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Article

The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles—Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways

by
Bergljót Magnadóttir
1,
Igor Kraev
2,
Alister W. Dodds
3 and
Sigrun Lange
4,*
1
Institute for Experimental Pathology at Keldur, University of Iceland, Keldnavegur 3, 112 Reykjavik, Iceland
2
Electron Microscopy Suite, Faculty of Science, Technology, Engineering and Mathematics, Open University, Milton Keynes MK7 6AA, UK
3
MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK
4
Tissue Architecture and Regeneration Research Group, Department of Biomedical Sciences, University of Westminster, London W1W 6UW, UK
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(2), 875; https://doi.org/10.3390/ijms22020875
Submission received: 24 December 2020 / Revised: 11 January 2021 / Accepted: 15 January 2021 / Published: 16 January 2021
(This article belongs to the Special Issue Extracellular Vesicles in Phylogeny)
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Abstract

:
Extracellular vesicles (EVs) are lipid bilayer vesicles which are released from cells and play multifaceted roles in cellular communication in health and disease. EVs can be isolated from various body fluids, including serum and plasma, and are usable biomarkers as they can inform health status. Studies on EVs are an emerging research field in teleost fish, with accumulating evidence for important functions in immunity and homeostasis, but remain to be characterised in most fish species, including halibut. Protein deimination is a post-translational modification caused by a conserved family of enzymes, named peptidylarginine deiminases (PADs), and results in changes in protein folding and function via conversion of arginine to citrulline in target proteins. Protein deimination has been recently described in halibut ontogeny and halibut serum. Neither EV profiles, nor total protein or deiminated protein EV cargos have yet been assessed in halibut and are reported in the current study. Halibut serum EVs showed a poly-dispersed population in the size range of 50–600 nm, with modal size of EVs falling at 138 nm, and morphology was further confirmed by transmission electron microscopy. The assessment of EV total protein cargo revealed 124 protein hits and 37 deiminated protein hits, whereof 15 hits were particularly identified in deiminated form only. Protein interaction network analysis showed that deimination hits are involved in a range of gene regulatory, immune, metabolic and developmental processes. The same was found for total EV protein cargo, although a far wider range of pathways was found than for deimination hits only. The expression of complement component C3 and C4, as well as pentraxin-like protein, which were identified by proteomic analysis, was further verified in EVs by western blotting. This showed that C3 is exported in EVs at higher levels than C4 and deiminated C3 was furthermore confirmed to be at high levels in the deimination-enriched EV fractions, while, in comparison, C4 showed very low detection in deimination-enriched EV fractions. Pentraxin was exported in EVs, but not detected in the deimination-enriched fractions. Our findings provide novel insights into EV-mediated communication in halibut serum, via transport of protein cargo, including post-translationally deiminated proteins.

Graphical Abstract

1. Introduction

Halibut is a teleost flatfish which belongs to the order Heteresomata (Pleuronectiformes). It is one of the largest teleost fish and endangered due to previous overfishing and slow rate of growth. The Atlantic halibut (Hippoglossus hippoglossus L.) is of considerable commercial value for aquaculture, where developmental abnormalities and viability in larval rearing have been one of the major obstacles [1,2]. Furthering understanding of immune, metabolic and developmental processes in commercially viable species, including halibut, is of great importance for the development of biomarkers associated to fish health and improved outcomes in aquaculture.
Peptidylarginine deiminases (PADs) are a calcium-dependent family of enzymes conserved throughout phylogeny with roles in physiological and pathophysiological processes [3,4,5,6]. PADs catalyse protein deimination/citrullination, which is an irreversible post-translational modification of protein arginine to citrulline, leading to structural and functional changes in target proteins [3,6,7]. Deimination can affect protein–protein interactions, as it modifies the protein structure and can cause protein denaturation or affect hydrogen bond formation [5,8]. Deimination can furthermore facilitate protein moonlighting, allowing one protein to carry out various functions within one polypeptide chain [9]. Intrinsically disordered proteins and β-sheets are most prone to undergo deimination and the position of the arginine within the protein plays roles as well [6,8,10]. While in fish, only one PAD form is present [11,12,13,14], mammals contain five tissue-specific PAD isozymes, with varying preferences for target proteins [3,4,5]. In other phyla, such as reptiles and birds, only three PAD forms are described [3,15,16], and PAD homologues are identified lower in the phylogeny tree [17], including in bacteria [18,19], fungi [20], parasites [21], as well as in Crustacea [22], Merostomata [23] and Mollusca [24]. PAD-mediated protein deimination has been reported in a range of taxa throughout the phylogeny tree, both in ontogeny, serum and plasma, as well as forming part of extracellular vesicle (EV) protein cargo [12,13,14,16,22,23,24].
EVs are lipid-bilayer vesicles in the size range of 50–1000 nm, released from most cells and participate in cellular communication in physiology and pathological processes. EVs are classified into small EVs (“exosomes”, <100 nm) and larger EVs (“microvesicles” 100–1000 nm), which are released from cells via different biogenesis pathways, including exocytosis or membrane blebbing [25,26]. Roles for PADs in the modulation of EV release have furthermore been described [27,28,29]. EVs carry a range of cargo, including proteins, enzymes, genetic material, long non-coding RNAs and microRNAs, derived from the cells of origin [25,26,27,28,29,30,31,32,33]. Protein EV cargo can furthermore consist of post-translationally modified proteins, which possibly contribute differently to cellular communication compared with non-modified protein forms. Therefore, it may be of considerable interest to gain insight into differences in such protein cargo in serum-EVs to further understanding of post-translational modifications (PTMs) in cellular communication.
While EV research has been an exponentially expanding field in the past decade in relation to human disease, less is known about EV communication in other taxa. The comparative field of EV research has recently been growing, including by studies from our group [14,16,19,22,23,24,32,33,34,35,36,37,38,39,40]. Therefore, there is currently great interest in expanding EV studies, also in relation to teleost fish and biomarker discovery for aquaculture [32,33,38,41]. Furthermore, fundamental research into EV communication across the phylogeny tree will allow for increased understanding of EV-mediated pathways in evolution.
This study aimed at characterising EVs from halibut sera, assessing both total proteomic cargo and deiminated protein cargo to gain insights into putative roles for protein deimination in the serum secretome.

2. Results

2.1. EV Profiling from Halibut Sera

Halibut serum EVs were characterised by NTA, revealing a poly-dispersed EV population in the size range of 50–600 nm, with the modal size of EVs falling at 138 nm (Figure 1A). The EVs were further characterised for two EV specific markers, CD63 and Flotillin-1 and found positive for both (Figure 1B). EV morphology was further confirmed by transmission electron microscopy (TEM), revealing typical EV morphology (see arrows) and confirming a polydispersed population (Figure 1C).

2.2. The Proteome and Citrullinome of Halibut Serum EVs

Total protein content, as well as F95 enriched protein content, representative of deiminated protein cargo in EVs (the “EV-citrullinome”), was identified by LC-MS/MS analysis. A range of proteins relating to innate and adaptive immunity, as well as gene regulation and cellular function, were identified as deiminated in EV cargo, and are listed in Table 1 (for full details on LC-MS/MS analysis, see Supplementary Table S1). Total EV protein cargo analysis revealed proteins relating to innate and adaptive immunity, nuclear proteins relating to gene regulation, proteins relating to cellular function and metabolism and are listed in Table 2 (for full details on LC-MS/MS analysis, see Supplementary Table S2). Total serum-EV proteins stained by silver staining are shown in Figure 2A, F95 enriched proteins from serum-EVs are shown in Figure 2B and the number of total EV proteins identified, overlapping with deiminated/citrullinated EV proteins identified are presented in the Venn diagram in Figure 2C.

2.3. Complement Component C3, C4 and Pentraxin-Like Protein Verified in Halibut EVs and F95 Enriched EV Protein Cargo Fractions Using Western Blotting

Three candidate proteins which were identified as part of EV total protein cargo by LC-MS/MS, namely complement component C3, C4 and pentraxin-like protein, were further assessed by western blotting in halibut serum-EVs (Figure 3A–C). Both total EV protein cargo as well as the F95 enriched protein cargo were assessed, using halibut-specific C3, C4 and pentraxin-like protein antibodies, respectively, which had previously been generated and validated in our laboratories [13,42]. Here, complement component C3 was verified to be present in total EV protein cargo, where it was strongly detected by western blotting, as well as at lower levels in the deiminated (F95-enriched) protein cargo (Figure 3A). This confirmed the hits identified by the LC-MS/MS analysis, showing that C3 is exported in EVs both in normal and deiminated form (Table 1 and Table 2). Complement component C4 was also confirmed to be exported in total EV cargo by western blotting, albeit at lower levels than C3, in accordance with the LC-MS/MS findings which identified C4 as a hit in total EV cargo. C4 was seen only at very low levels in deiminated form in the F95-enriched EV fraction by Wwestern blotting (Figure 3B), and was not identified as part of the F95-enriched cargo by LC-MS/MS. Pentraxin-like protein was strongly detected in total EV protein cargo by western blotting, but not in the F95-enriched EV protein fractions (Figure 3C), in accordance with the results from the LC-MS/MS analysis, which only detected pentraxin in total EV cargo (Table 2).

2.4. Protein–Protein Interaction Network Analysis for Halibut Serum-EV Protein Cargo: Deiminated and Total Protein Cargo

2.4.1. Protein Interaction Networks Enriched for Halibut Serum-EV Deiminated/Citrullinated Protein Cargo

For the generation of protein–protein interaction networks to further understanding of putative protein pathways regulated by deimination, deiminated (F95-enriched) protein hits from halibut EVs were assessed by STRING analysis. The protein hits were assessed using the general teleost STRING database, selecting the zebrafish (Danio rerio) database as a model database, as no specific database for halibut is available in STRING and zebrafish showed the highest identity with the teleost protein hits identified as deiminated in halibut serum-EVs. The protein–protein interaction networks showed a PPI enrichment p-value of 5.15 × 10−5, indicating significantly more interactions than expected from a random set of proteins (Figure 4).
Local network clusters enriched in deiminated proteins in EVs included: Histone H3/CENP-A, core histone H2A/H2B/H3/H4 network, post-translational protein phosphorylation and the regulation of IGF transport (Figure 4A).
UniProt keywords for deiminated proteins identified in serum-EVs included methylation, cytoskeleton, disulphide bond, cytoplasm and signalling (Figure 4A).
Reactome pathways enriched in deiminated proteins in the serum EVs included GRB2:SOS linkage to MAPK signalling for integrins, p130Cas linkage to MAPK signalling for integrins, MAP2K and MAPK activation, integrin signalling, the initial triggering of complement, L1CAM interactions, post-translational protein phosphorylation, the regulation of actin dynamics for phagocytic cup formation, the regulation of IGF transport, platelet degranulation, integrin cell surface interactions, cell junction organisation, clathrin-mediated endocytosis, VEGFA-VEGFR2 pathway, extracellular matrix organisation, developmental biology, innate immune system and neutrophil degranulation (Figure 4B).
PFAM protein domains for deiminated proteins identified in the serum EVs included alpha-macro-globulin tiolester bond-forming region, alpha-2-macroglobulin family N-terminal region, MG2 domain, alpha-2 macroglobulin family, a-macroglobulin complement component, a-macroglobulin receptor, UNC-6/NTR/C345C module, core histone H2A/H2B/H3/H4 and trypsin (Figure 4C).
SMART protein domains for deiminated EV proteins included alpha-macroglobulin family, alpha-2-macroglobulin, a-macroglobulin receptor, kazal type serine protease inhibitors, domains found in plexins, semaphorins and integrins and trypsin-like serine protease (Figure 4D).
Protein domains and features (InterPro) for deiminated proteins in serum-EVs included macroglobulin domain MG4 and MG3, tissue inhibitor of metalloproteinases-like, OB-fold, netrin module, non-TIMP type, netrin domain, PSI domain, peptidase S1A, chymotrypsin family, peptidase S1 PA clan, serine proteases trypsin family, histidine active site and serine active site (Figure 4E).

2.4.2. Protein Interaction Networks Enriched for Halibut Serum-EV Total Protein Cargo

The same approach for the generation of protein–protein interaction networks, selecting the zebrafish (D. rerio) STRING database as a representative database for teleost fish, was also applied for total protein EV cargo identified in halibut, showing a PPI enrichment p-value: <1.0 × 10−16 for the protein networks generated, indicating significantly more interactions than expected from a random set of proteins (Figure 5).
Local network clusters for total EV protein content included fibrinogen family, fibrinolysis, common pathway of fibrin clot formation, clotting cascade, ApoM domain, selenoprotein P, histone H3/CENP-A, histone H4, terminal pathway of complement, alternative complement activation, MG2 domain, terminal complement pathway, lectin pathway, plakophilin/delta catenin desmosomal, regulation of IGF transport, adherens junctions interactions, MHC class II antigen, MHC class II antigen presentation, post-translational protein phosphorylation, Histone H4, and core histone H2A/H2B/H3/H4 (Figure 5A).
Reactome pathways for total EV protein cargo included LDL remodelling, plasma lipoprotein assembly, remodelling and clearance, innate immune system, Toll-like receptor cascades, neutrophil degranulation, the regulation of complement cascade, terminal complement pathway, the activation of C3 and C5, the initial triggering of complement, platelet degranulation, platelet activation, GRB2:SOS linkage to MAPK, integrin signalling, integrin cell surface interactions, p130Cas linkage to MAPK signalling for integrins, MAP2K and MAPK activation, the activation of matrix metalloproteinases, chylomicron assembly, the common pathway of fibrin clot formation, the intrinsic pathway of fibrin clot formation, the formation of fibrin clot, clotting cascade, platelet aggregation (plug formation), the formation of the cornified envelope, the regulation of IGF transport, the binding and uptake of ligands by scavenger receptors, collagen degradation, the metabolism of vitamins and cofactors, retinoid metabolism and transport, clathrin-mediated endocytosis, peptide ligand-binding receptors, extracellular matrix organisation, G alpha signalling events, hemostasis, signalling and aggregation, developmental biology, GPCR downstream signalling, post-translational protein modification, signal transduction, metabolism of proteins (Figure 5B).
UniProt keywords for total EV protein content included methylation, kringle, nucleosome core, serine protease, secreted, chromosome, protease, disulphide bond, signalling (Figure 5C).
PFAM protein domains for total EV protein cargo included fibrinogen alpha/beta chain family, anaphylatoxin-like domain, vault protein inter-alpha-trypsin domain, MG2 domain, alpha-2-macroglobulin family, alpha-2-macroglobulin complement component, lipoprotein amino terminal region, UNC-6/NTR/C345C module, kringle domain, domain of unknown function (DUF1943), vWf type A domain, vWf type D domain, hemopexin, FYVE zinc finger, fibrinogen beta, gamma chains, C-terminal globular domain, trypsin-like peptidase domain, CUB domain, core histone H2A/H2B/H3/H4, and trypsin (Figure 5D).
SMART protein domains for total EV protein cargo included fibrinogen alpha/beta chain family, anaphylatoxin homologous domain, vault protein inter-alpha-trypsin domain, kringle domain, lipoprotein N-terminal domain, netrin C-terminal domain, large open beta-sheet protein family, alpha-2-macroglobulin family, alpha-2-macroglobulin receptor, vWf type A domain, vWf type D domain, hemopexin-like repeats, protein present in Fab1, YOTB, Vac1 and EEA1, fibrinogen related domains (FReDs), kazal type serine protease inhibitors, domain first found in C1r, C1s, uEGF and bone morphogenesis, and trypsin-like serine protease (Figure 5E).
Protein domains and features (InterPro) identified for total EV cargo included fibrinogen alpha/beta/gamma chain coiled-coil domain, complement C3/4/5, MG1 domain, anaphylatoxin, anaphylatoxin/fibulin, complement system, macroglobulin domain, alpha-macroglobulin TED domain, alpha-2-macroglobulin, VIT domain, kringle superfamily, terpenoid cyclases/protein prenyltransferase alpha-alpha toroid, zinc finger, FYVE related, FYVE zinc finger, immunoglobulin-like fold, lipid transport protein, beta-sheet shell, lipovitellin-phosvitin complex, superhelical domain, lipid transport protein, netrin domain, netrin module non-TIMP type, vWf type A and vWf type D domain, tissue inhibitor of metalloproteinases-like OB-fold, histone H2A/H2B/H3, histone-fold, peptidase S1, PA clam, fibrinogen alpha/beta/gamma chain C-terminal globular domain, fibrinogen-like C-terminal, serine proteases, trypsin family serine active site and histidine active site, sushi/SCR/CCP superfamily, and peptidase S1A chymotrypsin family (Figure 5F).

3. Discussion

This is the first study to assess EV profile signatures in halibut biofluids, identifying both total serum-EV protein cargo as well as deiminated protein cargo in serum-EVs. The size profiling of halibut serum-EVs by NTA showed vesicles in the range of 50–600 nm, which indicates a higher amount of larger EVs compared with human EVs, which typically fall in the size range of 30–300 nm. In comparison, while few teleost fish have been profiled for EVs, cod (Gadus morhua), serum-EVs were found to be in the size range of mainly 50–300 nm [33,38], while cod mucus-EVs are in the size range of 50–500 nm [32]. In other taxa across the phylogeny tree, differences in plasma or serum EV size profiles have indeed been reported. In elasmobranches (nurse shark Ginglymostoma cirratum) a higher abundance of small EVs in the 10–200 nm size range was observed [14]; in a group of eight pelagic seabird species, some species-specific differences were reported showing plasma-EVs at 50–200 nm size range for some birds and others showing larger EVs at 250–500 nm [37], while in reptile (alligator—Alligator mississippiesis), plasma EVs were in the size range of 50–400 nm [16]. In llama (Lama glama), plasma-EVs were reported at 40–400 nm [34], while Bos taurus plasma-EV showed size profiles of 70–500 nm [35]. Naked mole-rat (Heterocephelus glaber) plasma shows similar EV size profiles as human plasma at 50–300 nm [38], as does rat (Rattus norvegicus) plasma at 50–250 nm [43]. In sea mammals, such as pinnipeds and cetaceans, serum-EVs were observed at 50–600 nm in seals [40], similar to as observed in halibut in the current study. In four species of whale, EV profiles were seen in the ranges of 50–500 (minke whale Balaenoptera acutorostrata), 50–400 (fin whale Balaenoptera physalus), 80–300 (humpback whale Megaptera novaeangliae) and 90–300 nm (Cuvier’s beaked whale Ziphius cavirostris), respectively, while orca serum-EVs (Orcinus orca; dolphin family) were reported at 30–500 nm [39]. Reports of EV profiling of haemolymph from species lower in the phylogeny tree include Crustacea (lobster Homarus americanus) with EVs in the 10–500 nm size range (with the majority of EVs being small in the 22–115 nm size range) [22]; Mollusca haemolymph EVs at 50–300 nm (blue mussel, Mytilus edulis), 30–300 nm (soft shell clam Mya arenaria), 90–500 nm (Eastern oyster Crassostrea virginica) and 20–300 nm (Atlantic jacknife clam Ensis leei), respectively [24]; Arthropoda (horseshoe crab Limulus polyphemus) EVs at 20–400 nm (with the majority of EVs falling within 40–123 nm) [23]. In the protozoa Giardia intestinalis, two distinct size populations of EVs have been described (20–80 nm and 100–400 nm, respectively), which display different functions in host–pathogen interactions [21]. In Gram-negative and Gram-positive bacteria, with EV profiles described at 10–600 nm and 60–400 nm, respectively, EV profiles were shown to change in response to drug-treatment both with respect to size profile and EV cargo content [19,44]. This does indicate that EV size profiles differ between taxa and this may, amongst others, also have effects on EV cargo content, including proteomic, post-translationally modified proteomic cargo, as well as other genomic and non-coding RNA and mitochondrial-derived cargo [45]. Indeed, in teleost, it has been reported that changes in EV numbers and EV deimination protein and microRNA cargo can be a biomarker for environmental temperature factors [33] and, in response to other stressors, teleost plasma EVs have been found enriched with Hsp70 [46] and selected micro-RNAs [47]. In human parasitic disease, EV profiles can also be indicative of infection status [48]. Therefore, the characterisation of EVs across a wide range of taxa further highlights their potential for biomarker application or “EV-fingerprinting” for the assessment of animal health.
Analysing both whole proteomic and the deiminated protein content of halibut serum-EVs in the current study, some differences were found in protein-interaction pathways, while overall both the whole proteome and the EV-citrullinome involved a number of immune, metabolic and gene regulatory pathways.
When assessing protein-protein interaction networks for EVs enriched in deiminated proteins, these related to local network clusters for deiminated proteins in serum-EVs included histone H3/CENP-A, core histone H2A/H2B/H3/H4 network, post-translational protein phosphorylation and the regulation of IGF transport. In relation to such networks, UniProt keywords for deiminated proteins identified in serum-EVs included methylation, cytoskeleton, disulphide bond, cytoplasm and signalling. Reactome pathways enriched in deiminated proteins in the serum EVs included GRB2:SOS linkage to MAPK signalling for integrins, p130Cas linkage to MAPK signalling for integrins, MAP2K and MAPK activation, integrin signalling, initial triggering of complement, L1CAM interactions, post-translational protein phosphorylation, regulation of actin dynamics for phagocytic cup formation, regulation of IGF transport, platelet degranulation, integrin cell surface interactions, cell junction organisation, clathrin-mediated endocytosis, VEGFA–VEGFR2 pathway, extracellular matrix organisation, developmental biology, innate immune system and neutrophil degranulation. Correspondingly, PFAM protein domains for deiminated proteins identified in the serum EVs included alpha-macro-globulin tiolester bond-forming region, alpha-2-macroglobulin family N-terminal region, MG2 domain, alpha-2 macroglobulin family, alpha-macroglobulin complement component, alpha-macroglobulin receptor, UNC-6/NTR/C345C module, core histone H2A/H2B/H3/H4 and trypsin. SMART protein domains for deiminated EV proteins included alpha-macroglobulin family, alpha-2-macroglobulin, alpha-macroglobulin receptor, kazal-type serine protease inhibitors, domains found in plexins, semaphorins and integrins and trypsin-like serine. Protein domains and features (InterPro) for deiminated proteins in serum-EVs included macroglobulin domain MG4 and MG3, the tissue inhibitor of metalloproteinases-like OB-fold, netrin module, non-TIMP type, netrin domain, PSI domain, peptidase S1A, chymotrypsin family, peptidase S1 PA clan, serine proteases trypsin family, histidine active site and serine active site.
In comparison with deiminated EV protein content, more pathways were revealed for serum-EV total protein content, as would be expected due to only some of the proteins in the EV cargo being candidates for post-translational deimination and exported in EVs in deiminated form. Assessing protein interaction networks for total protein EV content showed local network clusters for fibrinogen family, fibrinolysis, the common pathway of fibrin clot formation, clotting cascade, ApoM domain, selenoprotein P, histone H3/CENP-A, histone H4, the terminal pathway of complement, alternative complement activation, MG2 domain, terminal complement pathway, lectin pathway, plakophilin/delta catenin desmosomal, the regulation of IGF transport, adherens junctions interactions, MHC class II antigen, MHC class II antigen presentation, post-translational protein phosphorylation, Histone H4, and core histone H2A/H2B/H3/H4.
The reactome pathways for total EV protein cargo included LDL remodelling, plasma lipoprotein assembly, remodelling and clearance, innate immune system, Toll-like receptor cascades, neutrophil degranulation, the regulation of the complement cascade, the terminal complement pathway, the activation of C3 and C5, the initial triggering of complement, platelet degranulation, platelet activation, GRB2:SOS linkage to MAPK, integrin signalling, integrin cell surface interactions, p130Cas linkage to MAPK signalling for integrins, MAP2K and MAPK activation, the activation of matrix metalloproteinases, chylomicron assembly, the common pathway of fibrin clot formation, the intrinsic pathway of fibrin clot formation, the formation of fibrin clot, clotting cascade, platelet aggregation (plug formation), the formation of the cornified envelope, the regulation of IGF transport, the binding and uptake of ligands by scavenger receptors, collagen degradation, the metabolism of vitamins and cofactors, retinoid metabolism and transport, clathrin-mediated endocytosis, peptide ligand-binding receptors, extracellular matrix organisation, G alpha signalling events, hemostasis, signalling and aggregation, developmental biology, GPCR downstream signalling, post-translational protein modification, signal transduction, and the metabolism of proteins.
UniProt keywords for total EV protein content included methylation, kringle, nucleosome core, serine protease, secreted, chromosome, protease, disulphide bond, signalling.
PFAM protein domains for total EV protein cargo included fibrinogen alpha/beta chain family, anaphylatoxin-like domain, vault protein inter-alpha-trypsin domain, MG2 domain, alpha-2-macroglobulin family, alpha-2-macroglobulin complement component, lipoprotein amino terminal region, UNC-6/NTR/C345C module, kringle domain, the domain of unknown function (DUF1943), vWf type A domain, vWf type D domain, hemopexin, FYVE zinc finger, fibrinogen beta, gamma chains, C-terminal globular domain, trypsin-like peptidase domain, CUB domain, core histone H2A/H2B/H3/H4, and trypsin.
SMART protein domains for total EV protein cargo included fibrinogen alpha/beta chain family, anaphylatoxin homologous domain, vault protein inter-alpha-trypsin domain, kringle domain, lipoprotein N-terminal domain, netrin C-terminal domain, large open beta-sheet protein family, alpha-2-macroglobulin family, alpha-2-macroglobulin receptor, vWf type A domain, vWf type D domain, hemopexin-like repeats, protein present in Fab1, YOTB, Vac1 and EEA1, fibrinogen related domains (FReDs), kazal type serine protease inhibitors, domain first found in C1r, C1s, uEGF and bone morphogenesis, and trypsin-like serine protease.
Protein domains and features (InterPro) identified for total EV protein cargo included fibrinogen alpha/beta/gamma chain coiled-coil domain, complement C3/4/5, MG1 domain, anaphylatoxin, anaphylatoxin/fibulin, complement system, macroglobulin domain, alpha-macroglobulin TED domain, alpha-2-macroglobulin, VIT domain, kringle superfamily, terpenoid cyclases/protein prenyltransferase alpha-alpha toroid, zinc finger, FYVE related, FYVE zinc finger, immunoglobulin-like fold, lipid transport protein, beta-sheet shell, lipovitellin-phosvitin complex, superhelical domain, lipid transport protein, netrin domain, netrin module non-TIMP type, vWf type A and vWf type D domain, the tissue inhibitor of metalloproteinases-like OB-fold, histone H2A/H2B/H3, histone-fold, peptidase S1, PA clam, fibrinogen alpha/beta/gamma chain C-terminal globular domain, fibrinogen-like C-terminal, serine proteases, trypsin family serine active site and histidine active site, sushi/SCR/CCP superfamily, peptidase S1A chymotrypsin family.
Proteomic analysis using LC-MS/MS, identified a range of innate and adaptive immune proteins to be exported in serum-EVs, including in deiminated form, as listed above. This also included a range of complement components, whereof C3 and C5 were detected as deiminated in serum EVs, while in total EV cargo, C1, C3, C4, C5, C6, C7, C8 and C9 were also identified as hits, as well as factor B and factor H. This correlates with previous findings reporting C3 to be deiminated in teleost fish, both in halibut and cod [13,32,33]. Furthermore, a proteomic analysis of deiminated target proteins in halibut serum identified C5, C7, C8 C9 and C1-inhibitor to be deiminated in whole halibut serum [13]. These findings, and the current study, indicate that not all complement components are exported in EVs in deiminated form, and some are found in deiminated form only in whole serum, while being exported in non-deiminated form in serum-EVs. Recent studies assessing protein deimination across the phylogeny tree have indeed identified various complement components as deimination candidates in a range of taxa [14,16,32,33,34,35,37,39,40]. Furthermore, C5 has been verified to be a deimination candidate by bacterial arginine deiminase, allowing for immune modulation of the host and bacterial immune evasion [18].
In the current study we furthermore evaluated by western blotting some key complement proteins identified by LC/MS-MS in EV total protein cargo and deimination-enriched protein cargo. For this purpose, we used halibut-specific antibodies against C3, C4 and pentraxin-like protein, previously developed and described by our group [13,42]. Using western blotting analysis, we verified the presence of C3, C4 and pentraxin-like protein in halibut serum-EVs, showing that these are indeed exported in EVs, as also identified by LC-MS/MS anlaysis. The C3 antibody also reacted strongly with the F95 enriched protein eluate from the serum EVs, while a lower signal was seen for C4, indicating that C3 is present at higher levels in deiminated form in serum-EVs, compared with C4. Pentraxin-like protein was only observed in total protein cargo of serum-EVs, but not the F95 enriched serum-EV eluate and this corresponds with the LC-MS/MS analysis which revealed hits with a pentaxin for the total protein cargo analysis of serum-EVs, but not the F95-enriched fraction. Our current findings in halibut serum-EV cargo also correspond to our previous analysis on serum EVs and mucus EVs in Atlantic cod, where C3 was detected at higher levels in serum-EVs than C4, both for total protein as well as in the F95-enriched eluate for a putative deiminated form [32,33,38]. Furthermore, cod serum and mucus EVs were also found to contain pentraxin-like protein (CRP-like), which was not detected in deiminated form in the cod EVs, similar to as observed for pentraxin-like protein in halibut serum-EVs in the current study.
Overall, our and others’ findings indicate that the complement system can be modulated by deimination both by the host and by pathogen interactions. Understanding of post-translational regulation of complement components via deimination is still in its infancy and requires in depth investigation as deimination may facilitate multifaceted functions of complement proteins in immunity and tissue remodelling in health and disease, also across phylogeny. Such regulation via deimination may furthermore allow for targeted modulation in relation to a range of pathological processes, including infection and autoimmune diseases, where PADs, EVs and the complement system all play important roles.
Besides differences in EV cargo for complement components, proteins that were only identified in whole protein cargo (and not in the F95 eluate) related to a range of innate and adaptive immune factors as well as metabolic and gene regulatory function. These included Apolipoprotein Bb, Apolipoprotein M, Ig-like domain-containing protein, Ig heavy chain Mem5-like, IGv domain-containing protein, Immunoglobulin light chain, nattectin, SERPIN domain-containing protein, Lysozyme, ceruloplasmin, vitellogenin, apoptosis-stimulating of p53 protein 2 Bcl2-binding protein, plasminogen, keratin, type I cytoskeletal 13-like, EGF-like domain-containing protein, hephaestin-like protein 1, desmoglein-2, carboxypeptidase Q, beta 1-globin, antithrombin-III, collagen alpha-1(XII) chain, desmoplakin, biotinidase, collagenase 3, cathepsin L1-like, prothrombin, putative insulin-like growth factor binding protein, sushi domain-containing protein 2 isoform 2, 14_3_3 domain-containing protein, catechol O-methyltransferase domain-containing protein 1, pleckstrin, hyaluronan-binding protein 2, retrotransposon-derived, FH2 domain-containing protein 1-like, protein-tyrosine-phosphatase, thyroid hormone receptor interactor 11, roundabout-like axon guidance receptor protein 2, protein Z-dependent protease inhibitor-like, myosin phosphatase Rho interacting protein, multidrug and toxin extrusion protein, FH2 domain containing 4, nesprin-2, histone H3-trimethyl-L-lysine(9) demethylase, centrosomal protein of 290 kDa and titin-like protein. These all relate to the protein-interaction networks identified for whole EV protein cargo, listed above and shown in Figure 5.
Furthermore, some protein candidates were only detected in the F95 eluate, indicating that they were exported in deiminated form only in the serum EVs. This included cytoplasmic 2 actin, tubulin alpha chain, keratin 93, trypsin-3-like, centrosomal protein of 162 kDa, 2-phospho-D-glycerate hydro-lyase, integrin beta and myosin_tail_1 domain-containing protein. Compared with a previous analysis from our group on deiminated proteins in whole halibut serum [13], deiminated candidates found here to be exported specifically in EVs are cytoplasmic 2 actin, tubulin alpha chain, centrosomal protein of 162 kDa, 2-phospho-D-glycerate hydro-lyase, integrin beta and myosin_tail_1 domain-containing protein. This indicates that there are differences in deiminated protein cargo in serum-EVs compared with whole serum, and this corresponds to findings from other comparative studies analysing differences in KEGG (Kyoto encyclopedia of genes and genomes) and GO (gene ontology) enrichment pathways for deiminated proteins in whole serum/plasma versus EVs in diverse taxa, including in cow, camelid, alligator, rat, naked mole-rat, shark and cod [14,16,33,34,35,36,43]. Furthermore, differences in deimination signatures in whole serum versus EV cargo have been reported to relate to immune/growth trade off in response to environmental temperature in teleost cod [33]. Such findings, including the findings reported in our current study, emphasise that variations in EV cargo, including via the transport of deiminated proteins, may play hitherto under-recognized and important roles in cellular communication in health and disease across the phylogeny tree.

4. Materials and Methods

4.1. Fish and Sampling

Blood was collected from four adult halibut (Hippoglossus hippoglossus L.; weight 4.5–5.0 kg), which were obtained from the experimental fish farm Fiskeldi Eyjafjardar hf, Thorlakshofn, Iceland (under licence from the Institute for Experimental Pathology, University of Iceland, number #0002 kt-650269—4549, approved by the central animal ethics committee in Iceland (Icelandic Food Regulation Authority, MAST Matvælastofnun). Following 1–3 mL blood collection from a gill vessel, the blot was left to clot overnight at 4 °C, and thereafter serum collection was performed by centrifugation at 750 g for 10 min. Serum aliquots of 200 µL were stored at −20 °C until used. The health status of the fish at the fish farm was routinely examined at regular 3 monthly intervals by the Fish Disease Laboratory, Institute for Experimental Pathology, Keldur, Iceland, declaring the fish healthy and disease free.

4.2. EV Isolation and Nanoparticle Tracking (NTA) Analysis

EVs were isolated from halibut serum of four individual fish by step-wise centrifugation, according to previously established methods in our laboratory [14,22,39] and according to the guidelines of the International Society for Extracellular Vesicles (ISEV) [49]. Total EV isolates were prepared from the individual 100 μL serum aliquots (n = 4), which were diluted 1:5 in Dulbecco’s PBS (DPBS, which had previously been ultrafiltered using a 0.22 μm filter, before use) and then centrifuged at 4000 g for 30 min at 4 °C, to ensure the removal of aggregates and apoptotic bodies. The supernatants containing the EVs were collected and ultracentrifuged at 100,000 g for 1 h at 4 °C. The EV-enriched pellets were then resuspended in 1 mL DPBS (“washing step”) and ultracentrifuged again at 100,000 g for 1 h at 4 °C. The final EV-enriched pellets were then resuspended in 100 µL DPBS and analysed by NTA for size distribution profiles, using the NanoSight NS300 system (Malvern Panalytical Ltd, Malvern, UK), recording five 60 s videos for each sample. The number of particles per frame was kept in-between 40 to 60 and replicate histograms were generated from the videos, using the NanoSight software 3.0 (Malvern), representing mean and confidence intervals of the five recordings for each sample.

4.3. Transmission Electron Microscopy (TEM)

EVs were further characterised by Transmission Electron Microscopy (TEM) as follows: A pool of EVs, isolated from serum of the four individual animals as described above, was used for morphological analysis using TEM according to previously described methods [16,23,34]. In brief, 100 mM sodium cacodylate buffer (pH 7.4) was used to resuspend the EVs, which were then placed onto a glow discharged carbon support film on a grid and fixed at room temperature for 1 min in 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0). For the staining of EVs, 2% aqueous Uranyl Acetate (Sigma, Gillingham, UK) was used for 1 min, thereafter removing the excess stain. EV imaging was performed using a JEOL JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV at a magnification of 30,000× to 60,000×. Digital images were recorded using an AMT XR60 CCD camera (Deben, Bury St. Edmunds, UK).

4.4. Proteomic Analysis and Protein Identification

The isolation of deiminated/citrullinated proteins from serum-EVs was carried out by immunoprecipitation, using the Catch and Release® v2.0 Reversible Immunoprecipitation System (Merck, Feltham, UK) according to the manufacturer’s instructions in conjunction with the pan-deimination F95 antibody (MABN328, Merck), which specifically detects proteins modified by citrullination [50]. F95 enrichment was performed overnight at 4 °C on a rotating platform from a pool of sera (n = 4 individuals), followed by the elution of the F95 bound proteins under reducing conditions, according to the manufacturer’s instructions (Merck). The F95 eluate was diluted in 2 × Laemmli sample buffer for subsequent SDS-PAGE and western blotting analysis. The total F95 bound protein eluate, as well as total protein from serum-EVs, were also analysed by liquid chromatography–mass spectrometry (LC-MS/MS) (performed by Cambridge Centre for Proteomics, Cambridge, UK), by in-gel digestion, as previously described [32,34]. For the identification of deiminated protein hits, the files were submitted to the Mascot search algorithm (Matrix Science, London, UK) and searched against the UniProt database for Teleostei (CCP_Teleostei Teleostei_20201009; 4085639 sequences; 2121030378 residues). A search was also conducted against a common contaminant database (cRAP 20190401; 125 sequences; 41,129 residues). A significance threshold value of p < 0.05 and a peptide cut-off score of 53 were also applied (carried out by Cambridge Proteomics, Cambridge, UK).
In addition to the LC-MS/MS analysis, both total EV proteins and F95 enriched EV proteins were assessed specifically for halibut C3, C4 and pentraxin-like protein content, using mono-specific antibodies, which were previously prepared against these proteins by our group [13,42] (see Section 4.5).

4.5. Western Blotting

Serum EVs were pooled (n = 4), reconstituted 1:1 in 2 × Laemmli sample buffer and boiled at 100 °C for 5 min before separation by SDS-PAGE, using 4–20% TGX gels (BioRad, Watford, UK). Proteins were blotted onto 0.45 μm nitrocellulose membranes (BioRad, UK) using semi-dry transfer for 1 h at 15 V and even protein transfer was assessed using Ponceau S (Sigma, Gillingham, UK) staining. Membranes were blocked for 1 h at RT in 5% bovine serum albumin (BSA, Sigma) in Tris-buffered saline containing Tween20 (TBS-T). Primary antibody incubation was performed overnight at 4 °C on a shaking platform, diluting the antibodies in TBS-T. EVs were assessed for the EV-specific markers Flotillin-1 (ab41927, 1/1000) and CD63 (ab216130, 1/1000). EV cargo was assessed for halibut pentraxin-like protein (1/1000; [13]), halibut C3 (1/1000; [42]) and halibut C4 (1/1000; [13]), using halibut-specific antibodies previously generated in our laboratory [13,42]. Following primary antibody incubation, the membranes were washed three times in TBS-T and then incubated at room temperature for 1h in either the corresponding anti-mouse IgG (for anti-pentraxin, anti-C3 and anti-C4 antibodies) or anti-rabbit IgG (for CD63 and Flot-1 antibodies) HRP-conjugated secondary antibodies (1/3000; BioRad). Thereafter, the membranes were washed in TBS-T five times for 10 min and then visualised using a UVP BioDoc-ITTM System (Cambridge, UK) in conjunction with ECL (Amersham, Merck, UK).

4.6. Silver Staining

Total proteins isolated from serum EVs and the F95-enriched protein eluates from halibut serum EVs were assessed by silver staining following SDS-PAGE in 4–20% gradient TGX gels (BioRad) under reducing conditions. The BioRad Silver Stain Plus Kit (1610449, BioRad) was used to visualise the protein bands according to the manufacturer’s instructions (BioRad).

4.7. Protein–Protein Interaction Network Analysis

For the construction of protein–protein interaction networks for deiminated proteins identified in halibut serum-EVs and for total protein content from serum EVs, respectively, STRING analysis (Search Tool for the Retrieval of Interacting Genes/Proteins; https://string-db.org/) was applied. The protein networks were built based on the protein names, using the teleost fish STRING database and using the function of “search multiple proteins”. Settings were as “basic” and “medium confidence”. Colour lines connecting the nodes represent the following evidence-based interactions for the network edges: “known interactions” (this is based on experimentally determined data or curated databases); “predicted interactions” (this is based on gene co-occurrence, gene neighbourhood or gene fusion); “others” (this is based on co-expression, text mining or protein homology (see colour key for lines in Figure 4A). Networks were assessed for local network clusters, reactome pathways, PFAM and SMART protein domains and UniProt keywords. The zebrafish (Danio rerio) STRING database was used as representative for Teleostei for the creation of the networks as no specific halibut STRING database is available (due to lack of annotation available for halibut), and D. rerio showed the most hit number identity with the proteins identified in halibut EVs.

4.8. Statistical Analysis

The Nanosight 3.0 software (Malvern) was used for the generation of NTA curves, which represent mean and standard error of mean (SEM), indicated by confidence intervals. Significance for protein network analysis generated in STRING (https://string-db.org/) was considered as p ≤ 0.05.

5. Conclusions

This study is the first report of EV profile signatures in halibut, analysing total protein and specifically also deiminated protein cargo in serum-EVs. Halibut serum EVs showed a poly-dispersed population with EVs in the size range of 50–600 nm, positive for phylogenetically conserved EV markers. Proteomic analysis of EV total protein cargo revealed 124 protein hits and 37 deiminated protein hits, whereof 15 hits were particularly identified in deiminated form only. Protein interaction network analysis revealed GO pathways for EV mediated protein cargo transport, relating to a range of gene regulatory, immune, metabolic and developmental processes, some of which were enriched for deiminated proteins. Further assessment of key immune related proteins—complement components C3, C4 and pentraxin—identified that C3 is exported in serum-EVs at higher levels than C4, also in deiminated form, while pentraxin was found in whole protein EV content only, but not in deiminated form. Our findings emphasize the putative differences in cell communication mediated by EV protein versus post-translationally deiminated protein cargo (the “EV-citrullinome”), providing novel insights into EV-mediated communication in halibut serum. Our findings furthermore contribute to current understanding of EV signatures across the phylogeny tree, with the potential for biomarker development and EV “fingerprinting” for the assessment of animal health.

Supplementary Materials

The following are available online at https://www.mdpi.com/1422-0067/22/2/875/s1, Table S1: Deiminated proteins in serum-EVs of halibut (Hippoglossus hippoglossus L.), as identified by F95-enrichment in conjunction with LC-MS/MS analysis; full LC-MS/MS data. Table S2: Total proteins in serum-EVs of halibut (Hippoglossus hippoglossus L.); full LC-MS/MS data.

Author Contributions

Conceptualization, S.L.; methodology, S.L., A.W.D. and I.K.; validation, B.M., I.K., A.W.D. and S.L.; formal analysis, I.K. and S.L.; investigation, B.M. and S.L.; resources, B.M., I.K., A.W.D. and S.L.; data curation, S.L.; writing—original draft preparation, S.L.; writing—review and editing, B.M., A.W.D. and S.L.; visualization, I.K. and S.L.; supervision, S.L.; project administration, S.L.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work is partly based on previous support by the EC grant Fishaid QLK2-CT-2000-01076 (B.M. and S.L.), the Icelandic Ministry of Fisheries (to S.L.), the Icelandic Research Council (to S.L.) and the European Molecular Biology Organization (to S.L.). The work was furthermore supported by internal funding from the University of Westminster (to S.L.).

Institutional Review Board Statement

The study was conducted under license from the Institute for Experimental Pathology, University of Iceland, number #0002 kt-650269–4549, approved by the central animal ethics committee in Iceland (Icelandic Food Regulation Authority, MAST Matvælastofnun).

Data Availability Statement

Data is contained within the article and supplementary material.

Acknowledgments

The authors wish to thank Birgir Kristjánsson and the staff at Fiskeldi Eyjafjardar, Þorlákshöfn, Iceland, and the staff at Fiskey hf, Hjalteyri, Iceland for providing the halibut and sampling facilities. Thanks to Michael Deery and Yagnesh Umrania at the Cambridge Centre for Proteomics for performing the LC-MS/MS analysis. Thanks are due to The Guy Foundation for funding the purchase of equipment utilised in this work.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Halibut serum-extracellular vesicles (EV)s were characterised by: (A) Nanoparticle tracking analysis (NTA), showing size distribution profiles of EVs in the size range of 50–600 nm, with the modal size of vesicles at 138 nm; (B) Western blotting (WB) analysis shows that the EVs are positive for CD63 and Flotillin-1; (C) Transmission electron microscopy (TEM) showing EV morphology—see arrows pointing at EVs (scale bar is indicated at 20 nm).
Figure 1. Halibut serum-extracellular vesicles (EV)s were characterised by: (A) Nanoparticle tracking analysis (NTA), showing size distribution profiles of EVs in the size range of 50–600 nm, with the modal size of vesicles at 138 nm; (B) Western blotting (WB) analysis shows that the EVs are positive for CD63 and Flotillin-1; (C) Transmission electron microscopy (TEM) showing EV morphology—see arrows pointing at EVs (scale bar is indicated at 20 nm).
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Figure 2. The proteome and citrullinome of halibut serum-EVs. Silver-stained gels for: (A) total protein cargo in EVs and (B) F95 enriched (deiminated/citrullinated) proteins from EVs. The protein standard (std) is indicated in kilodaltons (kDa). (C) Venn diagram shows the number of candidate protein hits identified as cargo in total serum EVs (“The serum EV proteome”) as well as deiminated protein hits in EV cargo (the serum “EV citrullinome”).
Figure 2. The proteome and citrullinome of halibut serum-EVs. Silver-stained gels for: (A) total protein cargo in EVs and (B) F95 enriched (deiminated/citrullinated) proteins from EVs. The protein standard (std) is indicated in kilodaltons (kDa). (C) Venn diagram shows the number of candidate protein hits identified as cargo in total serum EVs (“The serum EV proteome”) as well as deiminated protein hits in EV cargo (the serum “EV citrullinome”).
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Figure 3. Complement component C3, C4 and pentraxin-like protein in halibut EVs and F95 enriched EV fractions. Western blotting showing (A) complement component C3 detection in total protein cargo of halibut serum-EVs (“EVs”) and in F95-enriched protein fractions from serum-EVs (“EVs_F95”), C3 α- and β-chains, as well as α-fragment (α-f) are indicated; (B) complement component C4 detection in total protein cargo of serum-EVs (“EVs”) and lower detection observed in F95-enriched EV protein fractions (“EVs_F95”), C4 α-, β- and γ-chains are indicated; (C) pentraxin-like protein detection in total EV protein cargo (“EVs”), which was not detected in the F95-enriched EV protein fractions (“EVs_F95”).
Figure 3. Complement component C3, C4 and pentraxin-like protein in halibut EVs and F95 enriched EV fractions. Western blotting showing (A) complement component C3 detection in total protein cargo of halibut serum-EVs (“EVs”) and in F95-enriched protein fractions from serum-EVs (“EVs_F95”), C3 α- and β-chains, as well as α-fragment (α-f) are indicated; (B) complement component C4 detection in total protein cargo of serum-EVs (“EVs”) and lower detection observed in F95-enriched EV protein fractions (“EVs_F95”), C4 α-, β- and γ-chains are indicated; (C) pentraxin-like protein detection in total EV protein cargo (“EVs”), which was not detected in the F95-enriched EV protein fractions (“EVs_F95”).
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Figure 4. (A) Protein interaction networks for deiminated proteins in halibut EVs. Local network clusters and UniProt keywords are indicated by the colour coded nodes. See colour key for nodes and interaction networks in the figure. (B) Reactome protein interaction networks for deiminated proteins in halibut EVs. Reactome pathways are indicated by the coloured nodes, as shown in the figure. (C,D) PFAM and SMART protein interaction networks for deiminated proteins in halibut EVs. PFAM and SMART protein domains are indicated by the coloured nodes, see colour code in the figure. (E) InterPro protein interaction networks for deiminated proteins in halibut EVs. InterPro protein domains and features are indicated by the coloured nodes; see colour code in the figure.
Figure 4. (A) Protein interaction networks for deiminated proteins in halibut EVs. Local network clusters and UniProt keywords are indicated by the colour coded nodes. See colour key for nodes and interaction networks in the figure. (B) Reactome protein interaction networks for deiminated proteins in halibut EVs. Reactome pathways are indicated by the coloured nodes, as shown in the figure. (C,D) PFAM and SMART protein interaction networks for deiminated proteins in halibut EVs. PFAM and SMART protein domains are indicated by the coloured nodes, see colour code in the figure. (E) InterPro protein interaction networks for deiminated proteins in halibut EVs. InterPro protein domains and features are indicated by the coloured nodes; see colour code in the figure.
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Figure 5. (A) Protein interaction networks for total protein cargo in halibut EVs, showing local network clusters. The coloured nodes indicate the different networks, respectively. (B) Reactome protein interaction networks for total proteins in halibut EV cargo, showing reactome pathways. Specific reactome pathways are indicated by the coloured nodes, respectively. (C) UniProt protein interaction networks for total proteins in halibut EV cargo, showing UniProt keywords. UniProt keywords are indicated by the coloured nodes, respectively. (D) PFAM protein interaction networks for total proteins in halibut EV cargo. The specific PFAM protein domains are indicated by the coloured nodes, respectively. (E) Protein interaction networks for total proteins in halibut EVs, showing SMART protein domains. The specific SMART protein domains are indicated by the coloured nodes, respectively. (F) InterPro protein interaction networks for total proteins in halibut EVs. The specific protein domains and features (InterPro) are indicated by the coloured nodes, respectively.
Figure 5. (A) Protein interaction networks for total protein cargo in halibut EVs, showing local network clusters. The coloured nodes indicate the different networks, respectively. (B) Reactome protein interaction networks for total proteins in halibut EV cargo, showing reactome pathways. Specific reactome pathways are indicated by the coloured nodes, respectively. (C) UniProt protein interaction networks for total proteins in halibut EV cargo, showing UniProt keywords. UniProt keywords are indicated by the coloured nodes, respectively. (D) PFAM protein interaction networks for total proteins in halibut EV cargo. The specific PFAM protein domains are indicated by the coloured nodes, respectively. (E) Protein interaction networks for total proteins in halibut EVs, showing SMART protein domains. The specific SMART protein domains are indicated by the coloured nodes, respectively. (F) InterPro protein interaction networks for total proteins in halibut EVs. The specific protein domains and features (InterPro) are indicated by the coloured nodes, respectively.
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Table
Table 1. Deiminated proteins in serum extracellular vesicles (EVs) of halibut (Hippoglossus hippoglossus L), as identified by F95-enrichment in conjunction with LC-MS/MS analysis. Deiminated proteins were isolated from serum-EVs from a pool of n = 4 fish, using immunoprecipitation with the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analysed by LC-MS/MS and peak list files submitted to Mascot, using the Teleost UniProt database. Peptide sequence hits are listed, showing the number of sequences for protein hits and total score. Species hit names are indicated. In the case of uncharacterised protein ID, proteins matching the same set of peptides are indicated in brackets. Protein hits highlighted in pink (*) are specific to the F95 enriched EV fraction only. Protein names are written in bold. A full list of protein sequence hits and peptides is further provided in Supplementary Table S1.
Table 1. Deiminated proteins in serum extracellular vesicles (EVs) of halibut (Hippoglossus hippoglossus L), as identified by F95-enrichment in conjunction with LC-MS/MS analysis. Deiminated proteins were isolated from serum-EVs from a pool of n = 4 fish, using immunoprecipitation with the pan-deimination F95 antibody. The resulting F95-enriched eluate was then analysed by LC-MS/MS and peak list files submitted to Mascot, using the Teleost UniProt database. Peptide sequence hits are listed, showing the number of sequences for protein hits and total score. Species hit names are indicated. In the case of uncharacterised protein ID, proteins matching the same set of peptides are indicated in brackets. Protein hits highlighted in pink (*) are specific to the F95 enriched EV fraction only. Protein names are written in bold. A full list of protein sequence hits and peptides is further provided in Supplementary Table S1.
Protein ID Species NameMatchesTotal Score
Protein NameCommon Name(Sequences)(p < 0.05) ⱡ
A0A6J2W3P0_CHACNChanos chanos16 (13)538
Uncharacterised protein (histone H3-like)Milkfish
A0A672ZYE0_9TELESphaeramia orbicularis12 (9)451
Uncharacterised proteinOrbiculate cardinalfish
A0A0A1G3Q1_9TELE Oxyeleotris marmorata10 (10)427
Beta-actinMarble goby
A0A3P8Y5X6_ESOLUEsox Lucius26 (8)356
IF rod domain-containing proteinNorthern pike
* W5ZLY1_9TELECampylomormyrus compressirostris8 (8)336
Cytoplasmic 2 actinElephantfish
A0A3B4ZTX8_9TELEStegastes partitus8 (7)324
Uncharacterized protein (NTR domain-containing protein; Complement component C3)Bicolour damselfish
A0A3B4THR8_SERDUSeriola dumerili9 (8)299
Uncharacterized protein (NTR domain-containing protein; anaphylatoxin-like, Complement component C3)Greater amberjack
A0A6G0HQ07_LARCRLarimichthys crocea8 (6)281
Histone H4Yellow croaker
A0A3Q3IVX9_MONALMonopterus albus8 (7)276
Uncharacterized protein (Complement C3)Asian swamp eel
A0A3P9BEG5_9CICHMaylandia zebra6 (6)273
Uncharacterized protein (Anaphylatoxin-like, complement C3)Zebra mbuna
A0A484CCU5_PERFVPerca flavescens8 (7)271
Uncharacterized protein (complement C3)Yellow perch
A5JV31_HIPHIHippoglossus hippoglossus7 (7)261
PhosvitinAtlantic halibut
* A0A087XQB5_POEFOPoecilia formosa6 (5)256
Tubulin alpha chainAmazon molly
A0A6F9CZC7_9TELECoregonus sp. ‘balchen’5 (4)251
Uncharacterized protein (tubulin alpha-chain)Whitefish, salmonidae
* Q1RLR3_DANREDanio rerio8 (5)237
Keratin 93Zebrafish
A0A1S5XZE7_9TELELipogramma levinsoni7 (7)231
Histone H3Hourglass basslet
A3F5V1_ORENIOreochromis niloticus7 (7)222
Beta actin (Fragment)Nile tilapia
A0A5N5KJN7_PANHPPangasianodon hypophthalmus6 (4)185
IF rod domain-containing proteinIridescent shark
A0A4W6CP97_LATCALates calcarifer5 (4)179
Uncharacterized protein (Alpha-2-macroglobulin)Barramundi/Asian sea bass
* H2MSJ5_ORYLAOryzias latipes5 (4)159
Uncharacterized proteinMedaka/Japanese rice fish
A0A060WDP8_ONCMYOncorhynchus mykiss3 (3)136
Elongation factor 1-alphaRainbow trout
A0A671UYU7_SPAAUSparus aurata3 (1)117
Uncharacterized protein (A2M_recep domain-containing protein)Gilt-head bream
G3Q4A0_GASACGasterosteus aculeatus2 (2)116
Fibrinogen beta chainThree-spined stickleback
A0A0F8AH88_LARCRLarimichthys crocea2 (2)107
Ig heavy chain V region 5AYellow croaker
A0A4W6FLR7_LATCALates calcarifer3 (3)104
Uncharacterized protein (NTR domain-containing protein; anaphylatoxin like; A2M_N_2 domain-containing; complement C5)Barramundi/Asian sea bass
A0A4Z2B138_9TELETakifugu bimaculatus3 (3)99
Anaphylatoxin-like domain-containing proteinPufferfish
Q4KVK3_HIPHIHippoglossus hippoglossus2 (2)94
Complement component c3 (Fragment)Atlantic halibut
* A0A5J5C7F1_9PEROEtheostoma spectabile2 (2)94
Uncharacterized protein (Fragment)Orangethroat darter
* A0A0P7WL38_SCLFOScleropages formosus4 (2)93
Trypsin-3-likeAsian arowana
Q5DVG8_PLAFEPlatichthys flesus3 (2)84
Apolipoprotein AIEuropean flounder
A0A0F8ABH4_LARCRLarimichthys crocea3 (1)82
Granzyme B(G,H)Yellow croaker
A0A484D989_PERFVPerca flavescens3 (2)71
Peptidase S1 domain-containing proteinYellow perch
* A0A5N5Q536_PANHPPangasianodon hypophthalmus2 (2)70
Centrosomal protein of 162 kDaIridescent shark
* A0A0P7UEW6_SCLFOScleropages formosus1 (1)69
2-phospho-D-glycerate hydro-lyaseAsian arowana
A0A060YWU0_ONCMYOncorhynchus mykiss4 (2)68
Peptidase S1 domain-containing proteinRainbow trout
* A0A1A7WRH6_9TELEIconisemion striatum2 (2)64
Integrin betaKillifish
A0A3B5M528_9TELEXiphophorus couchianus1 (1)64
SerotransferrinMonterrey platyfish
A0A060Z3N3_ONCMYOncorhynchus mykiss2 (2)63
Ig-like domain-containing proteinRainbow trout
A0A060W543_ONCMYOncorhynchus mykiss2 (2)62
Histone H2ARainbow trout
A0A0R4IU44_DANREDanio rerio1 (1)61
Inter-alpha-trypsin inhibitor heavy chain 3bZebrafish
HV05_CARAUCarassius auratus2 (1)60
Ig heavy chain V region 5AGoldfish
* A0A060XD44_ONCMYOncorhynchus mykiss4 (2)60
Uncharacterized proteinRainbow trout
A0A4W5L5T6_9TELEHucho hucho1 (1)57
ThioredoxinDanube salmon
* A0A3Q3LZB0_9TELEMastacembelus armatus1 (1)57
Uncharacterized proteinZig-zag eel/Spiny eel
* A0A5J5DS23_9PEROEtheostoma spectabile1 (1)57
Uncharacterized proteinOrangethroat darter
* A0A3B3QST7_9TELEParamormyrops kingsleyae1 (1)55
Uncharacterized proteinElephantfish
* A0A0E9RVI6_ANGANAnguilla Anguilla1 (1)53
Uncharacterized proteinEuropean eel
* A0A3Q3SSB4_9TELEMastacembelus armatus1 (1)53
* Myosin_tail_1 domain-containing proteinZig-zag eel/Spiny eel
ⱡ Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores > 53 indicate identity or extensive homology (p < 0.05). Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.
Table
Table 2. Total protein cargo in serum-EVs of halibut (Hippoglossus hippoglossus L), as identified by LC-MS/MS analysis from serum-EVs isolated from a pool of sera from n = 4 fish. Peak list files were submitted to Mascot, using the Teleost UniProt database. Peptide sequence hits are listed, showing the number of sequences for protein hits and total score. Species hit names are indicated. In the case of uncharacterised protein ID, proteins matching the same set of peptides are indicated in brackets. Protein hits highlighted in blue (*) were not identified in the F95 enriched fraction. Protein names are written in bold. A full list of protein sequence hits and peptides is further provided in Supplementary Table S2.
Table 2. Total protein cargo in serum-EVs of halibut (Hippoglossus hippoglossus L), as identified by LC-MS/MS analysis from serum-EVs isolated from a pool of sera from n = 4 fish. Peak list files were submitted to Mascot, using the Teleost UniProt database. Peptide sequence hits are listed, showing the number of sequences for protein hits and total score. Species hit names are indicated. In the case of uncharacterised protein ID, proteins matching the same set of peptides are indicated in brackets. Protein hits highlighted in blue (*) were not identified in the F95 enriched fraction. Protein names are written in bold. A full list of protein sequence hits and peptides is further provided in Supplementary Table S2.
Protein ID Species NameMatchesTotal Score
Protein NameCommon Name(Sequences)(p < 0.05) ⱡ
A5JV31_HIPHIHippoglossus hippoglossus145 (56)3616
PhosvitinAtlantic halibut
A5JV30_HIPHIHippoglossus hippoglossus90 (52)3303
PhosvitinAtlantic halibut
Q4KVK3_HIPHIHippoglossus hippoglossus69 (25)1690
Complement component c3 (fragment)Atlantic halibut
A0A2U9BPE5_SCOMXScophthalmus maximus89 (24)1426
Complement component C3 isoform 2Turbot
A0A3B4THR8_SERDUSeriola dumerili79 (22)1269
Uncharacterized protein (NTR domain-containing protein, Complement C3-like, A2M_recep domain-containing protein)Greater amberjack
A0A3B4TYC3_SERDUSeriola dumerili65 (21)1250
NTR domain-containing proteinGreater amberjack
Q9PTY1_PAROLParalichthys olivaceus70 (22)1176
Complement component C3Olive flounder
G4WAB7_EPICOEpinephelus coioides57 (20)1145
Complement component c3Orange-spotted grouper
A0A3P9BEG5_9CICHMaylandia zebra66 (19)1120
Uncharacterized protein (Anaphylatoxin-like domain-containing protein; C3a)Zebra mbuna
* A0A669BPJ4_ORENIOreochromis niloticus71 (18)1097
Uncharacterized proteinNile tilapia
A0A671YHA0_SPAAUSparus aurata45 (17)904
Uncharacterized protein (C3)Gilt-head bream
* A0A6A5FQW4_PERFLPerca fluviatilis57 (16)885
Uncharacterized proteinEuropean perch
A0A484CCU5_PERFVPerca flavescens56 (17)879
Uncharacterized protein (Anaphylatoxin-like domain-containing protein)Yellow perch
F8R8R1_DICLADicentrarchus labrax59 (15)871
Complement component c3-2European bass
A0A484DL37_PERFVPerca flavescens42 (15)784
Anaphylatoxin-like domain-containing proteinYellow perch
A0A4W6E087_LATCALates calcarifer43 (15)744
Complement component c3a, duplicate 5Barramundi/Asian sea bass
A0A6A5FJW4_PERFLPerca fluviatilis14 (12)600
Uncharacterized protein (Integrase catalytic domain-containing protein, Alpha-2-macroglobulin-like)European perch
A0A2P9DTV2_SOLSESolea senegalensis16 (10)594
PhosvitinSenegalese sole
Q6QZI2_PSEAMPseudopleuronectes americanus37 (9)574
Complement component C3 (Fragment)Winter flounder
A0A3Q1ID66_ANATEAnabas testudineus25 (9)569
PhosvitinClimbing perch
* A0A4W6F6V9_LATCALates calcarifer15 (9)549
Apolipoprotein Bb, tandem duplicate 2Barramundi/Asian sea bass
A0A6G1PAV1_9TELEChanna argus38 (10)540
Complement C3 Complement C3 beta chain Complement C3 alpha chainNorthern snakehead
* A0A4P8JD10_9TELE Lateolabrax maculatus13 (9)532
Apolipoprotein Bb.1Spotted sea bass
* A0A6A5DT05_PERFLPerca fluviatilis16 (9)529
Vitellogenin domain-containing proteinEuropean perch
A0A673IJP2_9TELESinocyclocheilus rhinocerous48 (12)528
IF rod domain-containing proteinSinocyclocheilus cavefish (Cyprinid)
A0A4W6CMC4_LATCALates calcarifer14 (11)525
Uncharacterized protein (Alpha-2-macroglobulin)Barramundi/Asian sea bass
A0A3P8Y5X6_ESOLUEsox Lucius51 (11)499
IF rod domain-containing proteinNorthern pike
A0A6G1PQL3_9TELEChanna argus12 (10)497
Alpha-2-macroglobulinNorthern snakehead
A0A6A4SX26_SCOMXScophthalmus maximus51 (11)463
IF rod domain-containing proteinTurbot
Q5DVG8_PLAFEPlatichthys flesus26 (9)453
Apolipoprotein AIEuropean flounder
* A0A3B4T6U1_SERDUSeriola dumerili12 (9)440
Vitellogenin domain-containing proteinGreater amberjack
A0A665VQL3_ECHNAEcheneis naucrates9 (8)409
Uncharacterized protein (A2M_N_2 domain-containing protein)Live sharksucker
* A0A2U9D044_SCOMXScophthalmus maximus14 (8)407
Putative apolipoprotein B-100-like isoform 2Turbot
* Q9PVW6_PAROLParalichthys olivaceus14 (7)403
Complement component C9Olive flounder
A0A4W6FLR7_LATCALates calcarifer10 (8)386
Uncharacterized protein (Anaphylatoxin-like domain-containing, A2M_N_2 domain containing protein, NTR domain containing protein, Complement C5)Barramundi/Asian sea bass
A0A4W6CP97_LATCALates calcarifer17 (7)362
Uncharacterized protein (A2M_recep domain-containing protein, TED_complement domain-containing protein)Barramundi/Asian sea bass
* A0A3P8RR96_AMPPEAmphiprion percula12 (5)353
Complement component C9Orange clownfish
A0A3Q1HZ43_ANATEAnabas testudineus13 (9)336
Uncharacterized protein (Inter-alpha-trypsin inhibitor, VIT domain-containing protein)Climbing perch
* A0A3Q1H6Y9_ANATEAnabas testudineus8 (8)336
Complement component 8 subunit betaClimbing perch
A0A6G1PI27_9TELEChanna argus13 (7)324
Inter-alpha-trypsin inhibitor heavy chain H3Northern snakehead
A0A6A5FLM2_PERFLPerca fluviatilis10 (6)323
Uncharacterized protein (alpha-2-macroglobulin-like, A2M_recep domain-containing protein)European perch
A0A6A5FFR2_PERFLPerca fluviatilis15 (7)323
Anaphylatoxin-like domain-containing proteinEuropean perch
A0A484DIJ5_PERFVPerca flavescens11 (7)321
Uncharacterized protein (Alpha-2-macroglobulin)Yellow perch
A0A6A5FE70_PERFLPerca fluviatilis11 (7)318
Uncharacterized protein (A2M_recep domain-containing, MG2 domain-containing protein)European perch
A0A6J2W3P0_CHACNChanos chanos8 (7)312
uncharacterized protein LOC115819396 (Histone H4, Histone H3, Histone H2B)Milkfish
* A0A665V532_ECHNAEcheneis naucrates8 (6)310
PlasminogenLive sharksucker
A0A3Q3L7G2_9TELEMastacembelus armatus6 (6)308
Complement component c3b, tandem duplicate 2Zig-zag eel/Spiny eel
* CO8B_PAROLParalichthys olivaceus5 (5)304
Complement component C8 beta chainOlive flounder
A0A671PIL3_9TELESinocyclocheilus anshuiensis17 (6)301
IF rod domain-containing proteinSinocyclocheilus cavefish (Cyprinoid)
* A0A3Q3E5X5_9LABRLabrus bergylta7 (4)298
Uncharacterized protein (C1q domain-containing protein)Ballan wrasse
* A0A3Q0S0V4_AMPCIAmphilophus citrinellus18 (5)292
Uncharacterized proteinMidas cichlid
A0A6A4SU52_SCOMXScophthalmus maximus7 (7)291
Uncharacterized protein (Complement component c3b)Turbot
* A0A3P8TA20_AMPPEAmphiprion percula11 (7)290
Zgc:112265Orange clownfish
A0A096MDQ7_POEFOPoecilia formosa11 (6)288
PhosvitinAmazon molly
Q5XVQ2_FUNHEFundulus heteroclitus17 (5)288
Apolipoprotein A1 (Fragment)Atlantic killifish, mud minnow
* Q6QZI9_PSEAMPseudopleuronectes americanus12 (5)284
Complement component C9 (Fragment)Winter flounder
* A0A4U5UPP9_COLLUCollichthys lucidus7 (5)283
Apolipoprotein B-100(Big head croaker)
A0A3Q1EMN2_9TELEAcanthochromis polyacanthus8 (5)280
Uncharacterized protein (beta actin, actin cytoplasmic-1)Spiny chromis damselfish
* A0A6J2P874_COTGOCottoperca gobio7 (5)280
plasminogenChannel bull blenny
A0A3B4VID4_SERDUSeriola dumerili9 (5)279
Uncharacterized protein (MG2 domain-containing protein)Greater amberjack
* A0A3Q3M9S2_9TELEMastacembelus armatus12 (4)278
Uncharacterized proteinZig-zag eel/Spiny eel
W5ZMG9_9TELECampylomormyrus compressirostris7 (4)267
Cytoplasmic 1 actinElephantfish
A0A553Q7M4_9TELEDanionella translucida6 (6)262
Uncharacterized protein (Histone H2A, H2B putative, H3)Micro glassfish (Cyprinid)
A0A3Q1H0X2_ANATEAnabas testudineus5 (5)260
Complement component c3b, tandem duplicate 2Climbing perch
* A0A6A4SHP5_SCOMXScophthalmus maximus12 (5)258
Uncharacterized proteinTurbot
* G3NNM8_GASACGasterosteus aculeatus6 (6)256
Uncharacterized proteinThree-spined stickleback
* A0A0P7YVM9_SCLFOScleropages formosus10 (5)251
Keratin, type I cytoskeletal 13-likeAsian arowana
* A0A6A4SWR2_SCOMXScophthalmus maximus7 (6)251
EGF-like domain-containing proteinTurbot
A0A2U9B3I5_SCOMXScophthalmus maximus13 (6)247
Alpha-2-macroglobulinTurbot
A0A4Z2BCD9_9TELETakifugu bimaculatus6 (5)242
Uncharacterized proteinPufferfish
(Complement C5 C3 and PZP-like alpha-2-macroglobulin domain-containing protein)
A0A671TD78_SPAAUSparus aurata5 (5)238
Complement component c3b, tandem duplicate 2Gilt-head bream
* A0A0A0QKL5_OPLFAOplegnathus fasciatus6 (5)234
Complement component 4Striped beakfish
* A0A6A4RUD7_SCOMXScophthalmus maximus6 (6)233
Vitellogenin domain-containing proteinTurbot
A0A672YA60_9TELESphaeramia orbicularis7 (6)232
Uncharacterized protein (inter-alpha-trypsin inhibitor heavy chain)Orbiculate cardinalfish
* A0A672JL95_SALFASalarias fasciatus5 (5)232
Uncharacterized protein (complement C7)Lawnmower blenny
* A0A3B4Y8X6_SERLLSeriola lalandi10 (4)231
Uncharacterized protein (Hephaestin-like protein 1, Desmoglein-2)Yellowtail amberjack
* A0A3B4UHS2_SERDUSeriola dumerili4 (4)229
Uncharacterized proteinGreater amberjack
* A0A087YMZ0_POEFOPoecilia formosa11 (6)229
Uncharacterized protein (Ceruloplasmin)Amazon molly
A0A3Q4G4S3_NEOBRNeolamprologus brichardi11 (5)229
Uncharacterized protein (NTR domain-containing protein)Lyretail cichlid
* A0A3Q1EBE7_9TELEAcanthochromis polyacanthus4 (4)228
Vitellogenin domain-containing proteinSpiny chromis damselfish
A0A3P9A8D3_ESOLUEsox Lucius8 (4)218
Uncharacterized protein (Alpha-2-macroglobulin, A2M_recep domain-containing)Northern pike
* A0A3P8WZ01_CYNSECynoglossus semilaevis7 (4)217
Vitellogenin domain-containing proteinTongue sole
* A0A3B4F9T0_9CICHPundamilia nyererei3 (3)215
Carboxypeptidase QCichlid
* A0A6J2QSS9_COTGOCottoperca gobio3 (2)209
complement component C9Channel bull blenny
* A0A672GNQ4_SALFASalarias fasciatus7 (4)208
Vitellogenin domain-containing proteinLawnmower blenny
* A0A3B4ULR2_SERDUSeriola dumerili9 (5)207
Zgc:112265Greater amberjack
A0A3B4THN2_SERDUSeriola dumerili4 (4)205
Fibrinogen beta chainGreater amberjack
* A0A2U9BK85_SCOMXScophthalmus maximus3 (3)203
Putative complement component C8 alpha chainTurbot
* G8DP14_PLAFEPlatichthys flesus4 (4)201
Beta 1-globinEuropean flounder
* A0A0F8C5A6_LARCRLarimichthys crocea6 (5)200
Antithrombin-IIIYellow croaker
* A0A2U9CEJ2_SCOMXScophthalmus maximus4 (4)200
Complement component 7Turbot
A0A5C6MX12_9TELETakifugu flavidus17 (5)196
Complement C3Yellowbelly pufferfish
* Q6QZI5_PSEAMPseudopleuronectes americanus4 (4)194
Complement component C8 beta chainWinter flounder
* A0A3B3BJ38_ORYMEOryzias melastigma7 (4)192
Vitellogenin domain-containing proteinMarine medaka
* A0A6J2S534_COTGOCottoperca gobio5 (5)190
apolipoprotein B-100Channel bull blenny
* A0A3Q1JFY5_ANATEAnabas testudineus5 (3)187
Uncharacterized protein (ceruloplasmin)Climbing perch
A0A672I1M9_SALFASalarias fasciatus6 (4)186
Uncharacterized protein (Inter-alpha-trypsin inhibitor heavy chain, VIT domain-containing protein)Lawnmower blenny
A0A3B5AT07_9TELEStegastes partitus7 (4)185
IF rod domain-containing proteinBicolour damselfish
* A0A4Z2CEC7_9TELETakifugu bimaculatus4 (4)183
Uncharacterized protein (complement C4)Pufferfish
* A0A3Q3II57_MONALMonopterus albus5 (4)183
Uncharacterized proteinAsian swamp eel
* A0A3Q3FIH8_KRYMAKryptolebias marmoratus7 (4)180
Uncharacterized proteinMangrove rivulus(killilfish)
* A0A2U9AYP3_SCOMXScophthalmus maximus5 (3)177
Complement component 4Turbot
* A0A6J2RDF1_COTGOCottoperca gobio4 (4)176
complement C4-B-likeChannel bull blenny
A0A4W6ERJ2_LATCALates calcarifer5 (4)173
Fibrinogen gamma chainBarramundi/Asian sea bass
* A0A2I4C034_9TELEAustrofundulus limnaeus3 (3)167
collagen alpha-1(XII) chainKillifish
* A0A6I9PPD4_9TELENotothenia coriiceps4 (4)167
complement C4-likeBlack rockcod/Antarctic yellowbelly rockcod
* H3BWT7_TETNGTetraodon nigroviridis5 (3)163
CeruloplasminGreen spotted puffer
* Q4SXM5_TETNGTetraodon nigroviridis5 (4)160
Chromosome 12 SCAF12357, whole genome shotgun sequenceGreen spotted puffer
A0A1A8F2V0_9TELENothobranchius korthausae5 (2)160
Uncharacterized protein (Alpha2-macroglobulin)Killifish
* A0A3B5BD88_9TELEStegastes partitus4 (4)159
Vitellogenin domain-containing proteinBicolour damselfish
A0A6G1QB31_9TELEChanna argus9 (2)159
SerotransferrinNorthern snakehead
* A0A060WU48_ONCMYOncorhynchus mykiss2 (2)157
Uncharacterized protein (Desmoplakin)Rainbow trout
*A0A3Q3FAE5_9LABRLabrus bergylta4 (3)155
Complement component 8 subunit betaBallan wrasse
A0A6J2Q526_COTGOCottoperca gobio4 (3)155
fibrinogen gamma chainChannel bull blenny
* A0A3B4UV22_SERDUSeriola dumerili6 (4)154
Antithrombin-IIIGreater amberjack
* A0A3Q2QAA5_FUNHEFundulus heteroclitus4 (3)154
Uncharacterized proteinAtlantic killifish, mud minnow
* A0A6J2P7B9_COTGOCottoperca gobio3 (2)153
apolipoprotein B-100-likeChannel bull blenny
* A0A484D0P7_PERFVPerca flavescens6 (5)153
Uncharacterized protein (ceruloplasmin)Yellow perch
* A0A3B4TA89_SERDUSeriola dumerili3 (3)149
Uncharacterized proteinGreater amberjack
* A0A673XMC1_SALTRSalmo trutta3 (3)148
Uncharacterized protein (complement C4, C4-B)Brown trout
F8U8N8_CHELBChelon labrosus4 (3)146
Alpha 2 macroglobulin (fragment)Thicklip grey mullet
F2Y9S5_MORSAMorone saxatilis3 (3)145
PhosvitinStriped bass
* A0A3P9Q7U6_POEREPoecilia reticulate4 (4)144
Complement component C9Guppy
A0A0F8AH88_LARCRLarimichthys crocea9 (3)143
Ig heavy chain V region 5AYellow croaker
* A0A667Y3E0_9TELEMyripristis murdjan6 (3)142
Vitellogenin domain-containing proteinBlacktipped soldierfish
* A0A672QEF7_SINGRSinocyclocheilus graham8 (4)141
Uncharacterized proteinGolden-line barbell
* A0A3B5B7I8_9TELEStegastes partitus5 (4)141
Antithrombin-IIIBicolour damselfish
* A0A0B6VKQ1_ORYCLOryzias celebensis3 (3)139
B5 proteinCelebes medaka
* A0A671TKG8_SPAAUSparus aurata4 (2)138
Uncharacterized proteinGilt-head bream
* A0A4P8JCG0_9TELELateolabrax maculatus3 (2)136
Apolipoprotein Bb.2Spotted sea bass
* A0A3B4FS46_9CICHPundamilia nyererei4 (3)132
IGv domain-containing proteinCichlid
A0A3P9H4Z3_ORYLAOryzias latipes9 (3)132
Uncharacterized protein (A2M_N_2 domain-containing protein, anaphylatoxin-like domain)Medaka/Japanese rice fish
A0A0F8AKQ4_LARCRLarimichthys crocea5 (3)131
Alpha-2-macroglobulinYellow croaker
A0A3B4TIN1_SERDUSeriola dumerili3 (3)130
PhosvitinGreater amberjack
B6RUP0_ORYDNOryzias dancena4 (3)129
Beta-actin (Fragment)Indian ricefish
* A0A484CD54_PERFVPerca flavescens3 (3)129
Uncharacterized protein (Complement C7)Yellow perch
A0A3Q4FXR7_NEOBRNeolamprologus brichardi4 (3)128
Ig-like domain-containing proteinLyretail cichlid
Q5SET8_9TELEBembras japonica3 (3)128
Histone H3 (Fragment)Red flathead
A0A3Q1IXI9_ANATEAnabas testudineus3 (3)128
Uncharacterized protein (A2M_recep domain-containing protein)Climbing perch
* A0A4Z2H8W0_9TELELiparis tanakae2 (2)126
BiotinidaseTanaka’s snailfish
A0A6G1PSN0_9TELEChanna argus6 (5)126
Alpha-2-macroglobulinNorthern snakehead
A0A669DKF1_ORENIOreochromis niloticus4 (3)125
Uncharacterized protein (Ig-like domain-containing protein)Nile tilapia
A0A3B1JCF6_ASTMXAstyanax mexicanus6 (3)123
IF rod domain-containing proteinMexican tetra/blind cave fish
* A0A3Q2YHX2_HIPCMHippocampus comes3 (3)122
Complement component 8 subunit betaTiger tail seahorse
A0A3Q3IC70_MONALMonopterus albus1 (1)121
Ig-like domain-containing proteinAsian swamp eel
* A0A0F8AI97_LARCRLarimichthys crocea2 (2)121
Collagenase 3Yellow croaker
A0A6J2PEG5_COTGOCottoperca gobio2 (2)120
complement C5-likeChannel bull blenny
A0A6A4TFM7_SCOMXScophthalmus maximus3 (2)119
Ig-like domain-containing proteinTurbot
* A0A3Q0R4Z0_AMPCIAmphilophus citrinellus5 (3)119
Complement component C9Midas cichlid
A0A6I9NNH1_9TELENotothenia coriiceps3 (2)118
inter-alpha-trypsin inhibitor heavy chain H2Black rockcod/Antarctic yellowbelly rockcod
A0A437D6V7_ORYJAOryzias javanicus2 (2)114
ChitinaseJavanese ricefish
A0A3Q3EEY5_9LABRLabrus bergylta3 (3)113
Fibrinogen C-terminal domain-containing proteinBallan wrasse
* A0A1S3SMN1_SALSASalmo salar1 (1)111
cathepsin L1-likeAtlantic salmon
* A0A3Q3IZL2_MONALMonopterus albus4 (3)111
Uncharacterized proteinAsian swamp eel
* A0A3P8YF02_ESOLUEsox Lucius6 (3)109
Vitellogenin domain-containing proteinNorthern pike
* A0A3B3CJZ7_ORYMEOryzias melastigma3 (2)109
Complement 4B (Chido blood group)Marine medaka
* A0A2U9CVZ8_SCOMXScophthalmus maximus2 (2)108
Putative complement component C8 gamma chainTurbot
A0A3Q3RJX0_9TELEMastacembelus armatus2 (1)108
Ig-like domain-containing proteinZig-zag eel/Spiny eel
* A0A3Q4HZS4_NEOBRNeolamprologus brichardi3 (3)108
Uncharacterized protein (Ceruloplasmin)Lyretail cichlid
A0A6G1PYT4_9TELEChanna argus4 (3)108
Complement C5 C3 and PZP-like alpha-2-macroglobulin domain-containing protein 4Northern snakehead
* A0A2U9AV20_SCOMXScophthalmus maximus2 (2)107
ProthrombinTurbot
A0A4W6EWH0_LATCALates calcarifer3 (3)107
Peptidase S1 domain-containing proteinBarramundi/Asian sea bass
* H3C6P0_TETNGTetraodon nigroviridis2 (2)106
PlasminogenGreen spotted puffer
A0A3P8SDE5_AMPPEAmphiprion percula14 (2)105
SerotransferrinOrange clownfish
A0A3B4BP10_PYGNAPygocentrus nattereri10 (2)105
Uncharacterized proteinRed-bellied piranha
* A0A3B4TQB5_SERDUSeriola dumerili1 (1)105
SERPIN domain-containing proteinGreater amberjack
* D5A7I1_DICLADicentrarchus labrax4 (2)104
HemopexinEuropean bass
* A0A2U9CU10_SCOMXScophthalmus maximus3 (3)103
Putative insulin-like growth factor-binding protein complex acid labile subunitTurbot
* A0A6J2PA80_COTGOCottoperca gobio3 (3)103
histone H2B 1/2-likeChannel bull blenny
* A0A3P8SSL4_AMPPEAmphiprion percula2 (2)102
Uncharacterized protein (Ig-like domain-containing protein, Nattectin)Orange clownfish
* G3NN36_GASACGasterosteus aculeatus4 (3)99
Uncharacterized proteinThree-spined stickleback
A0A4W4FLR8_ELEELElectrophorus electricus3 (2)99
Fibrinogen beta chainElectric eel
A0A671TDU8_SPAAUSparus aurata2 (2)97
Ig-like domain-containing proteinGilt-head bream
A0A6I9PPY0_9TELENotothenia coriiceps2 (2)96
fibrinogen gamma chainBlack rockcod/Antarctic yellowbelly rockcod
A0A671TNW0_SPAAUSparus aurata4 (3)96
Histone H3Gilt-head bream
* A0A3B4XVK3_SERLLSeriola lalandi dorsalis2 (2)96
Vitellogenin domain-containing proteinYellowtail amberjack
* A0A3Q3L1F9_9TELEMastacembelus armatus1 (1)95
Complement component 1, r subcomponentZig-zag eel/Spiny eel
A0A1A8AN27_NOTFUNothobranchius furzeri3 (3)95
Fibrinogen, gamma polypeptideturquoise killifish
* A0A2D0QC28_ICTPUIctalurus punctatus2 (2)93
Ig heavy chain Mem5-likeChannel catfish
A0A3P8R4C1_ASTCAAstatotilapia calliptera6 (2)96
Uncharacterized protein (Ig-like domain-containing protein)Eastern happy/eastern river bream
A0A3B4H9E9_9CICHPundamilia nyererei3 (2)93
Ig-like domain-containing proteinCichlid
A0A3B4UNU3_SERDUSeriola dumerili4 (2)93
Ig-like domain-containing proteinGreater amberjack
* A0A060XWP2_ONCMYOncorhynchus mykiss 92
SERPIN domain-containing proteinRainbow trout
* A0A1A8CRV1_9TELENothobranchius kadleci8 (2)91
Uncharacterized proteinKillifish
* A0A2U9CFI3_SCOMXScophthalmus maximus2 (2)90
Putative sushi domain-containing protein 2 isoform 2Turbot
A0A5C6NS08_9TELETakifugu flavidus5 (2)90
Ig heavy chain V region VH558 A1/A4Yellowbelly pufferfish
* A0A4W4DXU4_ELEELElectrophorus electricus3 (3)89
14_3_3 domain-containing proteinElectric eel
* A0A0F8B5M5_LARCRLarimichthys crocea1 (1)88
Catechol O-methyltransferase domain-containing protein 1Yellow croaker
* A0A5N5KRW8_PANHPPangasianodon hypophthalmus3 (3)88
Uncharacterized protein (pleckstrin homology domain-containing family)Iridescent shark
* A0A5C6NRB2_9TELETakifugu flavidus2 (2)87
Apolipoprotein B-100Yellowbelly pufferfish
A0A2D0RGG9_ICTPUIctalurus punctatus3 (2)87
catenin beta-1 isoform X3Channel catfish
* A0A6I9P4Q9_9TELENotothenia coriiceps1 (1)86
apolipoprotein B-100-likeBlack rockcod/Antarctic yellowbelly rockcod
* A0A087XVJ8_POEFOPoecilia formosa2 (1)86
Uncharacterized protein (IGv domain-containing protein)Amazon molly
* H1AB41_PLASAPlatichthys stellatus4 (2)85
LysozymeStarry flounder
* A0A4P8JEC9_9TELELateolabrax maculatus2 (2)84
Apolipoprotein BaSpotted sea bass
A0A3Q4ACH4_MOLMLMola mola2 (2)84
Inter-alpha-trypsin inhibitor heavy chain 3Ocean sunfish
* A0A484CC61_PERFVPerca flavescens3 (1)84
Uncharacterized protein (Hyaluronan-binding protein 2)Yellow perch
A0A060Z3N3_ONCMYOncorhynchus mykiss3 (2)86
Ig-like domain-containing proteinRainbow trout
* A0A3B4ZU87_9TELEStegastes partitus3 (2)83
Uncharacterized protein (complement factor H-like)Bicolour damselfish
A0A3B3QDE5_9TELEParamormyrops kingsleyae2 (1)83
Ig-like domain-containing proteinElephantfish
A0A3B3CFL8_ORYMEOryzias melastigma5 (2)83
Ig-like domain-containing proteinMarine medaka
* A0A3Q3W6Q7_MOLMLMola mola2 (2)82
Sushi domain containing 2Ocean sunfish
A0A4W5L5T6_9TELEHucho hucho3 (2)82
ThioredoxinDanube salmon
* G1DHP8_GOBRAGobiocypris rarus2 (2)81
Vitellogenin (Fragment)Rare gudgeon/rare minnow
* A0A3B3QP35_9TELEParamormyrops kingsleyae3 (2)80
Uncharacterized proteinElephantfish
* A0A3B4Z082_9TELEStegastes partitus2 (2)80
Uncharacterized protein (complement C6)Bicolour damselfish
A0A669CCK4_ORENIOreochromis niloticus6 (2)80
Uncharacterized protein (Ig-like domain-containing protein)Nile tilapia
* A0A484C6M0_PERFVPerca flavescens1 (1)80
Uncharacterized proteinYellow perch
* A0A3P8U2B4_AMPPEAmphiprion percula4 (2)80
Keratin 98Orange clownfish
* A0A060WHH8_ONCMYOncorhynchus mykiss2 (2)79
Junction plakoglobinRainbow trout
A0A3B4ULY5_SERDU Seriola dumerili4 (2)78
Ig-like domain-containing proteinGreater amberjack
H3C0U1_TETNGTetraodon nigroviridis3 (2)77
Ig-like domain-containing proteinGreen spotted puffer
A0A087X4F8_POEFOPoecilia formosa1 (1)77
Uncharacterized protein (Ig-like domain-containing protein)Amazon molly
A0A3P9IRN4_ORYLAOryzias latipes2 (2)77
Ig-like domain-containing proteinMedaka/Japanese rice fish
A0A060W543_ONCMYOncorhynchus mykiss2 (2)77
Histone H2ARainbow trout
A0A3B4UFJ1_SERDUSeriola dumerili2 (1)75
Ig-like domain-containing proteinGreater amberjack
A0A0F8ABH4_LARCRLarimichthys crocea5 (1)75
Granzyme B(G,H)Yellow croaker
* A0A3B4UPX8_SERDUSeriola dumerili1 (1)74
Zona pellucida sperm-binding protein 3Greater amberjack
* A0A3P8U813_AMPPEAmphiprion percula2 (2)73
Si:ch1073-416d2.3Orange clownfish
* A0A3Q1KAD2_ANATEAnabas testudineus2 (2)72
SERPIN domain-containing proteinClimbing perch
* A0A4W5RID4_9TELEHucho hucho2 (1)71
RRM domain-containing proteinDanube salmon
* A0A3Q2QNZ9_FUNHEFundulus heteroclitus2 (2)71
Uncharacterized protein (Sushi domain containing 2)Atlantic killifish, mud minnow
* A0A4W5LQ29_9TELEHucho hucho8 (2)70
ATP-synt ab_N domain-containing proteinDanube salmon
A0A3Q2PS35_FUNHEFundulus heteroclitus5 (2)70
Ig-like domain-containing proteinAtlantic killifish, mud minnow
* A0A6G1QID3_9TELEChanna argus2 (2)70
Complement component C6Northern snakehead
* A0A3B3X986_9TELEPoecilia Mexicana1 (1)70
Uncharacterized protein (F-BAR domain-containing protein)Atlantic (shortfin) molly
* A0A498LNY2_LABROLabeo rohita6 (2)70
Retrotransposon-derived PEG10Rohu
* A0A6G1PD67_9TELEChanna argus2 (2)70
Apoptosis-stimulating of p53 protein 2 Bcl2-binding proteinNorthern snakehead
* A0A1S3L2W1_SALSASalmo salar5 (2)70
FH2 domain-containing protein 1-likeAtlantic salmon
A0A3B3HM39_ORYLAOryzias latipes1 (1)69
Ig-like domain-containing proteinMedaka/Japanese rice fish
* A0A3Q1HK94_ANATEAnabas testudineus6 (2)69
Protein-tyrosine-phosphataseClimbing perch
A0A3Q3JUN7_MONALMonopterus albus2 (2)68
IF rod domain-containing proteinAsian swamp eel
* A0A671X983_SPAAUSparus aurata3 (2)68
Uncharacterized protein (Early endosome antigen 1, FYVE-type domain-containing protein)Gilt-head bream
* A0A3B3DTR8_ORYMEOryzias melastigma3 (2)68
Uncharacterized proteinMarine medaka
* A0A3Q3XI23_MOLMLMola mola3 (2)67
Zgc:112265Ocean sunfish
A0A671YT10_SPAAUSparus aurata2 (2)67
Uncharacterized protein (Immunoglobulin like and fibronectin type III domain containing 1, tandem duplicate 2)Gilt-head bream
* A0A3B5ACM2_9TELEStegastes partitus6 (2)66
Uncharacterized proteinBicolour damselfish
A0A3P9H0Y9_ORYLAOryzias latipes2 (2)65
Ig-like domain-containing proteinMedaka/Japanese rice fish
* A0A5C6N3H2_9TELETakifugu flavidus4 (2)65
Keratin, type I cytoskeletal 18Yellowbelly pufferfish
* A0A3B5L5A5_9TELEXiphophorus couchianus3 (2)65
Thyroid hormone receptor interactor 11Monterrey platyfish
* Q2PZ29_SOLSESolea senegalensis2 (1)65
LysozymeSenegalese sole
A0A667YBU1_9TELEMyripristis murdjan5 (2)65
Ig-like domain-containing proteinBlacktipped soldierfish
* A0A672GWK0_SALFASalarias fasciatus2 (2)64
Uncharacterized protein (Complement factor B-like)Lawnmower blenny
* A0A3B4CEW8_PYGNAPygocentrus nattereri2 (2)64
Uncharacterized protein (Roundabout-like axon guidance receptor protein 2)Red-bellied piranha
* A0A3B4EX20_9CICHPundamilia nyererei6 (2)64
Uncharacterized protein (Apolipoprotein M)Cichlid
* A0A2I4BMF1_9TELEAustrofundulus limnaeus1 (1)63
protein Z-dependent protease inhibitor-likeKillifish
* A0A3Q3EPX4_9LABRLabrus bergylta2 (2)62
Vitellogenin domain-containing proteinBallan wrasse
* A0A3B4T5U4_SERDUSeriola dumerili3 (2)62
Uncharacterized protein (Myosin phosphatase Rho interacting protein)Greater amberjack
A0A3B3T2D8_9TELEParamormyrops kingsleyae1 (1)62
Ig-like domain-containing proteinElephantfish
* A0A3Q1FWV1_9TELEAcanthochromis polyacanthus2 (2)62
Multidrug and toxin extrusion proteinSpiny chromis damselfish
A0A3B4YHZ5_SERLLSeriola lalandi dorsalis1 (1)61
IGv domain-containing proteinYellowtail amberjack
* R4I5B0_EPICOEpinephelus coioides3 (2)61
Immmunoglobulin light chainOrange-spotted grouper
* A0A3Q0R568_AMPCIAmphilophus citrinellus3 (2)61
FH2 domain containing 4Midas cichlid
A0A3B4WXW5_SERLLSeriola lalandi dorsalis2 (2)60
Ig-like domain-containing proteinYellowtail amberjack
G3PK20_GASACGasterosteus aculeatus3 (2)60
SerotransferrinThree-spined stickleback
* A0A484DB45_PERFVPerca flavescens1 (1)60
Uncharacterized protein (Pentaxin)Yellow perch
* A0A671SV95_9TELESinocyclocheilus anshuiensis2 (2)60
FERM domain-containing proteinSinocyclocheilus cavefish (Cyprinoid)
A0A023REA6_9TELEMenidia estor1 (1)60
Elongation factor 1-alphaPike silverside
* A0A6J2PC09_COTGOCottoperca gobio2 (2)60
nesprin-2Channel bull blenny
* A0A0S7MGP3_9TELEPoeciliopsis prolifica3 (2)59
ZN287 (Fragment)Blackstripe livebearer
* A0A3Q3VSX4_MOLMLMola mola1 (1)59
Uncharacterized proteinOcean sunfish
* A0A553Q8B1_9TELEDanionella translucida3 (2)58
Uncharacterized proteinMicro glassfish (Cyprinid)
* A0A0P7TM62_SCLFOScleropages formosus1 (1)58
Keratin, type I cytoskeletal 18-likeAsian arowana
* A0A060XKV1_ONCMYOncorhynchus mykiss3 (2)58
[Histone H3]-trimethyl-L-lysine(9) demethylaseRainbow trout
* E7F6Y7_DANREDanio rerio4 (2)58
DNA polymerase kappaZebrafish
* F8W5U5_DANREDanio rerio2 (2)58
Centrosomal protein of 290 kDaZebrafish
* A0A2U9CTT6_SCOMXScophthalmus maximus7 (2)57
Putative utrophinTurbot
* A0A3B3BVC4_ORYMEOryzias melastigma2 (2)57
Uncharacterized proteinMarine medaka
* A0A3B4UZF1_SERDUSeriola dumerili1 (1)57
[Histone H3]-lysine(4) N-trimethyltransferase Greater amberjack
A0A060VW86_ONCMYOncorhynchus mykiss1 (1)56
Uncharacterized protein (Tubulin alpha, tubulin domain containing)Rainbow trout
* A0A671TLU7_SPAAUSparus aurata3 (2)56
Reverse transcriptaseGilt-head bream
A0A3Q4H8B0_NEOBRNeolamprologus brichardi1 (1)56
Ig-like domain-containing proteinLyretail cichlid
* A0A0U2ERZ3_CORCLCoregonus clupeaformis6 (1)56
Glyceraldehyde 3-phosphate dehydrogenaseLake whitefish
* A0A0R4IVM1_DANREDanio rerio11 (2)55
LSM14A mRNA-processing body assembly factor bZebrafish
* A0A3P8VC95_CYNSECynoglossus semilaevis1 (1)54
Uncharacterized proteinTongue sole
* Q9DFN6_GILMIGillichthys mirabilis1 (1)54
Glyceraldehyde-3-phosphate dehydrogenase
* A0A3B3BWJ2_ORYMEOryzias melastigma2 (2)54
Uncharacterized proteinMarine medaka
* A0A6A4SGZ4_SCOMXScophthalmus maximus1 (1)54
C1q domain-containing proteinTurbot
* A0A3B4EJ56_PYGNAPygocentrus nattereri2 (2)54
von Willebrand factorRed-bellied piranha
* A0A1S3RE28_SALSASalmo salar1 (1)53
uncharacterized protein LOC106602330 isoform X1Atlantic salmon
* A0A2I4CMN8_9TELEAustrofundulus limnaeus2 (2)53
titin-likeKillifish
ⱡ Ions score is −10*Log(P), where P is the probability that the observed match is a random event. Individual ions scores > 53 indicate identity or extensive homology (p < 0.05). Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.
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MDPI and ACS Style

Magnadóttir, B.; Kraev, I.; Dodds, A.W.; Lange, S. The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles—Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways. Int. J. Mol. Sci. 2021, 22, 875. https://doi.org/10.3390/ijms22020875

AMA Style

Magnadóttir B, Kraev I, Dodds AW, Lange S. The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles—Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways. International Journal of Molecular Sciences. 2021; 22(2):875. https://doi.org/10.3390/ijms22020875

Chicago/Turabian Style

Magnadóttir, Bergljót, Igor Kraev, Alister W. Dodds, and Sigrun Lange. 2021. "The Proteome and Citrullinome of Hippoglossus hippoglossus Extracellular Vesicles—Novel Insights into Roles of the Serum Secretome in Immune, Gene Regulatory and Metabolic Pathways" International Journal of Molecular Sciences 22, no. 2: 875. https://doi.org/10.3390/ijms22020875

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