A complete workflow for discovering small bioactive peptides in foods by LC-MS/MS: A case study on almonds

[Display omitted] •Small peptide sequencing is crucial for comprehensive bioactive peptide discovery.•A new LC-MS/MS-based method for small peptide identification was established.•Dimethyl labeling effectively aided in small peptide full-length sequencing.•Dimethyl labeling is inexpensive, rapid, simple, and applicable to any food matrix.•Over 200 small bioactive peptides derived from almond proteins were identified. Identification of bioactive peptides is an increasingly important target for food chemists, particularly in consideration of the widespread application of proteolytic enzymes in food processing. Because the characterization of small peptides by LC-MS/MS is challenging, we optimized a dimethyl labeling technique to facilitate small peptide identification, using almond proteins as a model. The method was validated by comparing the MS/MS spectra of standards and almond-derived peptides in their nonderivatized and derivatized forms. Signal enhancement of a1 ions was proved to effectively aid in the full-length sequencing of small peptides. We further validated this method using two industrially-relevant protein-rich extracts from almond flour: 1737 medium-sized peptides (5–39 amino acids) and 843 small peptides (2–4 amino acids) were identified. The use of an online bioactive peptide database, complemented by the existing literature, allowed the discovery of 208 small bioactive peptides, whereas for medium-sized peptides, only one was reported being bioactive.