High-Resolution Mass Spectrometry-Based Proteomic Profiling of Lemna minor: Insights into Protein Composition, Extraction Strategies and Allergenicity Risk

Lemna minor L. (LM) has recently gained considerable interest as a sustainable protein-rich novel food; however, information on its proteome remains limited due to the scarcity of species-specific sequence databases and the lack of comprehensive mass spectrometry-based investigations. This study provides the first extensive proteomic characterization of LM using high-resolution tandem mass spectrometry and evaluates the influence of different extraction strategies on proteome coverage. LM was cultivated under controlled conditions and harvested after 10 days, yielding 1.00 ± 0.15 kg/m2 and a protein content of 35% on a dry weight basis. To maximize proteome coverage, proteins were extracted at different pH values (7, 9, and 11) by ultrasound-assisted extraction and by thermostatic extraction under shaking. Although all protocols showed comparable protein contents (8.2–9.1 mg/mL) and extraction efficiencies (58.6–65.1%), hierarchical clustering, Gene Ontology analysis, SDS-PAGE profiling, and in-gel protein identification revealed differences in the recovery of specific protein classes, supporting the integration of data from multiple extraction approaches. Bottom-up shotgun proteomics identified 1211 proteins and 4149 validated peptides, representing the most comprehensive LM proteome dataset reported to date. Most proteins were associated with photosynthesis, carbon fixation, energy metabolism, cellular structure, and stress-response pathways. An in silico allergenicity assessment identified 40 proteins that were homologous to known food allergens, with chlorophyll a/b-binding protein showing the highest abundance in peak signal and similarity to the celery allergen Api g 3. Overall, this work provides a valuable resource for future nutritional, functional, and safety evaluations of duckweed-derived foods.