SPROUTS_DB: An Implemented Database of Contaminants for Extracellular Vesicle Proteomics Studies

Current proteomics techniques allow rapid identification and quantification of proteins within any given biological source. However, LC-MS/MS proteomics is vulnerable to laboratory and sample-associated contaminants. Therefore, accurate identification and annotation of such contaminants is crucial for development of reliable databases, especially for secretome and extracellular vesicle studies. When working in ex vivo/in vitro settings, proteins from fetal bovine serum (FBS) interfere with the proteome analysis. To address this issue, we designed SPROUTS_DB, Serum Protein Repository Of Unwanted Target(ed) Sequences DataBase, a dedicated resource to catalog serum-derived contaminants. Starting from media with EV-depleted FBS, we simulated cell growth conditions-without cells-followed by ultracentrifugation. LC-MS/MS analysis resulted in the identification of a novel set of 1288 contaminant proteins. SPROUTS_DB contains primarily soluble proteins linked to the Extracellular Region and Space, in line with the nature of the starting sample. In contrast, few membrane-associated proteins were found, confirming minimal vesicle contamination from the use of EV-depleted FBS. Finally, we demonstrated that SPROUTS_DB outperforms existing contaminants' databases, ensuring that only peptide spectra relevant to the examined sample are retained as true positive data. It is, to our knowledge, the most up-to-date resource for proteomic analysis of secretomes and EV-containing samples.