Suspect screening-based LC-HRMS characterization of short-chain peptide hydrolysates from marine macroalgae

Marine macroalgae are increasingly recognized as sustainable and chemically diverse sources of valuable biomolecules. However, the short-chain peptide fraction (2-4 amino acids) of edible and invasive seaweeds remains poorly explored, mainly because conventional proteomics workflows are generally optimized for longer peptide sequences. In this study, an untargeted LC-HRMS suspect screening workflow was developed to characterize short-chain peptides generated by hydrolysis with Alcalase®, an alkaline protease from Bacillus licheniformis hydrolysis in 14 marine macroalgae species belonging to green, brown, and red phyla collected along the Atlantic coast of southern Spain. Overall, 333 short-chain peptides, predominantly dipeptides, were putatively annotated through accurate-mass filtering and manual MS/MS interpretation. Multivariate and univariate analyses revealed distinct peptide distribution patterns among algal groups, with brown algae showing a higher abundance of hydrophobic dipeptides associated with predicted bioactive properties according to in silico screening tools. Notably, invasive brown algae displayed peptide profiles largely comparable to edible species, supporting their potential valorization as sustainable sources of short-chain peptides. Exploratory in silico analyses further suggested the occurrence of peptide sequences potentially associated with antioxidant, antihypertensive, antidiabetic, sensory, and antifungal properties. Several peptides were also predicted to contribute to umami-related flavor attributes, highlighting the relevance of these matrices for future investigation of food-related peptide functionality. Although the proposed annotations and predicted bioactivities require orthogonal confirmation and experimental validation, the results provide a comprehensive exploratory overview of marine macroalgal short peptidomes. Overall, this study expands current knowledge on algae-derived short-chain peptides and demonstrates the applicability of untargeted LC-HRMS workflows for the characterization of underexplored peptide fractions in complex marine matrices.