Identification and quantification of plasma free salusin-β, an endogenous parasympathomimetic peptide

Salusin-β is an endogenous parasympathomimetic proatherosclerotic peptide. Salusin-β was initially predicted from bioinformatic analyses and later immunologically detected in human biofluids. However, elucidation of salusin-β bioactivity has faced enormous challenges because of its unique physicochemical characteristics that cause it to strongly adhere to laboratory apparatus materials. In the strictest sense, the discovery of bioactive peptides is not complete until their exact native sequences have been confirmed in the peripheral circulation. In this study, we determined the plasma molecular form and levels of free salusin-β to determine its pathophysiological significance. Ultra-high-yield enrichment and preseparation of non-tryptic human plasma was followed by LC-MS/MS, and full-length salusin-β and seven different endogenous fragment sequences were identified. We established a new ELISA that specifically detects plasma free salusin-β without cross-reacting with any of its identified endogenous fragments. Free salusin-β levels exhibited a profound early morning nadir and rapidly decreased in response to parasympathetic nervous augmentation. Our technical advance in plasma native peptide analysis successfully identified a hard-to-detect bioactive peptide, salusin-β, together with its formerly unrecognized fragments, and further suggests that conventional immunological measurements of target peptides may not be fully representative.