Pittala, M. G. G.; Leggio, L.; Paterno, G.; Giusto, E.; Civiero, L.; Cunsolo, V.; Vivarelli, S.; Di Francesco, A.; Alpi, E.; Saletti, R.; Iraci, N.

Background Current proteomics techniques allow rapid identification and quantification of proteins within any given biological source. In particular, nanoUHPLC/High-Resolution nanoESI-MS/MS enables the characterization of proteins in complex biological samples due to its high sensitivity, accuracy, and scalability. However, LC-MS/MS proteomics might still be susceptible to laboratory and sample-associated contaminants, which can significantly compromise the quality and reliability of data. Therefore, an accurate identification and annotation of such contaminants is crucial for the development of robust proteomics databases and spectral-libraries related search engines. This approach is of special interest in the field of secretome and extracellular vesicles (EVs), membrane-enclosed nanostructures that contain a variety of proteins crucial for cell-to-cell communication and translational applications. Results When working in ex vivo/in vitro settings, proteins from fetal bovine serum (FBS), commonly employed in standard cell culture media, may interfere with the proteome analysis. To address this issue, we conceived and designed SPROUTS_DB, Serum Protein Repository Of Unwanted Target(ed) Sequences DataBase, a dedicated resource to catalog serum-derived contaminants. Starting from media supplemented with EV-depleted FBS, we simulated cell growth conditions - in the absence of cells - followed by ultracentrifugation. LC-MS/MS analysis of these samples resulted in the identification of a novel set of 1,288 contaminant proteins, which has been deposited in the ProteomeXchange repository (identifier PXD044137). SPROUTS_DB contains primarily soluble proteins, mainly related to the Gene Ontology categories Extracellular Region and Extracellular Space, in line with the nature of the starting sample. In contrast, only a small fraction of the contaminants is classified as membrane-associated proteins, supporting the limited vesicle contamination in the complete medium, due to the use of EV-depleted FBS. Of note, we demonstrated that SPROUTS_DB outperforms existing contaminants databases, ensuring that only peptide spectra relevant to the examined sample are retained and identified as true positive data. Conclusions Considering that even proteins from phylogenetically distant organisms share extensive stretches of sequences, SPROUTS_DB is designed to discern contaminants from real sample proteins of interest, minimizing false positive identifications. To the best of our knowledge, SPROUTS_DB is the most updated database of contaminants useful for proteomics investigations of cellular secretomes and EV-containing samples.