Protein Deimination and Extracellular Vesicle Profiles in Antarctic Seabirds

Pelagic seabirds are amongst the most threatened of all avian groups. They face a range of immunological challenges which seem destined to increase due to environmental changes in their breeding and foraging habitats, affecting prey resources and exposure to pollution and pathogens. Therefore, the identification of biomarkers for the assessment of their health status is of considerable importance. Peptidylarginine deiminases (PADs) post-translationally convert arginine into citrulline in target proteins in an irreversible manner. PAD-mediated deimination can cause structural and functional changes in target proteins, allowing for protein moonlighting in physiological and pathophysiological processes. PADs furthermore contribute to the release of extracellular vesicles (EVs), which play important roles in cellular communication. In the present study, post-translationally deiminated protein and EV profiles of plasma were assessed in eight seabird species from the Antarctic, representing two avian orders: Procellariiformes (albatrosses and petrels) and Charadriiformes (waders, auks, gulls and skuas). We report some differences between the species assessed, with the narrowest EV profiles of 50–200 nm in the northern giant petrel Macronectes halli, and the highest abundance of larger 250–500 nm EVs in the brown skua Stercorarius antarcticus. The seabird EVs were positive for phylogenetically conserved EV markers and showed characteristic EV morphology. Post-translational deimination was identified in a range of key plasma proteins critical for immune response and metabolic pathways in three of the bird species under study; the wandering albatross Diomedea exulans, south polar skua Stercorarius maccormicki and northern giant petrel. Some differences in Gene Ontology (GO) biological and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins were observed between these three species. This indicates that target proteins for deimination may differ, potentially contributing to a range of physiological functions relating to metabolism and immune response, as well as to key defence mechanisms. PAD protein homologues were identified in the seabird plasma by Western blotting via cross-reaction with human PAD antibodies, at an expected 75 kDa size. This is the first study to profile EVs and to identify deiminated proteins as putative novel plasma biomarkers in Antarctic seabirds. These biomarkers may be further refined to become useful indicators of physiological and immunological status in seabirds—many of which are globally threatened.


Introduction
Peptidylarginine deiminases (PADs) are calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner. This protein deimination can lead to structural and functional changes in target proteins [1][2][3][4]. Structures most prone to deimination are beta-sheets and intrinsically disordered proteins, and identified deiminated targets to date include nuclear, cytoplasmic and mitochondrial proteins [2,[4][5][6][7][8][9][10][11]. Protein deimination can affect gene regulation and cause generation of neo-epitopes [5,12] but may also allow for protein moonlighting, facilitating several physiologically relevant functions from within one polypeptide chain [13,14]. PADs have been identified in diverse taxa from bacteria to mammals, with five tissue-specific PAD isozymes in mammals, three in the chicken Gallus gallus domesticus, one in bony fish [1,6,7,15,16] and PAD homologues in bacteria, protozoa and fungi [17][18][19][20]. Although PADs are well known to have pathophysiological roles in cancer, autoimmune and central nervous system (CNS) diseases [4,5,12,16,21,22], much less is known about their involvement in physiological processes. Recent comparative animal studies have therefore focused on elucidating roles for post-translational deimination in immunological and metabolic pathways in a wide range of animal species [6][7][8][9][10][11]23,24]. In birds, PADs have been implicated in tissue regeneration of the chicken CNS, including via inflammatory pathways [16], but their roles in physiology and immunology of birds in general remain to be fully understood.
PADs play crucial roles in the cellular release of extracellular vesicles (EVs) in diverse taxa [18,19,[25][26][27]. EVs are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material [5,[28][29][30][31]. EVs isolated from a range of bodyfluids, including plasma, have been identified as usable biomarkers for assessment of health and can be indicative of pathological processes [32,33]. Hitherto, the main body of EV research has been in the context of human pathologies; however, recent studies assessing EVs in comparative animal models reflect an increasing interest in elucidating roles for EVs throughout the phylogenetic tree [8][9][10][11]19,24,34,35]. Differences in EV profiles among taxonomic groups have indeed been reported in a range of taxa. Human EVs are generally observed in a narrow size range from 30 to 300 nm [36] and similar size-ranges of EV size profiles have been reported in naked mole-rats (Heterocephalus glaber) [11]. In teleost fish EVs were reported in higher abundance at 300-500 nm [8,35], while in elasmobranches higher abundance of small EVs in the 10-200 nm size range are reported [9]. In the protozoa Giardia intestinalis, two distinct EV size populations with different functions in host-pathogen interactions have been described [19]. In bacteria, EV profiles from Gram-negative and Gram-positive bacteria have been described in the size range of 10-600 nm and 60-400 nm and were also shown to change with respect to size profile and EV cargo in response to drug-treatment [18,37]. In camelids EVs are reported in llama (Lama glama) plasma in the 40-400 nm range [10]. In human cancer studies, cellular EV profiles vary between cancer types and change in response to drug-treatment, both with respect to EV size distribution and cargo [25][26][27]37]. A recent study assessing serum EVs from teleost fish (cod, Gadus morhua L.), reported changes in EV release and cargo (deiminated proteins and microRNAs) related to immunological status and growth in response to change in water temperature during rearing [38]. Hitherto, no studies on EVs have been carried out in seabirds, despite the potential for assessments of physiological status or the level of environmental or immunological challenges.
Seabirds are subject to a range of natural and anthropogenic pressures, including from incidental mortality (bycatch) in fisheries, overfishing, invasive species and exposure to pathogens and contaminants [39][40][41]. In addition, global climate change affects prey abundance and distribution at sea, increases the frequency of extreme weather (storms, high winds, rainfall or heatwaves) and possibly the likelihood or severity of disease outbreaks [42][43][44]. Numerous studies have examined levels of a range of heavy metal and other contaminants [39,[45][46][47]. Similarly, a range of seabird species have been screened for specific pathogens [48], including for the agent of avian cholera (Pasteurella multocida) [49][50][51][52], avian pox [53] as well as other bacterial [54], viral [55,56] and parasitic infections [57][58][59][60][61]. However, less research has been carried out on immunological markers, which should be indicative of general health in seabirds [62][63][64][65][66]. This compares with poultry, for example, Biology 2020, 9, 15 3 of 44 in which acute-phase proteins have been studied because of commercial interests in minimizing disease outbreaks on farms [67][68][69][70][71]. As seabirds provide an ideal model for assessment of environmental changes and belong to the most globally threatened of all groups of birds [40,41], the identification of novel biomarkers to assess their health status is of pivotal importance.
In the current baseline study, plasma EV profiles were assessed in one individual from eight seabird species representing two avian orders: Procellariiformes (albatrosses and petrels) and Charadriiformes (waders, auks, gulls and skuas). Furthermore, deiminated protein profiles were assessed in plasma of three species. Our findings indicate some differences in EV profiles and reveal that a range of key immune and metabolic proteins are post-translationally deiminated in plasma of seabirds. Our findings further current understanding of moonlighting functions of such proteins both in physiological and pathophysiological processes in birds. In addition, EVs and deimination profiles have potential value as novel biomarkers to assess immunological and general health status of seabirds.

Sampling of Seabird Plasma
Blood was collected from one adult individual of each of the following eight seabird species during the breeding season: wandering albatross (Diomedea exulans), grey-headed albatross (Thalassarche chrysostoma), black-browed albatross (Thalassarche melanophris), southern giant petrel (Macronectes giganteus), northern giant petrel (Macronectes halli), white-chinned petrel (Procellaria aequinoctialis) and brown skua (Stercorarius antarcticus) at Bird Island, South Georgia (54 • 00 S, 38 • 03 W), and south polar skua (Stercorarius maccormicki) at Rothera Point, Adelaide Island (67 • 04 S, 68 • 07 W). All individuals appeared to be good health at the time of sampling. Sample collection was approved by the British Antarctic Survey Animal Welfare and Ethical Review Committee and conducted under permits from the Government of South Georgia and the South Sandwich Islands, and UK Foreign and Commonwealth Office. Volumes of 1.0-2.0 mL of blood per bird (one bird per species) were collected in lithium heparin paediatric tubes, and plasma was separated by centrifuging at 750× g for 10 min. Sampling conditions, procedures and processing were similar in all cases, and should therefore not contribute to sample variation. Plasma was immediately frozen at −20 • C until further use. EVs isolated from the individual bird plasma sample were characterised by size exclusion using nanoparticle tracking analysis (NTA), by Western blotting, using EV-specific protein markers and by morphological analysis using transmission electron microscopy (TEM).

Extracellular Vesicle Isolation and NTA Analysis
Plasma samples from individual birds, were thawed and EVs isolated by step-wise centrifugation according to established protocols using ultracentrifugation and the recommendations of MISEV2018 (the minimal information for studies of extracellular vesicles 2018; [72]). The plasma was diluted 1:4 in ultrafiltered (using a 0.22 µm filter) Dulbecco's PBS (250 µL plasma added to 750 µL DPBS) and then centrifuged at 4000× g for 30 min at 4 • C for removal of aggregates and apoptotic bodies. The supernatant was collected and centrifuged at 100,000× g for 1 h at 4 • C. The resulting EV-enriched pellet was resuspended in DPBS, centrifuged again at 100,000× g for 1 h at 4 • C and thereafter resuspended in 100 µL DPBS and frozen at −80 • C until further analysis. For nanoparticle tracking analysis (NTA), each EV pellet was diluted 1/100 in DPBS (10 µL EV pellet diluted in 990 µL DPBS) and analysed by NTA, based on Brownian motion of particles in suspension [73], using the NanoSight NS300 system (Malvern Panalytical Ltd., Malvern, UK). The NanoSight system was used in conjunction with a syringe pump to ensure continuous flow of the sample, with approximately 40-60 particles per frame and videos recorded for 5 × 60 s. Replicate histograms generated from the recordings were averaged using the Nanosight NS300 software (Malvern).

Transmission Electron Microscopy (TEM)
The EV pellets obtained from plasma, as described above for each individual, were fixed with 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer (pH 7.0) for 1 h at 4 • C. EVs were then resuspended in 100 mM sodium cacodylate buffer (pH 7.0) and placed on to a grid with a glow-discharged carbon support film. The EVs were stained with 2% aqueous Uranyl Acetate (Sigma-Aldrich, Gillingham, UK) and imaged by using transmission electron microscopy (TEM) with a Morada CCD camera (EMSIS GmbH, Münster, Germany), processed via iTEM (EMSIS).

Immunoprecipitation and Protein Identification
Total deiminated proteins were isolated by immunoprecipitation from plasma of the following three species, representing three taxonomic families: wandering albatross (Diomedeidae), northern giant petrel (Procellariidae) and south polar skua (Stercorariidae). The Catch and Release ® v2.0 immunoprecipitation kit (Merck, Watford, UK) was used together with the F95 pan-deimination antibody (MABN328, Merck), which has been developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination/deimination [74]. For F95 enrichment, 50 µL of plasma was used from each bird and immunoprecipitation was carried out on a rotating platform overnight at 4 • C, according to the manufacturer's instructions (Merck). The F95 bound proteins were eluted using denaturing elution buffer (Merck), according to the manufacturer's instructions, and thereafter analysed by Western blotting and by liquid chromatography with tandem mass spectrometry (LC-MS/MS) (Cambridge Proteomics, Cambridge, UK). For LC-MS/MS, the F95-enriched eluates were run 0.5 cm into a 12% TGX gel (BioRad) and each cut out as one band. The 1D gel bands were transferred into a 96-well PCR plate. The bands were cut into 1 mm 2 pieces, destained, reduced (DTT) and alkylated (iodoacetamide) and subjected to enzymatic digestion with trypsin overnight at 37 • C. After digestion, the supernatant was pipetted into a sample vial and loaded onto an autosampler for automated LC-MS/MS analysis. All LC-MS/MS experiments were performed using a Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scientific Inc., Waltham, MA, USA) system and a QExactive Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Separation of peptides was performed by reverse-phase chromatography at a flow rate of 300 nL/min and a Thermo Scientific reverse-phase nano Easy-Spray column (Thermo Scientific PepMap C18, 2 µm particle size, 100 A Biology 2020, 9, 15 5 of 44 pore size, 75 µm i.d. × 50 cm length). Peptides were loaded onto a pre-column (Thermo Scientific PepMap 100 C18, 5 µm particle size, 100 A pore size, 300 µm i.d. × 5 mm length) from the Ultimate 3000 autosampler with 0.1% formic acid for 3 min at a flow rate of 10 µL/min. After this period, the column valve was switched to allow elution of peptides from the pre-column onto the analytical column. Solvent A was water + 0.1% formic acid and solvent B was 80% acetonitrile, 20% water + 0.1% formic acid. The linear gradient employed was 2-40% B in 30 min. The LC eluant was sprayed into the mass spectrometer by means of an Easy-Spray source (Thermo Fisher Scientific Inc.). All m/z values of eluting ions were measured in an Orbitrap mass analyzer, set at a resolution of 70,000 and was scanned between m/z 380 and 1500. Data dependent scans (Top 20) were employed to automatically isolate and generate fragment ions by higher energy collisional dissociation (HCD, NCE:25%) in the HCD collision cell and measurement of the resulting fragment ions was performed in the Orbitrap analyser, set at a resolution of 17,500. Singly charged ions and ions with unassigned charge states were excluded from being selected for MS/MS and a dynamic exclusion window of 20 s was employed. Post-run, the data was processed using Protein Discoverer (version 2.1., Thermo Scientific). Briefly, all MS/MS data were converted to mgf files and the files were then submitted to the Mascot search algorithm (Matrix Science, London, UK) and due to low annotation of species-specific databases the hit search was carried out against the UniProt Aves database: CCP_Aves_class Aves_class_20190709 (876,224 sequences; 364,491,521 residues) and a common contaminant sequences database (123 sequences; 40,594 residues). The peptide and fragment mass tolerances were set to 20 ppm and 0.1 Da, respectively. A significance threshold value of p < 0.05 and a peptide cut-off score of 20 were also applied.
Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis (https://string-db.org/) was used for the identification of putative protein-protein interaction networks for the deiminated proteins identified in northern giant petrel, south polar skua and wandering albatross. Due to lack of species-specific proteins in the STRING database, protein-interaction network analysis was based on human protein identifiers. Protein networks were built by using the function of "search multiple proteins" in STRING and applying basic settings and medium confidence, with colour lines between nodes indicating evidence-based interactions for network edges as follows: known interactions (based on curated databases, experimentally determined), predicted interactions (based on gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology. Coloured nodes in the analysis represent query proteins and first shell of interactors; white nodes represent second shell of interactors.

Statistical Analysis
Histograms and Nanosight graphs were prepared using GraphPad Prism version 7 (GraphPad Software, San Diego, CA, USA) and the Nanosight NS300 software (Malvern, UK). Histograms represent mean of data and standard error of mean (SEM) is indicated by the error bars.

Extracellular Vesicle Analysis in Seabird Plasma
EVs from the individual seabird plasma were characterised by size exclusion using NTA ( Figure 1A-H), by Western blotting using EV-specific protein markers ( Figure 1A-H) and by morphological analysis using transmission electron microscopy (TEM) ( Figure 1I). A poly-dispersed population of EVs, overall in the size range of 30 to 500 nm, was observed in plasma of all eight individuals/species, with some differences observed in size distribution profiles ( Figure 1A-H). The main EV peaks in plasma were as follows: wandering albatross (48,120,198,280, 347 and 413 nm); grey-headed albatross (72,93,144,183, 297 and 355 nm); black-browed albatross (70, 87, 153,  with some differences observed in size distribution profiles ( Figure 1A-H). The main EV peaks in plasma were as follows: wandering albatross (48,120,198,280, 347 and 413 nm); grey-headed albatross (72,93,144,183, 297 and 355 nm); black-browed albatross (70,87,153, 257 and 310 nm); northern giant petrel (88, 201 and 289 nm); southern giant petrel (67, 119, 344 and 424 nm); whitechinned petrel (110,145, 238 and 380 nm); brown skua (136, 264 and 432 nm); south polar skua (104, 156, 211 and 239 nm); ( Figure 1A-H). Western blotting analysis confirmed that the plasma EVs isolated from all 8 species were positive for the EV-specific markers CD63 and Flot-1 ( Figure 1A-H, see inserted WB figures). Comparing the EV profiles, modal size ranged from 80 to 140 nm; the largest EVs were found in the brown skua ( Figure 2A). The yield of EVs isolated from the seabird plasma also varied and was highest in the brown skua (9 × 10 10 particles/mL), while the proportionally lowest EV yield was found in the wandering albatross and white-chinned petrel (1.5 × 10 10 particles/mL) ( Figure 2B). Comparing the EV profiles, modal size ranged from 80 to 140 nm; the largest EVs were found in the brown skua ( Figure 2A). The yield of EVs isolated from the seabird plasma also varied and was highest in the brown skua (9 × 10 10 particles/mL), while the proportionally lowest EV yield was found in the wandering albatross and white-chinned petrel (1.5 × 10 10 particles/mL) ( Figure 2B).

Deiminated Proteins and PAD in Seabird Plasma
Total deiminated proteins in the seabird plasma (one representative individual per species) were detected using the F95 pan-deimination antibody, revealing a range of proteins between 10 and 250 kDa by Western blotting analysis ( Figure 3A). PAD homologues were identified in seabird plasma by Western blotting ( Figure 3B) via cross reaction with anti-human PAD2 and PAD3 antibodies and detected at an expected approximate 75 kDa size (Figure 3(B.1,B.2)). The plasma-derived EVs were positive for deiminated proteins as assessed by Western blotting, using the pan-deimination F95 antibody ( Figure 3C), and therefore confirming EV-mediated export of deiminated proteins.

Deiminated Proteins and PAD in Seabird Plasma
Total deiminated proteins in the seabird plasma (one representative individual per species) were detected using the F95 pan-deimination antibody, revealing a range of proteins between 10 and 250 kDa by Western blotting analysis ( Figure 3A). PAD homologues were identified in seabird plasma by Western blotting ( Figure 3B) via cross reaction with anti-human PAD2 and PAD3 antibodies and detected at an expected approximate 75 kDa size (Figure 3(B.1,B.2)). The plasma-derived EVs were positive for deiminated proteins as assessed by Western blotting, using the pan-deimination F95 antibody ( Figure 3C), and therefore confirming EV-mediated export of deiminated proteins.

Deiminated Proteins and PAD in Seabird Plasma
Total deiminated proteins in the seabird plasma (one representative individual per species) were detected using the F95 pan-deimination antibody, revealing a range of proteins between 10 and 250 kDa by Western blotting analysis ( Figure 3A). PAD homologues were identified in seabird plasma by Western blotting ( Figure 3B) via cross reaction with anti-human PAD2 and PAD3 antibodies and detected at an expected approximate 75 kDa size (Figure 3(B.1,B.2)). The plasma-derived EVs were positive for deiminated proteins as assessed by Western blotting, using the pan-deimination F95 antibody ( Figure 3C), and therefore confirming EV-mediated export of deiminated proteins.  Deiminated protein candidates were further identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, following F95 enrichment, in three of the bird species under study, with 26, 53 and 67 deimination protein candidate hits (including unidentified protein hits) identified for wandering albatross, northern giant petrel and south polar skua, respectively, whereof 15 hits were shared between all three species ( Figure 4). Biology 2020, 9, x FOR PEER REVIEW 8 of 42 in plasma-EVs isolated from all eight bird species tested in this study. This confirms EV-mediated export of deiminated proteins.
Deiminated protein candidates were further identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, following F95 enrichment, in three of the bird species under study, with 26, 53 and 67 deimination protein candidate hits (including unidentified protein hits) identified for wandering albatross, northern giant petrel and south polar skua, respectively, whereof 15 hits were shared between all three species ( Figure 4). Details for deiminated protein hits identified in plasma of the three bird species, with homology to the Aves database, are listed in Tables 1-3 (and Supplementary Tables S1-S3), respectively. Table 1. Deiminated protein hits identified by F95 enrichment in plasma of northern giant petrel (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pandeimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in northern giant petrel only, and not in wandering albatross or south polar skua are listed first and highlighted in light green and with an asterix (*). For full LC-MS/MS data analysis, see Supplementary Table S1  Details for deiminated protein hits identified in plasma of the three bird species, with homology to the Aves database, are listed in Tables 1-3 (and Supplementary Tables S1-S3), respectively. Table 1. Deiminated protein hits identified by F95 enrichment in plasma of northern giant petrel (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in northern giant petrel only, and not in wandering albatross or south polar skua are listed first and highlighted in light green and with an asterix (*). For full LC-MS/MS data analysis, see Supplementary Table S1.

Protein Name
Species Name Common Name Total Score (p < 0.05) Biology 2020, 9, x FOR PEER REVIEW 8 of 42 in plasma-EVs isolated from all eight bird species tested in this study. This confirms EV-mediated export of deiminated proteins.
Deiminated protein candidates were further identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, following F95 enrichment, in three of the bird species under study, with 26, 53 and 67 deimination protein candidate hits (including unidentified protein hits) identified for wandering albatross, northern giant petrel and south polar skua, respectively, whereof 15 hits were shared between all three species ( Figure 4). Details for deiminated protein hits identified in plasma of the three bird species, with homology to the Aves database, are listed in Tables 1-3 (and Supplementary Tables S1-S3), respectively. Table 1. Deiminated protein hits identified by F95 enrichment in plasma of northern giant petrel (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pandeimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in northern giant petrel only, and not in wandering albatross or south polar skua are listed first and highlighted in light green and with an asterix (*). For full LC-MS/MS data analysis, see Supplementary Table S1 (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pandeimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in northern giant petrel only, and not in wandering albatross or south polar skua are listed first and highlighted in light green and with an asterix (*). For full LC-MS/MS data analysis, see Supplementary Table S1. (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pandeimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in northern giant petrel only, and not in wandering albatross or south polar skua are listed first and highlighted in light green and with an asterix (*). For full LC-MS/MS data analysis, see Supplementary Table S1. Ions score is −10. * Log (P), where P is the probability that the observed match is a random event. Individual ions scores > 40 indicated identity or extensive homology (p < 0.05). Protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. Table 2. Deiminated proteins identified by F95 enrichment in plasma of south polar skua (Stercorarius maccormicki). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are listed and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in south polar skua only, and not in northern giant petrel or wandering albatross are listed first and highlighted in light blue and with an asterix (*). For full LC-MS/MS data analysis, see Supplementary Table S2.

Protein Name
Species Name Common Name Total Score (p < 0.05) Biology 2020, 9, x FOR PEER REVIEW 8 of 42 in plasma-EVs isolated from all eight bird species tested in this study. This confirms EV-mediated export of deiminated proteins.
Deiminated protein candidates were further identified by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, following F95 enrichment, in three of the bird species under study, with 26, 53 and 67 deimination protein candidate hits (including unidentified protein hits) identified for wandering albatross, northern giant petrel and south polar skua, respectively, whereof 15 hits were shared between all three species (Figure 4). Details for deiminated protein hits identified in plasma of the three bird species, with homology to the Aves database, are listed in Tables 1-3 (and Supplementary Tables S1-S3), respectively.      (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pandeimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in northern giant petrel only, and not in wandering albatross or south polar skua are listed first and highlighted in light green and with an asterix (* Ions score is −10. * Log (P), where P is the probability that the observed match is a random event. Individual ions scores > 22 indicated identity or extensive homology (p < 0.05). Protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. Table 3. Deiminated proteins identified by F95 enrichment in plasma of wandering albatross (Diomedea exulans). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are listed and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits that were identified as deiminated in wandering albatross only, but not in northern giant petrel or south polar skua, are listed first and highlighted in pink and with an asterix (*). For full LC-MS/MS data analysis, see Supplementary Table S3.

Protein Name
Species Name Common Name Total Score (p < 0.05) Details for deiminated protein hits identified in plasma of the three bird species, with homology to the Aves database, are listed in Tables 1-3 (and Supplementary Tables S1-S3), respectively.  (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pandeimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown and total score is reported. Protein hits with Aves are indicated, including species name. Protein hits which were identified as deiminated in northern giant petrel only, and not in wandering albatross or south polar skua are listed first and highlighted in light green and with an asterix (*    Ions score is −10. * Log (P), where P is the probability that the observed match is a random event. Individual ions scores > 22 indicated identity or extensive homology (p < 0.05). Protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits.

Protein-protein Network Interaction Analysis for Deiminated Proteins in Seabird Plasma
Based on Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, the PPI enrichment p-value for the deiminated proteins identified in wandering albatross, northern giant petrel and south polar skua was < 1.0 × 10 −16 for all three species. Human protein homologues were used for the assessment of the protein interaction networks (Figures 5-7) due to a lack of annotations of species-specific bird protein annotations in STRING. For all three seabird species, some of the same biological GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified for deiminated proteins and these included vesicle-mediated transport, protein metabolic processes, response to wounding and wound healing, stress and immune system processes, including complement coagulation cascade, bacterial infection defence pathways (Staphylococcus aureus) and cholesterol metabolism (Figures 5-7). There were some species-specific differences observed for deiminated protein candidates, as biological GO pathways for signal transduction and KEGG pathways for deiminated proteins involved in fat digestion and absorption were observed in northern giant petrel only ( Figure 5A,B).
In south polar skua only, deiminated proteins involved in organonitrogen compound metabolic process were identified, as well as pathways for pantothenate and Coenzyme A (CoA) biosynthesis ( Figure 6A,B).
In the wandering albatross only, specific deimination positive pathways were identified involving biological GO pathways for post-translational modification, plasma lipoprotein particle remodelling and KEGG pathways for Chagas disease, regulation of actin cytoskeleton and extracellular matrix (ECM) receptor interaction ( Figure 7A,B).
Some pathways were common for two of the species under study, such as KEGG pathways for platelet activation as well as vitamin digestion and absorption for northern giant petrel and south polar skua; while KEGG pathways for prion diseases, systemic lupus erythematousus, pertussis were common to both south polar skua and wandering albatross (Figures 6 and 7); and peroxisome proliferator-activated receptor (PPAR) signalling pathway common for northern giant petrel and wandering albatross (Figures 5 and 7).
(Staphylococcus aureus) and cholesterol metabolism (Figures 5-7). There were some species-specific differences observed for deiminated protein candidates, as biological GO pathways for signal transduction and KEGG pathways for deiminated proteins involved in fat digestion and absorption were observed in northern giant petrel only ( Figure 5A,B).  Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines and for nodes indicating the specific GO and KEGG pathways included in the figure). complement and coagulation cascade; platelet activation; PPAR signalling pathway; fat digestion and absorption; Staphylococcus aureus infection; cholesterol metabolism and vitamin digestion and absorption. Coloured nodes represent query proteins and first shell of interactors; white nodes are second shell of interactors. Coloured lines indicate whether protein interactions are identified via known interactions (curated databases, experimentally determined), predicted interactions (gene neighbourhood, gene fusion, gene co-occurrence) or via text mining, co-expression or protein homology (see the colour key for connective lines and for nodes indicating the specific GO and KEGG pathways included in the figure).
In south polar skua only, deiminated proteins involved in organonitrogen compound metabolic process were identified, as well as pathways for pantothenate and Coenzyme A (CoA) biosynthesis ( Figure 6A,B).

Discussion
The current study describes, for the first time, extracellular vesicle (EV) and deiminated protein profiles in the plasma of a range of Antarctic seabirds, representing three families from two orders (Procellariiformes and Charadriiformes), and two breeding locations (South Georgia and Adelaide Island). Although the analysis was of only one individual from each species (while representing eight species from two avian orders) and therefore the level of intraspecific variation is unknown, our findings nevertheless highlight novel aspects of post-translational deimination in key proteins with functions in innate and adaptive immunity, wound healing and signal transduction, as well as proteins involved in a range of metabolic pathways. As studies on protein deimination in birds are mainly limited to CNS regeneration studies in Gallus gallus [16], the current findings provide a first baseline for putative protein moonlighting functions in seabirds via protein deimination. These are hitherto unidentified contributors to different physiological and immunological responses, in the target protein identified in these seabirds, and provide novel insights also into putative species-specific differences. Furthermore, EV profiles in plasma of these seabirds were analysed, showing EVs positive for phylogenetically conserved EV-specific markers and displaying typical EV morphology. We observed some differences in EV size distribution profiles in the diverse seabird species under study, including for example, narrower EV profiles of 50-200 nm in the northern giant petrel, and a higher abundance of larger EVs in the brown skua.
Using antibodies against human PAD2 and PAD3, respectively, peptidylarginine deiminase (PAD) homologues were identified in the seabird plasma by Western blotting for PAD2, which is the phylogenetically most conserved PAD form [1,6], as well as for PAD3, at an expected 70-75 kDa size, similar to that observed for mammalian PADs and Gallus gallus PAD3 [16]. This indicates the presence of more than one PAD isozyme in these birds and is in line with previous studies in Gallus gallus [1,16]. A range of deiminated proteins identified in the seabird plasma in our study, using F95 enrichment and LC-MS/MS analysis, included key proteins involved in immunity, protein synthesis, response to infection, cell signalling and metabolism.
Based on Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, the PPI enrichment p-value for the deiminated proteins identified in northern giant petrel, south polar skua and wandering albatross, respectively, was <1.0 × 10 −16 for all three species. Such an enrichment value indicates that the identified network of proteins has significantly more interactions than expected for a random set of proteins of similar size, drawn from the genome. Such an enrichment indicates that the proteins as a group are at least partly connected in terms of their biological function. For all three seabird species assessed, some of the same biological GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were identified for deiminated proteins; however, some species-specific differences between the three birds for GO and KEGG pathways were also observed ( Figures 5-7). This indicates that target proteins for deimination may differ between species and possibly contribute to a range of physiological functions including metabolism and immunity, as well as to some key defence mechanisms. Such post-translational deimination may play important roles for their moonlighting roles in physiological and pathophysiological processes. Shared proteins identified to be deiminated in all three species of seabirds assessed were serum albumin, apolipoprotein A-I, fibrinogen, kininogen-1, alpha-2-macroblogulin, complement C3, Factor H, comlement C1q, immunoglobulin, ceruloplasmin, fibronectin, ovotransferrin, alpha-1-antiproteinase 2 and selenoprotein P. Main proteins identified here, and their key roles in immunity and metabolism, are as follows and discussed in relation to Aves where information is available, as well as from a comparative angle with regard to human pathologies. Shared hits between the three seabird species are listed first: Serum albumin is a known glycoprotein in some species and is a major acidic plasma protein in vertebrates and serves as a transport molecule for fatty acids, bilirubin, steroids, amino acids and copper, as well as having roles in maintaining the colloid osmotic pressure of blood [75]. Albumin belongs to acute phase proteins that have been studied in birds, particularly chicken (Gallus gallus) [67].
Apolipoprotein A-I, Apolipoprotein AI-V and Apolipoprotein B-100 were all found to be deiminated in seabird plasma. Apolipoprotein A-I is primarily involved in lipid metabolism and associated with regulation of mitochondrial function and bioenergetics [76,77]. ApoA-I has a regulatory role in the complement system in various species [78][79][80]. ApoA-IV is a lipid binding protein, primarily synthesized in the small intestine and involved in a range of physiological proteins including lipid absorption and metabolism, glucose homeostasis, platelet aggregation and thrombosis [81]. ApoB-100 is synthesised by the liver, plays a part in innate immune responses [82] (Peterson et al., 2008), and is associated with endoplasmic reticulum (ER) stress and insulin resistance [83], as well as lipid metabolism disorders [84].
Fibrinogen is a glycoprotein, synthesised in liver and forms part of the acute phase response as part of the coagulation cascade [85]. Fibrinogen has diverse functions, including roles in the immune defence and has for example been associated with host defences against pathogens, as well as in acute phase and stress responses and in toxicity [86,87]. In humans, various fibrinogen disorders are known, related to coagulopathies, ischemic stroke, cancer, liver disease or post-translational modifications [88]. Fibrinogen is a known deimination candidate and this post-translational modification contributes for example to its antigenicity in autoimmune diseases [89,90]. In birds, fibrinogen is a known acute phase protein, and particularly studied in chickens, also in response to immune challenge and infection [67].
Kininogen-1 forms part of the acute phase response. In mammals, elevated levels of kininogen are linked to sepsis [91]. Roles in inflammatory and oxidative stress pathways have also been described [92].
A range of complement components was deiminated in the plasma of our study species, including complement components C3, C4 and C9, which are central to the the alternative and classical pathways and participate in formation of the membrane attack complex (MAC). Furthermore, regulatory factors of the complement system, receptors and recognition molecules, including Factor H, C4b-binding protein, complement receptor type 2, Complement C1q tumour necrosis factor-related protein 3 isoform A and C4b-binding protein alpha chain were also deiminated. These complement proteins are involved both in the alternative and classical complement pathway and contribute to cell lysis as well as being implicated in clearance of apoptotic cells, tissue remodelling and host-defences against pathogens and in infection [7,[97][98][99][100]. The identification of post-translational modification of these complement components, which include central complement components, recognition molecules and complement regulatory proteins, is of considerable interest in the light of the multifaceted functions of complement components and the diversification of the complement system throughout phylogeny [98,101,102]. Recently such post-translational deimination of a range of complement components was recognized in both bony and cartilaginous fish [6,7,9,35]. There was a difference in complement component deimination hits observed between the seabird species assessed here, as C1q, C3 and Factor H were identified in all three bird species, while C4, which belongs to the classical pathway, was only found deiminated in wandering albatross, and C9, which forms part of the final complement lysis MAC complex, was only found deiminated in south polar skua. Complement receptor type 2 was found deiminated in northern giant petrel and south polar skua, but not in wandering albatross, while C4b-binding protein was found deiminated in south polar skua only. The role of post-translational deimination for complement protein moonlighting in birds, and its contribution to diverse functions of the complement system in physiological and pathophysiological processes, as well as complement diversification throughout phylogeny remains to be elucidated.
A range of immunoglobulins was found to be deiminated in all three seabird species tested. This included IgGFc-binding protein, Ig lambda-1 chain C regions, Ig gamma-1 chain C region, membrane-bound form and Ig heavy chain V-III region KOL. Ig's are key molecules in adaptive immunity and studied in diverse taxa. Post-translational deimination of Ig's and roles in Ig function have hitherto received little attention except in teleosts and cartilaginous fish [6,7,9], as well as in the llama Lama glama [10] and cetaceans [24]. In human patients with bronchiectasis and RA, the IgG Fc region is post-translationally deiminated [103]. Given the increased interest in furthering understanding of Ig diversity throughout the phylogenetic tree [104][105][106][107] our current finding of deimination of bird Ig's highlights a novel concept of diversification of Ig function via post-translational deimination.
Ceruloplasmin was found to be deiminated in all three seabird species tested. It is a serum ferroxidase with antioxidative function and highly conserved throughout vertebrate evolution. It carries the majority of copper in plasma and has roles in iron homeostasis [108,109]. In birds it has been identified as an inflammatory marker associated with trauma and infection [110] and studied as an acute-phase protein biomarker in broiler breeding lines [67,70].
Fibronectin is an important part of the extracellular matrix and is a hepatic glycoprotein protein which constitutes a major protein component of blood plasma. It has major roles in cell migration, differentiation, migration and growth and plays important roles in wound healing, as well as in embryogenesis [111,112]. Fibronectin is associated with a number of pathologies, including cancer and fibrosis [113]. Fibronectin has been previously found to be deiminated in various sites, which has been related to autoimmunity [114], and also found to support wound healing [115]. In birds, fibronectin is an acute phase protein in chickens (Gallus gallus), responding to infection and changes in temperature [67].
Ovotransferrin is an iron-binding glycoprotein, belonging to the transferring family of iron-binding glycoproteins. In birds, ovotransferrin is the only form and present in both plasma and egg albumen, while in mammals two forms of transferrin (serum transferrin and lactoferrin), with different functions exist [116]. As ovotransferrin has multifaceted functions and plays major roles in avian natural immunity [67,70,116,117], post-translational deimination may contribute to its diverse functions.
Alpha-1-antiproteinase 2 belongs to the serpin superfamily, is a protease inhibitor protecting tissues from enzymes of inflammatory cells, and an acute-phase protein, levels of which rise upon acute inflammation [118][119][120]. While it is a known glycoprotein [121], post-translational deimination has not been reported before.
Selenoprotein P (Sepp1) is a plasma glycoprotein, mainly secreted from liver but also other tissues and contains most of the selenium in plasma [122]. It has antioxidant properties [122] and serves in homeostasis and distribution of selenium [123]. In birds, selenoprotein has been shown to be important in immune responses [124] and to be protective against growth inhibition, including nutritional muscular dystrophy [125], as well as oxidative damage and apoptosis in response to fluorine [126]. Phylogenetically, Sepp1 is believed to have appeared in early metazoan species [127]. While Sepp1 is known to be glycosylated, little is understood about roles for post-translational deimination for its function.
Hemoglobin was found deiminated in northern giant petrel and south polar skua plasma. It is a key molecule in molecular oxygen transport in the bloodstream. In the south polar skua, two haemoglobins had peculiar functional features including additional phosphate binding sites, possible as an adaption to extreme environmental conditions [128,129]. Post-translational modifications, including deimination identified here, may further add to such functional adaptions.
Vitamin D-binding protein was found deiminated in wandering albatross plasma. It is a multifaceted protein mainly produced in the liver, where its regulation is influenced by oestrogen, glucocorticoids and inflammatory cytokines [130]. It is secreted into the blood circulation and is able to bind the various forms of vitamin D [131]. It is at higher levels in geese during the laying than pre-laying period, indicative of roles in lipid metabolism related to egg formation [132]. In humans, VDBP is implicated in cancer and coronary artery disease [133,134]. VDBP has previously been identified to be glycosylated [135] and post-translational deimination identified here may further add to its functional diversity.
Vitronectin (VTN) was found deiminated in wandering albatross plasma. It is a glycoprotein of the hemopexin family, which is abundant in serum, the extracellular matrix and in bone. In mammals, VTN is a key controller of tissue repair and remodelling activity [136]. It promotes cell adhesion and spreading, and furthermore inhibits the membrane-damaging effect of the terminal cytolytic complement pathway and binds to several serine protease inhibitors [137,138]. Roles for VTN in haemostasis and tumour malignancy have also been described [139,140]. Noelin (olfactomedin 1 or pancortin) was found deiminated in wandering albatross plasma. It is a member of the olfactomedin domain-containing superfamily and a highly expressed neuronal glycoprotein important for nervous system development [141]. It binds a range of secreted proteins and cell surface-bound receptors for induction of cell signalling processes and its structure has been described in detail [142]. Noelin also plays important roles in synaptic plasticity [143]. It is also related to growth and metastasis suppression of colorectal cancer [144] and linked to epithelial-mesenchymal transition in the chick embryonic heart [145]. Deimination of noelin identified here has not been studied before and provides a new aspect of multifaceted functions of noelin via such post-translational modification.
Histidine-rich glycoprotein was found deiminated in plasma of northern giant petrel and south polar skua. It is a multifaceted glycoprotein which is synthesised in the liver and also white blood cells [146] and is located in plasma and platelets, where it binds amongst other heme and metal ions [147]. It has numerous biological functions including in immunity, vascularisation and coagulation [148,149]. Due to roles in angiogenesis, which can be both pro-and anti-angiogenic, it is also implicated in cancer [150]. Furthermore, it also plays roles in infection and has selective antibacterial activity [151].
Protein NEL also known as protein kinase C-binding protein, was found deiminated in south polar skua plasma only. It has a broad array of cellular functions [152]. It is a cytoplasmic glycoprotein involved in cell growth regulation and differentiation, and roles in neural function and development have been described in the chick [153,154].
Plasma serine protease inhibitor was found deiminated in south polar skua plasma. It belongs to the serpins, which have multifaceted roles via protease inhibition activity, including in blood clotting, inflammatory and immune responses [119,155,156]. As the protease inhibitor effects of serpins are achieved through conformational changes, also involving beta-sheets [157], this may be of considerable interest as beta-sheets belong to structures prone to post-translational deimination [2].
Glutathione peroxidase was found deiminated in south polar skua and wandering albatross plasma. It forms part of the glutathione (GSH) biosynthesis pathway involved in homeostasis and cellular maintenance and also acts as a potent antioxidant [158].
Pantetheinase, also known as non-inflammatory molecule-1 (vanin 1), was found deiminated in south polar skua plasma. It has physiological roles related to coenzyme A (CoA) metabolism, lipid metabolism, and energy production [159,160]. It also has a range of roles in relation to oxidative stress and inflammation in developmental, repair and inflammatory processes, contributing to tissue tolerance to stress, and is related to a range of human pathologies [160,161].
Vascular non-inflammatory molecule 3 also belongs to the vanin family of encoding pantetheinase isoforms and was found deiminated in south polar skua plasma. It has physiological roles in metabolising proteins, carbohydrates and fats [162]. It also has roles in inflammatory pathways via neutrophils and the induction of proinflammatory cytokines [163,164], and has been identified as a biomarker in acute graft-versus-host disease [165].
Beta-2-glycoprotein 1 (β2GPI) was found deiminated in south polar skua plasma. It is a circulating blood protein with several essential physiological roles, including in haemostasis, homeostasis and immunity [166]. It furthermore is associated with autoimmunity [167] and has anti-bacterial effects [168,169]. The diverse function of β2GPI, including its dual capability to up-and down-regulate the complement and coagulation systems depending upon external stimulus [170] may reflect hitherto unrecognised structural modifications via post-translational deimination, and be related to glycolysis [167].
Inter-alpha-trypsin inhibitor was found deiminated in south polar skua plasma. It is an acute inflammatory marker [171] which functions as a protease inhibitor and is linked to a range of inflammatory responses [172], oxidative stress [173] and infection [174]. Furthermore, inter-α inhibitor proteins play roles in maintaining the resting state of neutrophils by regulating shape and reducing ROS production [175].
Leucine-rich repeat-containing protein 49 was here identified as post-translationally deiminated in south polar skua plasma. Leucine-rich repeat-containing proteins are linked to a range of functions including mitochondrial transcription [176] and inflammatory responses [177].
Deleted in malignant brain tumours 1 protein was found deiminated in wandering albatross plasma. It is a glycoprotein containing multiple scavenger receptor cysteine-rich (SRCR) domains and is related to cellular immune defences and mucosal immunity, as well as to regeneration [178][179][180]. It has dual functions in viral transmission [181] and displays a broad calcium-dependent binding spectrum against a range of bacterial pathogens [180,182]. Absence of the protein or glycosylation has been described in cancer [183][184][185][186], while roles for post-translational deimination in its multifaceted functions remain unknown. This may be of particular interest as DMBT1 shows a pattern recognition activity for poly-sulfated and poly-phosphorylated ligands, including nucleic acids, and also has the ability to aggregate ligands-properties which have made it a protein of interest for targeted nano-delivery [187]. Therefore, indication of structural changes of this protein via post-translational deimination may be of considerable interest.
Soluble scavenger receptor cysteine-rich domain-containing protein ssc5d-like was found deiminated in wandering albatross plasma. It plays a role in the innate defence and homeostasis [188]. It binds to extracellular matrix proteins and acts as a pattern recognition receptor (PRR) by binding to pathogen-associated molecular patterns (PAMPs) present on the cell walls of bacteria and fungi, subsequently inhibiting PAMP-induced cytokine release [189]. It is implicated in arthritis [190].
Corticosteroid binding globulin (CBG) was found deiminated in wandering albatross plasma. It is the primary cortisol binding protein capable of conformational change from a high cortisol-binding affinity form to a low affinity form [191]. The main role of CBG is in acute, severe inflammation where depletion is associated with mortality, and to chronic inflammation where defects in cortisol delivery may perpetuate inflammation [191,192]. Furthermore, it has roles in metabolism and neurocognitive function, implying that CBG is a multifaceted component in the mechanisms of hypothalamic-pituitary-adrenal axis related homeostasis [191]. In free-living birds, corticosterone may be involved in delaying the onset of breeding including via altering hormone titers, negative feedback regulation, plasma binding globulin concentrations, intracellular receptor concentrations, enzyme activity and interacting hormone systems [193]. It is also implicated in corticosterone regulation in the songbird brain [194]. Such diverse functions may indeed be facilitated via post-translational changes, including deimination recognised here.
Retinol-binding protein 4 (RBP4) was found deiminated in south polar skua and wandering albatross plasma. It is mainly synthesized in the liver and circulates in the bloodstream bound to retinol in a complex with transthyretin. It delivers retinol from the liver stores to the peripheral tissues [195]. RBP4 has recently been described as an adipokine that contributes to insulin resistance and diabetes [196], partly via activation of antigen-presenting cells [197]. RBP4 is also secreted by adipocytes of the fat tissue in a smaller portion and acts as a signal to surrounding cells, when there is a decrease in plasma glucose concentration [198]. While some post-translationally processed forms of human RBP4 have been implicated in in chronic renal failure [199], deimination has not been assessed.
Ubiquitin carboxyl-terminal hydrolase (UCH) was found deiminated in wandering albatross plasma. It is a deubiquitinating enzyme, essential for a variety of biological processes including cell growth, differentiation, transcriptional regulation, and oncogenesis [200]. It is highly specific to neurones and to cells of the diffuse neuroendocrine system, required for the maintenance of axonal integrity, and its dysfunction is implicated in neurodegenerative disease [201]. Furthermore, it is a reliable serum biomarker for outcome prediction in traumatic brain injury [202]. UCH also plays roles in protecting neurones against ZnO particle-induced neurotoxicity via modulation of the NF-κB signalling pathway [203]. While UCH has been found to have low expression in other healthy tissues, it is highly expressed in several forms of cancer. Interestingly, UCH enzymes can act both as a tumour suppressor and tumour promotor and influence several signalling pathways that play crucial roles in oncogenesis, tumour invasion, and migration [200]. There are also indications that UCHL1 contributes to metabolic response following thermal injury [204]. In birds, UCH enzymes have been described in chick muscle [205]. To our knowledge, post-translational deimination of UCH enzymes has not been described and may well contribute to the moonlighting functions of these hydrolases.
Collagen alpha-4 (VI) chain was found deiminated in wandering albatross plasma. It is an extracellular matrix protein [206], is found in lymphoid tissues [207] and has roles, amongst others, in cellular and mucosal immunity and inflammatory diseases such as ulcerative colitis and membranous glomerulonephritis [208,209]. In birds, it has roles in CNS development relating to plasticity and axon growth in the chicken [210].
SET and MYND domain-containing protein 4 was found deiminated in wandering albatross plasma. It belongs to the Smyd Family of Methyltransferases, which is recognized in diverse taxa [211]. SMYD can methylate histones and non-histone proteins and have diverse roles in chromatin remodelling as well as normal development, in cell growth and differentiation and in the regulation of a series of pathophysiological processes, including cardiac and skeletal muscle physiology and pathology and cancer [212][213][214]. As PADs cause deimination of several histones, the deimination of histone regulatory proteins, such as SMYD here, may be of considerable interest, particularly in the light of their multifaceted functions in regulating a range of histone and non-histone proteins, including histone H3 in Gallus gallus [16].
The current study describes for the first time post-translational deimination of a range of proteins involved in immunological and metabolic pathways in pelagic seabirds in the Antarctic. Besides novel insights into diverse protein functions through post-translational modifications in bird physiology, our findings also further knowledge of the translatable functions of PADs throughout the phylogenetic tree, informing comparative studies. Given the numerous and complex structural and functional changes that proteins can undergo via various post-translational modifications, the roles for post-translational deimination in protein moonlighting during physiological and pathophysiological processes, are a promising field for further studies. Similarly, roles for EV-mediated cellular communication in different animal groups is currently an expanding field of research.

Conclusions
Our findings unravel hitherto unrecognised biomarkers in Antarctic seabirds, which are likely to be indicative of immunological and metabolic functions, and possibly health status. The EV and deimination profiles generated in this study provide a suite of novel biomarkers with considerable potential for developing novel tools to assess seabird health status, as well as providing insights into phylogenetically conserved mechanisms in cellular communication via EV-mediated transport, further informing EV-mediated pathologies. Future research would involve assessing EV cargo, including deiminated EV cargo, in addition to overall plasma deimination biomarkers, in Antarctic seabirds at the individual level, in relation to environmental conditions, pollutant levels and past or recent immune challenges from pathogens. In addition, wider sampling within and between populations would provide further insights into effects of environmental variation, enabling comparisons with normal physiological protein deimination status and EV profiles. This would be particularly valuable for assessing natural and anthropogenic stresses in seabirds in general, many of which are declining and face increasing threats both on land and at sea related to changing climate [41]. While the current study lays a baseline for these novel biomarkers, future studies will need to further refine and develop these markers as an applicable tool in the evaluation of seabirds' health status.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-7737/9/1/15/s1. Table S1: LC-MS/MS analysis of F95-enriched protein hits identified in plasma of northern giant petrel (Macronectes halli). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown. Peptide sequences for individual protein hits, their m/z values and individual scores are listed. Table S2: LC-MS/MS analysis of F95-enriched protein hits identified in plasma of south polar skua (Stercorarius maccormicki). Deiminated proteins were isolated by immunoprecipitation using the pan-deimination F95 antibody. The F95-enriched eluate was analysed by LC-MS/MS and peak list files were submitted to mascot. Peptides matching with Aves_class_20190709 (876,224 sequences; 364,491,521 residues) are shown. Peptide sequences for individual protein hits, their m/z values and individual scores are listed. Table S3: