Abstract
As advanced mass spectrometry- (MS-) based hepcidin analysis offers to overcome the limitations in analytical methods using antihepcidin, further improvement of MS detection sensitivity for the peptide may enhance the diagnostic value of the hepcidin for various iron-related disorders. Here, improved MS detection sensitivity of hepcidin has been achieved by reducing the disulfide bonds in hepcidin, by which proton accessibility increased, compared to the intact hepcidin peptide. Comparing the ionization efficiencies of reduced and nonreduced forms of hepcidin, the reduced form of hepcidin showed an increase in ionization efficiency more than two times compared to the nonreduced form of hepcidin. We also demonstrated improved detection sensitivity of the peptide in SRM assay. We observed a significant improvement of detection sensitivity at the triple-quadrupole MS platform, that the ionization efficiency increased at least twice more, and that the limit of detection (LOD) increased more than 10 times in the concentration ranges of 1 fmol to 10 fmol of hepcidin. In this study, we demonstrated the usefulness of the hepcidin modification for overall enhancement of the ionization efficiencies of the hepcidin peptide in the MS-based quantitative measurement assay.
Highlights
Hepcidin, a folded 25-residue peptide hormone stabilized by four disulfide bonds, plays an important role in the regulation of iron metabolism in mammals, such as digestive iron absorption and macrophage iron recycling [1–3]
Anemia of inflammation and chronic kidney diseases are developed in various clinical settings, the most direct causes of the diseases are associated with the destruction of homeostasis of iron metabolism [4–6]
To improve MS detection sensitivity of hepcidin, the disulfide bonds of intact hepcidin peptide was reduced, and the resulting free thiols were subsequently modified by alkylation to linearize the peptide as described in Materials and Methods
Summary
A folded 25-residue peptide hormone stabilized by four disulfide bonds, plays an important role in the regulation of iron metabolism in mammals, such as digestive iron absorption and macrophage iron recycling [1–3]. Anemia of inflammation and chronic kidney diseases are developed in various clinical settings, the most direct causes of the diseases are associated with the destruction of homeostasis of iron metabolism [4–6]. Hepcidin has been considered as an important clinical utility for the diagnosis and management of a wide range of iron-related disorders [7–10]. Quantitative immunoassays based on the use of antihepcidin antibodies have been developed for quantitative assessment of hepcidin concentrations in the patient’s serum and urine [11–15]. Recent advances in mass spectrometry(MS-) based quantitative assays (e.g., SRM assay) for hepcidin complement the immunoassays by overcoming hepcidin (25-amino acids) detection specificity, reproducibility, and accuracy [18–22]. Absolute concentrations of hepcidin can be measured with synthetic hepcidin labeled with heavy stable isotopes in SRM assay [23, 24]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
