Challenges of MS-based small extracellular vesicles proteomics.

Proteomic profiling of small extracellular vesicles (sEV) is a powerful tool for discovering biomarkers of various diseases. This process most often assisted by mass spectrometry (MS) usually lacks standardization and recognition of challenges which may lead to unreliable results. General recommendations for sEV MS analyses have been briefly given in the MISEV2023 guidelines. The present work goes into detail for every step of sEV protein profiling with an overview of factors influencing such analyses. This includes reporting and defining the sEV source and vesicle isolation, protein solubilization and digestion, 'offline' and 'online' sample complexity reduction, the analysis type itself, and subsequent data analysis. Every stage in this process affects the others, which could result in different outcomes. Although characterization and comparisons of different sEV isolation methods are known and accessible and MS-based profiling details are provided for cell or tissue samples, no consensus work has been ever published to describe the whole process of sEV proteomic analysis. Reliable results can be obtained from sEV profiling provided that the analysis is well planned, prepared for, and backed by pilot studies or appropriate research.