Work

Profiling Protein Citrullination in Extracellular Vesicles by Single‐Molecule Detection Using Direct Stochastic Optical Reconstruction Microscopy

Year: 2025

Type: article

Abstract: 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.

Source: Journal of Biophotonics

DOI: 10.1002/jbio.202500483