Solvent-dependent proteome profiling in edible mushrooms (Hypsizygus marmoreus and Agaricus bisporus)

This study compared five solvents - urea, carbonate-bicarbonate, citrate, phosphate, and water - for their ability to extract proteins from beech (BM) ( Hypsizygus marmoreus ) and Swiss brown (SB) ( Agaricus bisporus ) mushrooms. Using mass spectrometry-based proteomics and bioinformatics, we found that urea produced the highest total protein identifications by data-dependent acquisition at 1% false discovery rate (FDR): BM: 1,739, SB: 1,907; and by data-independent acquisition: BM: 1,969, SB: 2,172 proteins, especially enriching cytosolic and ribosomal proteins. Carbonate-bicarbonate favoured the extraction of metabolic and translational proteins, while citrate targeted extracellular and membrane-associated proteins related to cell wall dynamics. Phosphate-enriched catalytically active proteins, and water extracted mainly soluble, simple proteins. Sequence-based enzyme profiling of urea extracts revealed an abundance of oxidoreductases, transferases, ligases, and translocases. Overall, extraction solvent selection strongly influences yield, diversity, and functional potential of mushroom proteomes, establishing a framework for targeted extraction of endogenous enzymes and functionally distinct proteins for food ingredient and nutraceutical formulations. • No single solvent captures the full proteome; each enriches specific proteins. • Solvent chemistry and cell wall structure influence protein extraction outcomes. • Urea yielded most proteins and enhanced oxidoreductase and transferase abundance. • Citrate, carbonate, phosphate, and water enriched distinct protein and enzyme sets. • Combining solvents can deepen and tailor macro-fungal proteome analysis.