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Needham, Sarah R., Davis, Benjamin M., Uysal‐Onganer, Pinar, Rolfe, Daniel J., Hristova, Mariya, Kraev, Igor, Inal, Jameel and Lange, Sigrun (2025) Profiling protein citrullination in extracellular vesicles by single-molecule detection using direct stochastic optical reconstruction microscopy. Journal of Biophotonics: e202500483. pp. 1-17. ISSN 1864-0648
Extracellular vesicles (EVs) are critical in cellular communication and pathological biomarkers. Post‐translationally deiminated/citrullinated proteins are reported in EV cargoes by LC–MS/MS but it is unknown in which EV sub‐types they are exported, as part of EVs' intraluminal cargo or on the EV surface. Here, dSTORM super‐resolution microscopy is used to co‐localise total citrullinated proteins (pan‐Cit), and citrullinated histone H3 (CitH3) to EV subtypes of three cancer cell lines, captured by tetraspanin trio (TT) or phosphatidylserine (PS). Permeabilised and non‐permeabilised EVs are analysed with a Bayesian framework using beta‐distributed posteriors for binomial outcomes. Pan‐Cit and CitH3 labelling is confirmed in EVs as intraluminal cargo and on the EV surface, with higher levels detected in the permeabilized EVs. Pan‐Cit staining is higher in TT‐bound EVs, but CitH3 staining higher in PS‐bound EVs. This study expands the landscape of EV‐associated post‐translational modifications with translational potential for EV‐citrullinome based liquid biopsy tools.
Available under License Creative Commons Attribution 4.0.
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