Project description:Amaranthus hypochondriacus is a highly nutritious alternative grain native to Central and South America but consumed worldwide. Increased interest in A. hypochondriacus has driven characterization of bioactive secondary metabolites and their impact on the human body. A. hypochondriacus seeds are known to contain bioactive small molecules but there is a dearth of knowledge regarding endogenous bioactive peptides, especially cysteine-rich peptides (CRPs) which may be resistant to human digestion. Here, 89 CRPs were predicted in silico from A. hypochondriacus, 13 of which were detected in a seed extract via bottom-up proteomics, providing direct evidence for the translation of snakins, hevein-like peptides, defensins, lipid transfer proteins, and α-hairpinins. Mature forms of four novel and two known CRPs were molecularly characterized via top-down mass spectrometry. Four peptides demonstrated resistance to in vitro gastrointestinal digestion, suggesting that A. hypochondriacus CRPs may exhibit bioactivity after consumption and should be prioritized for further characterization.