepcidin-25 (with 13C and 15N).17,18 In this case, although such assays are accurate and precise, their complexity, expense, and high level of operator expertise limit their implementation into most routine clinical laboratories. ICP-MS based strategies, based on the high S content of the molecule (8 cysteines and 1 methionine),19 have also been recently reported applied for hepcidin-25 quantification. Unfortunately, the achievable detection limits obtained by S monitoring with ICP-MS (even with the most sensitive instrumentation) and the high abundance of S-containing species in the urine did not allowed any significant analytical improvement over previously published methods. However, a current trend for improving sensitivity of the detection, aiming at quantitative protein/peptide determinations by ICP-MS, is the application of labeling methodologies.20 Labeling procedures have been introduced into proteomics workflows with the intention to visualize the analyte of interest from the biological matrix or to improve the detection of the biomolecule itself, for example, by introduction of multiple lanthanide ions per mol of labeling reagent.21 Although the great potential of protein labeling has been generally recognized and accepted, elemental mass spectrometric detection of these derivatives is still quite scarce. Two different strategies for labeling of proteins/peptides have been yet developed for its use in elemental mass spectrometric detection: (i) direct biomolecule labeling and (ii) antibody labeling with subsequent detection of antigen (protein/peptide)−antibody interaction. Thus, the simplest approach, direct biomolecule labeling has been explored here for hepcidin-25 determination in serum samples. Because of the great affinity of hepcidin-25 for Cu(II) ions, we have tried to establish a Cu-labeling strategy to permit, on the one hand, to lower hepcidin-25 detection limits and also to develop indirect hepcidin-25 determination via Cu quantification by ICP-MS. For this purpose, two different quantitative Cu speciation methods, based on isotope dilution analysis (IDA) in combination with HPLC-ICP-MS have been investigated. They have been critically compared and applied to the quantification of hepcidin-25 in human serum.
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METHODS AND INSTRUMENTATION Instrumentation. All ICP-MS experiments during this study were performed using a Thermo Element 2 (Thermo Fisher Scientific, Bremen, Germany) mass spectrometer, equipped with a double focusing sector field mass analyzer applying medium resolution (m/Δm = 4000). The optimized parameters of the Element 2 instrument are summarized in the Supporting Information. For hyphenation of the ICP-MS to the HPLC system the instrument was fitted with an interface based on a concentric nebulizer and a Scott double-pass spray chamber. For chromatographic analysis, a LC-20AD dual-piston HPLC pump (Shimadzu Corporation, Kyoto, Japan) was used. Sample injection was conducted with a dual mode injection valve from Rheodyne, model 9125 (Cotati, California, USA), fitted with a 50 μL PEEK injection loop (Upchurch Scientific, Oak Harbor, Washington, USA). Chromatographic separations were achieved by a Mono Q 5/50 GL anion exchange column (10 μm particle size, 50 × 5 mm) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) applying gradient elution at a flow rate of 1 mL min−1. The typical gradient applied consists of a linear 8134
dx.doi.org/10.1021/ac300578n | Anal. Chem. 2012, 84, 8133−8139
Analytical Chemistry
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Figure 1. Complex formation between hepcidin-25 and CuCl2 or CuSO4 showing the mass spectrum of the species formed at different molar ratios Cu:hepcidin-25 (0:1 spectrum A, 1:1 spectrum B, 4:1 spectrum C and 10:1).
vortexed and separated into aliquots for storage at −20 °C. An incubation time of 5 min was chosen for all complex formation reactions. This time proved to be sufficient for total complex formation using a molar excess Cu:hepcidin-25 (4:1). Serum sample preparation. To eliminate or reduce the concentration of high abundant serum proteins like HSA and IgG, different methodologies were tested. The optimal final methodology made use of 30 kDa ULTRA centrifugal filter devices: typically a volume of 350 μL serum, diluted with the 4fold volume of Milli-Q water was centrifuged at 7500 g for 20 min. For optimal recovery, the samples were washed with MilliQ water before the centrifugal step was repeated. Subsequently, the ultrafiltrate was preconcentrated by a gentle N2 stream to approximately the same volume (weighted on each case). Then, the corresponding volume of 10 mM ammonium acetate solution (pH 7) was added to obtain a constant final volume before Cu-labeling. For this purpose, a 5-fold molar excess of copper in 10 mM ammonium acetate, pH = 8.2 was added. For
incubation, a physiological serum hepcidin concentration of 20 ng mL−1 (∼7.2 nM) was assumed, as recently described in the literature.22 The final preconcentration factor is approximately 5-fold (as average since it is calculated individually for each sample). Synthesis of 65Cu:Hepcidin-25. The synthesis of the 65 Cu:hepcidin-25 complex was carried out by adding a 5-fold molar excess of a CuSO4 solution (10 mM ammonium acetate, pH = 8.2) to the 0.1 mM hepcidin stock. In the case of the 65 Cu:hepcidin-25 complex, a solution of isotopically enriched copper was diluted with 10 mM ammonium acetate to give a final concentration of 80 μM of the complex and adjusted to pH = 8.2. For the possible isotope exchange experiments, this standard was diluted to 10 μM (considering that an excess of free 65Cu accounting for 49 μM was still present in the solution) and a standard of natural CuSO4 was added to achieve a final concentration of 44.5 μM. 8135
dx.doi.org/10.1021/ac300578n | Anal. Chem. 2012, 84, 8133−8139
Analytical Chemistry
Article
Isotope Dilution Analysis. For postcolumn IDA experiments, the isotopically enriched copper was dissolved in 2% nitric acid and injected by a peristaltic pump, maintaining a constant flow rate of 0.3 mL min−1. The quantification of hepcidin by species specific IDA was conducted by mixing the 65 Cu:hepcidin-25 complex with the previously depleted and copper-incubated serum samples.
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RESULTS AND DISCUSSION
Characterization of the Cu:Hepcidin-25 Complex. For synthesis of the complex Cu:hepcidin-25, initial conditions were taken from the literature.9 However, in this work we evaluated the use of two Cu salts (CuSO4 and CuCl2) at increasing molar ratios (Cu:hepcidin-25), such as (1:1), (4:1), and (10:1). The simultaneous monitoring of the formed complex and the remaining free hepcidin-25 excess was done by ESI-q-Tof. Additionally, different sample solubilization conditions were assayed in order to address the stability of the complex at different pH values. Figure 1 shows the comparison of the obtained spectra using both Cu salts at increasing metal molar ratios. All the spectra correspond to the +3 ions (m/z 930.80 and 951.31 Da without and with Cu, respectively) corresponding to the most intense signals observed for the peptide. As can be seen in the plot, the complex is formed even at equimolar concentrations of Cu(II) and hepcidin-25 (1:1). In this case, the MS spectrum (Figure 1B) still shows the presence of unreacted peptide (m/z 930.3569 Da), although an important signal corresponding to the labeled peptide (m/z 950.2738) can be clearly seen. Additionally, the plot indicates that both Cu salts provide quantitative complex formation when using a Cu excess (4:1) in respect to the hepcidin-25 concentration. However, the spectrum using CuSO4 (see Figure 1C) did not report the presence of any trace of the unreacted hepcidin-25 while this species was still detectable (
