Identifying linear ultra-short peptides in Agrifood: Challenges and critical insights

Over the years, the agrifood industry has played a key role in valorizing protein-rich by-products generated during food processing. Fermentation and enzymatic hydrolysis of these by-products are efficient, industry-friendly, and cost-effective methods for generating protein hydrolysates which contain large-, medium/short, and ultra-short peptides (USPs). Despite their prevalence in such mixtures, the identification of USPs (2–5 amino acids) remains more challenging than other peptides. This review critically examines the technical challenges involved in USP identification, offering a detailed timeline of approximately forty key studies that have contributed to advancing the field. This research dedicated to USP peptidomics began only about twelve years ago. The total theoretical number of di-to penta-peptides is 3,200,000. We reviewed enrichment and clean-up protocols, the synthesis peptide sets used as standard over the last decade to demonstrate efficiency of developed analytical methods (including pre-analytical derivatization and post-column addition of chemical agents), mass spectrometry techniques (including the mass analyzers used, the fragmentation-data acquisition modes, and the instrumental techniques used to fragment peptides), and the bioinformatics approaches used for tandem mass spectrometry data processing in the context of USP identification. Finally, to our point of view, publishing the identity of USPs should require an international guideline and the deposition of tandem mass spectrometry raw data in online repositories. • Nearly 100 % of peptides identified by peptidomics are medium/short peptides. • Peptidomics is not well-suited for identifying ultra-short peptides (USPs). • Identifying USPs necessitates the development of dedicated peptidomics techniques. • Need of international guidelines for USP-identification and -reporting are mandatory.