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 extremely high therapeutic and diagnostic
potential. Mass spectrometry (MS)-based proteomics is a powerful technique
to profile and quantify the protein content in exosomes, but the current
methods require laborious and time-consuming multistep sample preparation
that significantly limit throughput. Herein, we report a one-pot exosome
proteomics method enabled by a photocleavable surfactant, Azo, to
simplify exosomal lysis, effectively extract proteins, and expedite
digestion. We have applied this method to exosomes derived from isolated
mammary fibroblasts and confidently identified 3466 proteins and quantified
2288 proteins using a reversed-phase liquid chromatography coupled
to trapped ion mobility spectrometry (TIMS) quadrupole time-of-flight
mass spectrometer. Here, 3166 (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 that this method could be generally applicable for exosome
proteomics applications in biomedical research, therapeutic interventions,
and clinical diagnostics.