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Title: A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification

Abstract

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 themore » 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.« less

Authors:
 [1];  [2];  [1];  [1];  [1];  [1]; ORCiD logo [1];  [2];  [2];  [1];  [1]
  1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  2. Univ. of Connecticut, Farmington, CT (United States)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1530848
Report Number(s):
PNNL-SA-138022
Journal ID: ISSN 2045-2322
Grant/Contract Number:  
AC05-76RL01830
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Moghieb, Ahmed M., Tesfay, Lia, Nie, Song, Gritsenko, Marina A., Fillmore, Thomas L., Jacobs, Jon M., Smith, Richard D., Torti, Frank M., Torti, Suzy, Shi, Tujin, and Ansong, Charles K. A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification. United States: N. p., 2019. Web. doi:10.1038/s41598-019-43756-9.
Moghieb, Ahmed M., Tesfay, Lia, Nie, Song, Gritsenko, Marina A., Fillmore, Thomas L., Jacobs, Jon M., Smith, Richard D., Torti, Frank M., Torti, Suzy, Shi, Tujin, & Ansong, Charles K. A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification. United States. doi:10.1038/s41598-019-43756-9.
Moghieb, Ahmed M., Tesfay, Lia, Nie, Song, Gritsenko, Marina A., Fillmore, Thomas L., Jacobs, Jon M., Smith, Richard D., Torti, Frank M., Torti, Suzy, Shi, Tujin, and Ansong, Charles K. Mon . "A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification". United States. doi:10.1038/s41598-019-43756-9. https://www.osti.gov/servlets/purl/1530848.
@article{osti_1530848,
title = {A Targeted Mass Spectrometric Assay for Reliable Sensitive Hepcidin Quantification},
author = {Moghieb, Ahmed M. and Tesfay, Lia and Nie, Song and Gritsenko, Marina A. and Fillmore, Thomas L. and Jacobs, Jon M. and Smith, Richard D. and Torti, Frank M. and Torti, Suzy and Shi, Tujin and Ansong, Charles K.},
abstractNote = {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.},
doi = {10.1038/s41598-019-43756-9},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United States},
year = {2019},
month = {5}
}

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