Exploratory Profiling of Extracellular Vesicles and Citrullinated Proteins in Plasma of Greenland Shark Somniosus microcephalus

The Greenland shark Somniosus microcephalus is the longest-living vertebrate on Earth. With an estimated lifespan of up to at least 272 years, slow metabolism and resistance to high pressure and cold temperature, its unusual physiology remains poorly understood. Protein citrullination (deimination) is a post-translational modification of arginine into citrulline, catalysed by peptidylarginine deiminases (PADs) in a calcium-dependent manner. Citrullination of proteins changes charge, conformation and downstream protein folding, affecting protein function and protein–protein interactions. Citrullination is also linked to extracellular vesicle (EV) communication, which is crucial in cell–cell interactions. This explorative study focussed on profiling F95-enriched pan-citrullinated proteins from plasma and identifying plasma EVs from two archived Greenland shark plasma samples (from one shark sampled in 2010, storage at −20 °C, and one pool of three sharks sampled in 2015, storage at −80 °C). EVs were labelled for EV markers and citrullinated proteins were confirmed to co-localise to EVs by single-molecule detection using super-resolution microscopy. Total EV-associated proteins from the EV-enriched fraction were identified by LC-MS/MS, revealing 76 (plus seven uncharacterised) proteins associated with several immune, oxidative stress, DNA replication, cell senescence and metabolic pathways, using pathway annotation analysis (10 KEGG, 37 Reactome and 30 Biological Process GO pathways). Furthermore, LC-MS/MS analysis of F95-enriched (pan-citrullinated) proteins from Greenland shark plasma identified 75 protein hits (plus six uncharacterised proteins), which were associated with nine KEGG, 38 Reactome and 25 Biological Process GO pathway annotations linked to gene regulation, stress and immune responses, chemical homeostasis, metabolism and cell signalling. As the Greenland shark is an extremely long-living species which also tolerates cold temperatures and high pressures, understanding of EV-mediated processes and target proteins of citrullination in this species may provide insights into its unusual physiological adaptations.