Hepcidin, a cysteine-rich peptide hormone, secreted mainly by the liver, plays a central role in iron metabolism regulation. Emerging evidence suggests that disordered iron metabolism is a risk factor for various types of diseases including cancers. However, it remains challenging to apply current mass spectrometry (MS)-based hepcidin assays for precise quantification due to the low fragmentation efficiency of intact hepcidin as well as synthesis difficulties for the intact hepcidin standard. To address these issues we recently developed a reliable sensitive targeted MS assay for hepcidin quantification that uses fully alkylated rather than intact hepcidin as the internal standard. Limits of detection and quantification were determined to be <0.5 ng/mL and 1 ng/mL, respectively, which are better than those from currently available MS-based hepcidin assays. Application of the alkylated hepcidin assay to 70 clinical plasma samples (42 normal and 28 ovarian cancer patient samples) enabled reliable detection of endogenous hepcidin from the plasma samples, as well as conditioned culture media. The hepcidin concentrations ranged from 0.0 to 95.6 ng/mL across normal and cancer plasma specimens. Interestingly, cancer patients were found to have significantly higher hepcidin concentrations compared to normal patients (mean: 20.6 ng/ml for cancer; 5.94 ng/ml for normal) with the p value of <0.001. Our results demonstrate that the newly developed hepcidin assay has better sensitivity and quantification accuracy than current MS-based hepcidin assays without the challenges in synthesis of intact hepcidin standard and accurately determining its absolute amount.

All samples were subjected to preparation work flow for precise detection and quantification of hepcidin-25 as shown in Supplementary Figure 1. First, samples were thawed in ice and then vortex mixed. In a 1.5 mL LoBind tube, 100 µl plasma proteins were mixed with 10 µl heavy internal standard (7.1 ng/ml), and then denatured with 8 M urea and reduced with 10 mM TCEP for 2h at 37° C. Protein cysteine residues were alkylated with 40 mM iodoacetamide for 2 h at room temperature in the dark. 4% of trichloroacetic acid (TCA) solution (1:1 ratio) was added. Samples were then vortexed for a few seconds and centrifuged at 18,000 x g for 5 minutes to obtain a clear supernatant. The supernatant was transferred into a new LoBind tube, then diluted with 0.1% formic acid in water for SPE cleaning using a 1 mL SPE C18 column (Phenomenex, Torrence, CA). After SPE cleaning, the samples were completely dried in a vacuum concentrator, resuspended with 40 µl 0.1% formic acid in water and then centrifuged at 18,000 x g for 3h at 4oC. The supernatant was transferred into LC vials (Waters) for MS analysis. For media, in 15 ml tube, 2 ml media was mixed with 10 µl internal hepcidin-25 standard solution (71.03 ng/ml), using the same protocol as described above for plasma samples with only one extra step of BSA blocking of SPE C18 columns prior to media sample cleaning. Liquid Chromatography (LC) Separation All samples were analyzed using a nanoACQUITY UPLC system (Waters Corporation, Milford, MA) coupled online to a TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA). Solvents used were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in 90% acetonitrile (mobile phase B). Peptide separations were performed at a mobile phase flow rate of 400 nL/min using an ACQUITY UPLC BEH 1.7 μm C18 column (100 μm i.d. × 10 cm), which was connected to a chemically etched 20 μm i.d. fused-silica emitter via a Valco stainless steel union. 0.5 µl sample was injected for LC-SRM using a binary gradient of 5-20 % B in 35 min, 20-25 % B in 10 min, 25-38 % B in 8 min, 38-95 % B in 1 min, and 95 % for 6 min for a total of 75 min. SRM Assay Configuration Mass spectrometric detection was performed using TSQ Vantage triple quadrupole mass spectrometer (Thermo Scientific). Scan width of 0.002 m/z and a dwell time of 75 ms were set for all SRM transitions. The synthesized fully alkylated hepcidin-25 peptides were further evaluated for peptide response and fragmentation pattern. Precursor ions followed were 651.3 m/z (z = 5) for the endogenous hepcidin-25 and 652.8 m/z (z = 5) for the heavy standard. The relative intensity ratios among the four final selected transitions for the SRM assay were predefined by internal standard heavy peptides. Matrix interference for a given transition that fell into the mass width of Q1 and Q3 from co-eluting peptides was determined by deviation from the expected relative intensity ratios between the transitions. While four transitions were monitored, the best two transitions with no matrix interference were used to generate calibration curve and hepcidin-25 quantification in media and clinical plasma samples. Data Analysis SRM data acquired on the TSQ Vantage were analyzed using Xcalibur 2.0.7 (Thermo Scientific). Peak detection and integration were determined based on two criteria: 1) same retention time; 2) approximately same relative SRM peak intensity ratios across multiple transitions between light peptide and heavy peptide standard. All data were manually inspected to ensure correct peak detection and accurate integration. Signal to noise ratio (S/N) was calculated by the peak apex intensity over the highest background noise in a retention time region of ± 15 s for the target peptides. The background noise levels were conservatively estimated by visually inspecting chromatographic peak regions. The LOD and LOQ were defined as the lowest concentration point at which the S/N of surrogate peptide was at least 3 and 10, respectively. For conservatively determining the LOQ values, in addition to the requirements of the S/N to equal or be above 10, two other criteria were applied: the coefficient of variation (CV) at the concentration point be less than 20%; surrogate peptide response over the protein concentration be within the linear dynamic range. The L/H SRM peak area ratio was used to generate the calibration curve and assess reproducibility. Microsoft Excel 2010 was used for statistical analysis and calibration curve plotting. The RAW data from TSQ Vantage were loaded into Skyline software 18 to create high resolution figures of extracted ion chromatograms (XIC) of multiple transitions monitored for hepcidin-25.

Generation of Calibration Curve Control plasma with negligible endogenous hepcidin-25 was used as the matrix for generating calibration curve. Lyophilized high-purity light alkylated hepcidin-25 peptide was reconstituted with H2O/ACN (70:30, v/v), then hepcidin-25 stock solutions (5, 50 and 500 fmol/µl) were diluted with the same solvent. Light alkylated hepcidin-25 was spiked into the control plasma with the final concentrations of 0, 0.5, 1, 2.5, 5, 10, 25, 50, 100 and 250 ng/mL. Heavy alkylated synthetic peptide was also spiked into each sample at a final concentration of 71.03 ng/ml. The spiked-in samples were processed according to the sample preparation described above. The generated calibration curve was used for calculating the endogenous hepcidin-25 concentrations in clinical samples, alkylated heavy synthetic peptide, and for determining the LOD and LOQ values. Human Plasma Specimens The use of human blood plasma samples was approved by the Institutional Review Boards of the University of Connecticut and Pacific Northwest National Laboratory in accordance with federal regulations. Clinical plasma samples were collected from the biorepository of UCHC (IRB IE-08-310-1). The control and the cancer plasma specimens were obtained from healthy people with no cancer diagnosis and female diagnosed with ovarian cancer, respectively. Media Samples HepG2 cells were obtained from American Type Culture Collection (ATCC) and cultured in Eagle's Minimum Essential Medium (EMEM medium) from ATCC containing 10% Fetal Bovine Serum (FBS) purchased from Gemini Bioproducts. For the induction of hepcidin, the cells were allowed to secrete for 48 hours in Fetal Bovine Serum-free EMEM medium in presence or absence of 10 ng/ml Bone Morphogemic Protein 6 (BMP6) obtained from R&D Systems.