Optimizing fungal extracellular vesicle proteomic profiling through combined analysis of in-solution and in-gel digestion

Proteomics offers a powerful tool to identify proteins within diverse microbial organisms, environments, and organelles, including extracellular vesicles (EVs). Fungal EVs are of particular interest due to their roles in cellular development and communication. While several methods exist to isolate EVs from cells, a universally accepted approach for EV protein characterization is lacking. This study investigated in-solution digestion (SD) and in-gel digestion (GD), for characterizing proteins from Neurospora crassa EVs, followed by LC-MS/MS analysis. GD identified three to four-times more proteins than SD while using the same number of unique peptides. Although GD requires a higher amount of starting sample, it offers a more comprehensive protein identification for fungal EVs, potentially preventing the omission of crucial data.