We have developed protocol called SPEA to allow us to identify and quantify peptides derived from active low abundance small-protein hormones in plasma (Harney et. al. 2019. Mol. Cell. Proteomics). This has allowed us to explore all components of this fraction using unbiased mass spectrometry-based analysis. Using the SPEA protocol, we detected three peptides from the uncharacterised 8 kDa protein (erusiolin) in human plasma samples from a clinical trial examining the response to intermittent fasting. A highly conserved peptide from erusiolin was significantly increased in abundance after 8-weeks of intermittent fasting. From this, we hypothesised that the abundance of erusiolin was induced by fasting, which we have now tested using human mixed meal tests and observed decreased abundance after food consumption. The mRNA encoding erusiolin is largely duodenum-specific in both humans and mice and immunohistochemistry analysis of both human and mouse tissues using an erusiolin-specific antibody has demonstrated a staining pattern consistent with expression in enteroendocrine cells. We have used the Quantitative Endocrine Network Interaction Estimation (QENIE) method in mice to identify potential target tissues for erusiolin, which showed the hypothalamus had many transcripts significantly linked to variation in the locus. Strikingly, a significant number of the transcripts were derived from the Prader-Willi Syndrome locus, which is a disease characterised by extreme hyperphagia (over-eating) and subsequent obesity. This leads us to hypothesise that the conserved peptide from erusiolin is secreted by the duodenum into blood plasma during fasting and acts on the hypothalamus to inhibit transcription of the PWS locus, leading to orexigenic (hunger) signals. We have now generated knock-out mice and are currently characterizing their phenotype compared to wildtype littermates for food-intake and related metabolic phenotypes.