One-Pot Exosome Proteomics Enabled by a Photocleavable Surfactant

Exosomes are small extracellular vesicles (EVs) secreted by all cells and found in biological fluids, which can serve as minimally invasive liquid biopsies with high therapeutic and diagnostic potential. Mass spectrometry (MS)-based proteomics is a powerful technique to profile and quantify the protein content of exosomes but the current methods require laborious and time-consuming multi-step sample preparation that significantly limit throughput. Herein, we report a one-pot exosome proteomics method enabled by a photocleavable surfactant, Azo, for rapid and effective exosomal lysis, protein extraction, and digestion. We have applied this method to exosomes derived from isolated mammary fibroblasts and confidently identified 3,466 proteins and quantified 2,288 proteins using reversed-phase liquid chromatography coupled to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight mass spectrometer. 3,166 (91%) of the identified proteins are annotated in the exosome/EVs databases, ExoCarta and Vesiclepedia, including important exosomal markers, CD63, PDCD6IP, and SDCBP. This method is fast, simple, and highly effective at extracting exosomal proteins with high reproducibility for deep exosomal proteome coverage. We envision this method could be generally applicable for exosome proteomics applications in biomedical research, therapeutic interventions, and clinical diagnostics. ### Competing Interest Statement The authors declare the following competing financial interest(s): The University of Wisconsin−Madison has filed a provisional patent application P180335US01, US serial number 62/682027 (7 June 2018) on the photocleavable surfactant. Y.G., S.J., and K.A.B. are named as inventors on the provisional patent application. The University of Wisconsin−Madison has filed a provisional patent application P220113US01 (1 November 2021) on this work. Y.G., S.J., K.M.B, and K.A.B are named as inventors on the provisional patent application.