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	Figure 1. Strongylocentrotus purpuratus coelomic fluid EV profile. (A) Nanoparticle tracking analysis (NTA) showing a representative size distribution profile of sea urchin coelomic fluid EVs, with the majority of EVs within a 30–300 nm range. (B) Transmission electron microscopy (TEM) from sea urchin coelomic fluid EVs; scale bar represents 50 nm. (C) Western blotting analysis (WB) showing sea urchin coelomic fluid EVs positive for Flot-1 and CD63, respectively; the molecular weight for the standard is indicated in kilodaltons (kDa).
	Figure 2. Protein–protein interaction networks of total EV protein cargo identified in purple sea urchin. (A) Functional protein networks are based on known and predicted interactions in Echinoidea using STRING analysis. Coloured nodes represent query proteins only. Coloured lines connecting nodes show the type of interactions between the nodes of the networks based on known interactions, predicted interactions, and others (including text-mining, co-expression, and protein homologue); colour code legend is provided in the figure. PPI enrichment p-value for the protein network is 9.25 × 10−5. (B) KEGG pathways identified from STRING analysis for EV total protein cargo (annotated hits). (C) Gene Ontology (GO) Biological processes identified from STRING analysis for total EV protein cargo (annotated hits). (D) GO Molecular Function pathways identified from STRING analysis for total EV protein cargo (annotated hits; protein names of hits are presented in the figures; additional interacting proteins are: LOC579085 = ATP synthase subunit gamma, mitochondrial; LOC587430 = ATP synthase subunit O, mitochondrial; LOC373382 = ATP synthase subunit alpha; LOC576006 = ATP synthase subunit delta, mitochondrial; LOC579751 = ATP synthase F(0) complex subunit B1, mitochondrial).
	Figure 3. Peptidylarginine deiminase (PAD)-like protein detection and deiminated proteins in sea urchin coelomic fluid. (A) Using the human PAD2-specific antibody, a strong positive band was detected in coelomic fluid (CF). (B,C) Coelomic fluid (CF) was silver-stained for total protein (B); the F95-enriched proteins from coelomic fluid (CF) are shown in (C)—protein bands for F95-enriched proteins are highlighted with arrows. (D) Showing total proteins as detected by silver staining in EVs isolated from sea urchin coelomic fluid (a pool of 3 samples was used for the EV enriched fraction) and citrullinated/deiminated proteins were isolated from the EVs using F95 enrichment (EV_F95 IP); arrows point at main F95-enriched protein bands. The molecular weight standard (Std) is shown on the left hand side of each blot/gel and indicated in kilodaltons (kDa).
	Figure 4. Protein–protein interaction networks of deiminated proteins identified in purple sea urchin coelomic fluid EVs. (A) Protein networks for deiminated proteins in purple sea urchin EVs based on known and predicted interactions in Echinoidea, using STRING analysis. Coloured nodes represent query proteins only. Coloured lines connecting nodes show the type of interactions between the nodes of the networks based on known interactions, predicted interactions, and others (including text-mining, co-expression, and protein homologue); colour code legend is provided in the figure. PPI enrichment p-value for the protein network is 1.11 × 10−16. (B) KEGG pathways identified from STRING analysis for the deiminated protein candidates in purple sea urchin EVs. (C) Gene Ontology (GO) Biological processes identified from STRING analysis for deiminated protein candidates in purple sea urchin EVs. (D) GO Molecular functions identified from STRING analysis for deiminated proteins candidates in purple sea urchin EVs. Protein names of hits listed in the tables are presented in the figures; additional interacting proteins are: LOC588440 = dnaJ homolog subfamily B member 11; LOC577666 = dnaJ homolog subfamily C member 1; LOC584585 = endoplasmic reticulum chaperone BiP; hyou1 = heat shock protein 70 family member; LOC586348 = uncharacterised protein(Calnexin-like); SPU_005691-tr = Protein disulfide-isomerase; SPU_010434-tr = annotation not available (part of calriticulin protein network); pdia4 = Protein disulfide-isomerase A4.
	Figure 5. Protein–protein interaction networks of deiminated proteins identified in purple sea urchin coelomic fluid. (A) Protein networks for deiminated proteins in purple sea urchin coelomic fluid based on known and predicted interactions in Echinoidea, using STRING analysis. Coloured nodes represent query proteins only. Coloured lines connecting nodes show the type of interactions between the nodes of the networks based on known interactions, predicted interactions, and others (including text-mining, co-expression, and protein homologue); colour code legend is provided in the figure. PPI enrichment p-value for the protein network is 5.31 × 10−5. (B) KEGG pathways identified from STRING analysis for the deiminated protein candidates in purple sea urchin coelomic fluid. (C) Gene Ontology (GO) Biological processes identified from STRING analysis for deiminated protein candidates in purple sea urchin coelomic fluid. (D,E) GO Molecular functions identified from STRING analysis for deiminated proteins candidates in purple sea urchin coelomic fluid. Protein names of hits listed in the tables are presented in the figures; additional interacting proteins are: LOC589934 = large subunit GTPase 1 homolog; LOC577725 = heat shock protein 83; LOC765087 = Hsp70/Hsp90-organising protein; LOC575643 = activator of heat shock 90kDa protein ATPase homolog 1; LOC763735 = Pyruvate kinase; LOC548623 = Fructose-bisphosphate aldolase; LOC592868 = Fructose-bisphosphate aldolase, non-muscle type like; SPU_012817-tr = Phosphoglycerate kinase; SPU_011119-tr = Glucose-6-phosphate isomerase.
	Figure 6. Deiminated protein hits and EV cargo and associated STRING protein network analysis of purple sea urchin coelomic fluid citrullinome and EV citrullinome, and of total EV protein cargo. (A) Venn diagram showing deiminated protein hits (“citrullinome”) identified in Strongylocentrotus purpuratus coelomic fluid and EVs, representing shared and unique proteins (uncharacterised proteins are indicated in brackets), identified using the Echinoidea UniProt database. (B) Venn diagram showing protein hits identified for total EV proteome cargo and deiminated EV cargo (EV citrullinome) protein hits. (C) Venn diagram showing KEGG and GO pathway analysis for deiminated proteins identified in coelomic fluid (coelomic fluid citrullinome) and EVs (EV citrullinome), respectively. (D) Venn diagram showing GO and KEGG pathways for total EV protein cargo and shared pathways with the EV citrullinome. The number of specific and overlapping pathways is indicated and relates to the STRING networks in Figure 2, Figure 4 and Figure 5.

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