Journal of Extracellular Vesicles (Nov 2022)

Characterizing single extracellular vesicles by droplet barcode sequencing for protein analysis

  • Mahsan Banijamali,
  • Pontus Höjer,
  • Abel Nagy,
  • Petra Hååg,
  • Elizabeth Paz Gomero,
  • Christiane Stiller,
  • Vitaliy O. Kaminskyy,
  • Simon Ekman,
  • Rolf Lewensohn,
  • Amelie Eriksson Karlström,
  • Kristina Viktorsson,
  • Afshin Ahmadian

DOI
https://doi.org/10.1002/jev2.12277
Journal volume & issue
Vol. 11, no. 11
pp. n/a – n/a

Abstract

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Abstract Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS‐Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV‐subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof‐of‐concept study we demonstrate that DBS‐Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV‐subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.

Keywords