Proteoform analysis by mass spectrometry reveals post-translational processing of legumins and vicilins in chickpeas (Cicer arietinum L.).

The most abundant proteins in pulse seed, including chickpea (Cicer arietinum L.), are the 7S globulins (vicilins) and 11S globulins (legumins), which play key roles in plant reproduction and human nutrition and are also recognized as potential allergens. During seed maturation, legumins and vicilins undergo limited proteolysis. Legumins are processed into α- and β-polypeptide chains that remain linked by a disulfide bond, whereas vicilins are cleaved at two sites, generating three lower-molecular-mass polypeptides (α-, β-, and γ-chains). An integrated mass spectrometry approach, combining bottom-up and top-down strategies, was applied to characterize the post-translational processing of legumins and vicilins. Our data suggest that (i) proteolytic cleavages in both legumins and vicilins occur at conserved sequence motifs; (ii) legumin processing is catalyzed by an asparaginyl endopeptidase, whereas vicilin processing is mediated by legumains, a family of cysteine endopeptidases; and (iii) one chickpea vicilin is N-glycosylated and carries a high-mannose oligosaccharide side chain. Limited proteolysis process represents a key physiological mechanism regulating storage protein assembly, stability, and mobilization during seed development and germination while preserving storage functions. Glycosylation of vicilins further modulates their structural and functional properties, although it may also influence digestibility and allergenic potential. The mass spectrometry data have been deposited with the ProteomeXchange with the identifier <PXD078377>.