Abstract
Assays for the detection of the iron regulatory hormone hepcidin in plasma or urine have not yet been widely available, whereas quantitative comparisons between hepcidin levels in these different matrices were thus far even impossible due to technical restrictions. To circumvent these limitations, we here describe several advances in time-of flight mass spectrometry (TOF MS), the most important of which concerned spiking of a synthetic hepcidin analogue as internal standard into serum and urine samples. This serves both as a control for experimental variation, such as recovery and matrix-dependent ionization and ion suppression, and at the same time allows value assignment to the measured hepcidin peak intensities. The assay improvements were clinically evaluated using samples from various patients groups and its relevance was further underscored by the significant correlation of serum hepcidin levels with serum iron indices in healthy individuals. Most importantly, this approach allowed kinetic studies as illustrated by the paired analyses of serum and urine samples, showing that more than 97% of the freely filtered serum hepcidin can be reabsorbed in the kidney. Thus, the here reported advances in TOF MS-based hepcidin measurements represent critical steps in the accurate quantification of hepcidin in various body fluids and pave the way for clinical studies on the kinetic behavior of hepcidin in both healthy and diseased states.
Highlights
Protein profiling by time-of flight mass spectrometry (TOF MS) is based on polypeptide enrichment by selective binding to achemical surface prior to TOF MS of retained proteins and peptides [1]
To overcome several of the technical limitations that previously interfered with the robustness of hepcidin measurements by TOF MS, we have systematically reconsidered all analytical steps and made improvements wherever possible
In this study we have described an updated TOF MS method for both serum and urine hepcidin with considerable improvements on sensitivity, reproducibility, value assignment and, importantly, quantitative abilities which are critical to allow the exchangeability of studies performed by the few other available methods and to study hepcidin kinetics
Summary
Protein profiling by time-of flight mass spectrometry (TOF MS) is based on polypeptide enrichment by selective binding to a (bio)chemical surface prior to TOF MS of retained proteins and peptides [1]. This technique is widely used to address several biomedical questions in the proteomics field, e.g. for the discovery of disease related biomarkers in biological fluids [2,3] for protein interaction studies [4] and for immunoproteomics-based approaches [5,6,7]. TOF MS-based assays have the potential to simultaneously distinguish and quantify multiple isoforms/variants of a particular protein/peptide in contrast to most ELISA-based assays [5,8,9,10,11]. Hepcidin deficiency plays a central role in the iron loading in hereditary hemochromatosis and thalassemia’s
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