Hepcidin is a small liver-derived peptide central in the regulation of systemic 
iron homeostasis. Although the gene regulation has been extensively studied at 
transcriptional level, the corresponding effects on the production of bioactive 
peptide are largely unknown. We therefore applied a proteomics-based approach by 
combining immunocapture with time-of-flight mass spectrometry to characterize 
hepcidin-25 produced by hepatocyte-derived cell lines. Similar to its 
transcriptional regulation, mature hepcidin-25 was strongly secreted upon 
stimulation with BMPs and IL-6. The immunocaptured peptide down-modulated 
iron-exporter ferroportin on the monocyte/macrophage surface. Further mass 
spectrometry-based analyses indicated that hepcidin-25 in its bioactive 
conformation was very stable in serum and urine and not converted into its 
smaller isoforms. Hepcidin-25 was processed in the Golgi apparatus from its 
precursor, while the unprocessed prohepcidin was secreted only when furin-like 
protease activity was intracellularly inhibited. Furthermore, the amounts of 
hepatocytic secretion of hepcidin-25 are highly correlated with the gene 
transcript levels. An unexpected observation was the synergistic effect of BMPs 
and IL-6 on hepcidin-25 secretion, which points towards cross-talk between iron 
and inflammatory stimuli. The study underscores hepcidin-25 quantification as a 
valuable tool to unravel regulatory pathways in iron metabolism.