Comprehensive proteomic profiling of Lemna minor L. by advanced MS analysis and in-silico assessment of allergen encoding sequences

The promotion of alternative protein sources has a fundamental role in a more sustainable diet as envisaged by the European Green Deal. Plant-based proteins, including underutilized pulses and weeds (terrestrial and aquatic), are being widely investigated for their pivotal role in the actualization of this dietary shift, and several valuable sources have been identified and characterized. Duckweeds are a diverse category of aquatic plants, primarily from Lemna and Wolffia genera. From a nutritional perspective, they contain a good and balanced amount of protein and essential amino acids. As for Lemna genus, there are few data and no curated proteome database available, with less than 500 accessions of full-length sequenced proteins in public databases. The knowledge about its allergenic potential mainly relies on in-silico evaluations and rare in-vitro tests. Multiple EFSA reports have been published in the last years following Novel Food (NF) applications for Lemna and Wolffia either as whole plants or derived products. As general opinion, the risk of allergenicity in humans is expected to be low. The aim of this investigation is to increase current knowledge about the proteomic profile of L. minor as whole plant material and its allergenic potential by advanced mass spectrometry (MS) and multiple bioinformatics tools. To address the informational gaps, a shot-gun bottom-up proteomic approach and protein identification by homologous sequence alignment within the Araceae family were accomplished. An optimized extraction protocol was applied to provide exhaustive protein extraction from lyophilized samples under basic buffered aqueous conditions. Both physical and chemical aids were included to achieve a satisfactory extraction yield, up to 65±5%. Discovery proteomics by high-resolution MS/MS was applied with commercial software for protein identification. A total of 1892 entries were retrieved at the highest confidence, described by 5310 tryptic peptides. A wide redundancy along the identification list was disclosed mainly due to highly conserved regions among homologous proteins from different species. The main identified protein belonged to the photosynthetic and metabolic pathways, such as RuBisCo chains, ATP synthase subunits, photosystems I/II, chlorophyll a-b binding proteins, among others. The collected data have been used to undertake an in-silico assessment of the allergenic potential according to the guidelines provided by FAO/WHO and EFSA, and by means of the web-server AllerCatPro 2.0 to predict cross-reactivity with known allergens. A curated list of water lentil proteins with putative allergenicity based on strong and weak evidence of cross-reactivity was obtained and will be presented as complementary support to the safety assessment of L. minor formulates as NF.