Mass Spectrometry Analysis of Shark Skin Proteins

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage in which proteins play a key role. While proteins in the skin mucus layer of various common bony fish species have been explored, the proteins of shark skin mucus remain unexplored. In this pilot study, we examine the protein composition of the skin mucus in spiny dogfish sharks and chain catsharks through mass spectrometry (NanoLC-MS/MS). Overall, we identified 206 and 72 proteins in spiny dogfish (Squalus acanthias) and chain catsharks (Scyliorhinus retifer), respectively. Categorization showed that the proteins belonged to diverse biological processes and that most proteins were cellular albeit a significant minority were secreted, indicative of mucosal immune roles. The secreted proteins are reviewed in detail with emphasis on their immune potentials. Moreover, STRING protein–protein association network analysis showed that proteins of closely related shark species were more similar as compared to a more distantly related shark and a bony fish, although there were also significant overlaps. This study contributes to the growing field of molecular shark studies and provides a foundation for further research into the functional roles and potential human biomedical implications of shark skin mucus proteins.


Introduction
Elasmobranchs, encompassing sharks as a prominent example, have garnered considerable research focus owing to conservation endeavors.However, their molecular biology remains a subject of immense scientific interest, despite the inherent challenges associated with experimental investigations.Prior investigations have yielded noteworthy findings with potential implications for human medicine.Notably, the liver and stomach of Atlantic spiny dogfish (Squalus acanthias) sharks unveiled the presence of the antibiotic squalamine [1], while research on chloride channels within the rectal gland of these sharks [2] has proven relevant to the study of cystic fibrosis.Furthermore, the denticle patterns found on the skin of shortfin mako sharks (Isurus oxyrinchus) have proven to be valuable in enhancing the aerodynamic properties of airplanes, specifically in terms of reducing drag and improving lift, which bears resemblance to how these denticle patterns enhance the swimming abilities of sharks in water [3].
One significant contrast between fish and mammalian skin is that the dead, keratinized protective layer of skin known as the stratum corneum is absent in nearly all fish species.Instead, fish epidermis is composed solely of living cells [4,5] and is shielded by a layer of mucus.This slimy substance comprises large glycoproteins called mucins that creates a scaffold for multiple other proteins and glycoproteins, some of which exhibit antimicrobial properties that aid in preventing the entry and establishment of pathogens [4,6,7].In mammalian infection models, increased susceptibility to infections have been demonstrated in animals lacking specific mucins [8][9][10][11].The mucus layer is created both through secretion by various secretory cell types present in the epidermis as well as sloughing of dead cells [12].
Despite the well-documented proteome of the mucus layer in certain common bony fish (Osteichthyes), likely due to the demands of the fish farming industry, our understanding of the proteome within elasmobranchs, the main subclass of cartilaginous fish (Chondrichthyes) including most sharks, remains limited.Shark skin boasts unique attributes, including its tooth-like denticles, suggesting the possibility of novel proteins in the mucus layer with distinctive properties and functions, such as pathogen defense.A characterization of the proteome in shark mucus represents a crucial initial stride toward unraveling its biological significance.Using liquid chromatography-electrospray ionization tandem mass spectrometry, we herein present the most comprehensive proteome description of shark mucus to date in two shark species representing different genera: Atlantic spiny dogfish (Squalus acanthias), one of the most common shark species worldwide, and the bottom-dwelling chain catshark (Scyliorhinus retifer).

Results and Discussion
Over the preceding decade, there has been a notable increase in the number of studies focused on Chondrichthyes, encompassing sharks and rays.These investigations have sought to elucidate their biological intricacies, primarily with a focus on conservationoriented research [13][14][15].Nevertheless, despite the significant importance of exploring Chondrichthyes for conservation and preservation efforts, as well as for translating their exceptional features for potential therapeutic applications, molecular understanding of sharks remains comparatively limited when juxtaposed with bony fish and mammals.
The skin mucus of the more prevalent bony fish (Osteichthyes) and its critical role in fish health has been examined utilizing proteomics methodologies in several studies.The primary emphasis has been on the identification and characterization of innate and adaptive immune system proteins [12,[16][17][18][19]. Sharks differ from bony fish in several aspects including cartilaginous skeleton and skin structure characterized by placoid scales (denticles) that reduce fluid friction, thereby enhancing swimming efficiency [20].The mucus layer of sharks is far less researched than in bony fish and is probably different due to dissimilar skin architecture as well as extensive evolutionary separation.In sharks, proteomics studies are scarce [21][22][23][24] perhaps due to the fact that less than 1% of Chondrichthyan species have a sequenced genome [25], which complicates analysis combined with practical experimental difficulties in handling sharks.In this work, we present, for the first time, the identification of proteins from shark skin mucus using mass spectrometry, with a focus on immune-relevant molecules.

Protein Categorization
Skin protein samples were harvested from spiny dogfish and chain catsharks, as shown in Figure 1 and described in Methods.By utilizing nanoscale liquid chromatography coupled with tandem mass spectrometry (NanoLC-MS/MS) a wide range of proteins were identified from the skin mucus of both shark species.The protein fragments were matched against the Swissprot human database and Uniprot Chondrichthyes database using Mascot 2.5.1, a tool useful for proteome analyses of relatively unexplored species such as sharks, which have very little molecular data available in public databases.Overall, we identified 206 and 72 proteins in spiny dogfish and chain catsharks, respectively, described in detail in Tables 1 and 2 as well as Supplementary Files S1 and S2.These proteins could, in principle, be from either cellular sloughing, a central process in skin physiology, or actively secreted to the mucus.The fact that more proteins were identified in spiny dogfish than catsharks is consistent with a previous study of ours in which less glycans were found in chain catshark skin and may be attributed to the tissue absorption sampling method, which was developed for teleost fish, or represent true biological differences such as a thinner mucus protein layer in chain catsharks [26].Due to the importance of the mucus as a defense barrier, we focused our attention on the proteins that may have immune system roles.Proteins were grouped into eight different clusters based on biological process annotation as described in Methods (Table 3 for dogfish and catsharks, respectively).Carbohydrate and protein metabolism represented almost 40% of the proteins detected, possibly reflective of active regulatory processes such as osmoregulation, respiration, nutrition, or locomotion, as well as defense against pathogens [27], while immune-related proteins represented almost 20%, conceivably reflecting mucus antimicrobial properties.These proportions resemble previously reported data in Atlantic cod [28].We further classified the proteins based on their type and found that 84% and 72% of the proteins in dogfish and catsharks, respectively, are cytoplasmic (including organelles and nucleus residential proteins) and that 19% and 31% are secreted proteins (Table 3 under "cellular location"), perhaps not surprising due to the fact that skin constantly turns over by sloughing.The secreted proteins are particularly interesting, as they include diverse classes of molecules such as mucins, immunoglobulins, proteases, and other proteins that have well-established roles in the immune system [29].In Tables 4 and 5 (secreted proteins), we characterize in detail the secreted proteins found in the sharks' skin mucus and below we discuss their potential roles, together with other immune-related proteins from other classified groups (see Tables 1 and 2).Table 3. Classification of proteins from the shark skin mucus.Proteins from spiny dogfish and from chain catsharks identified using LC-MS/MS.The proteins were clustered into different categories based on the gene ontology category "biological process".Further classification of protein type and cellular location) was carried out using UniProt data (www.oniprot.org)for individual proteins.Some proteins can be found in more than one cellular location and can also have more than one biological classification.Therefore, the sum of proteins from different classifications and locations can exceed the total number of proteins.Highly glycosylated and gel-forming macromolecular components of mucus secretions [30].Also named vWFD domain-containing protein, exhibiting an evolutionarily-conserved von Willebrand factor type D domain (vWD), found in mucins [31].

A0A401S584
Inter-alpha-trypsin inhibitor heavy chain H3 BBBS Heavy chain subunit of the pre-alpha-trypsin inhibitor complex.This complex stabilizes the extracellular matrix through its ability to bind hyaluronic acid, found in mucins (UniProt, [31]).

A0A4W3JXK3
Glucose-6-phosphate isomerase GS Induces immunoglobulin secretion [54] A0A4W3GT93 GDP-mannose 4,6dehydratase GS This enzyme converts GDP-mannose to GDP-4-dehydro-6-deoxy-D-mannose, the first of three steps for the conversion of GDP-mannose to GDP-fucose in animals, plants, and bacteria [55,56] Mucins are large glycoproteins that cover epithelial cell surfaces and form gel-like structures, thereby able to protect against harmful molecules and microorganisms.We found mucin-5B and 5B-like (catshark, dogfish, respectively), mucin-2-like (dogfish), as well as von Willebrand factor domain (vWFD)-containing protein (dogfish, catshark), which all are large, secreted gel-forming mucins harboring a cysteine-rich domain that strengthens the mucus barrier [60]; however, the vWFD can also be found in other, non-glycosylated, proteins.Thus, despite the sharks not appearing "slimy" as bony fish, they are indeed covered by mucins albeit with a thinner layer [26].A study from 2013 on gilthead sea bream (Sparus aurata) skin showed that mucin-2 and mucin-2-like are expressed at relatively low levels and that probiotics [61] and bacterial infection [62] increased mucin-5B expression.Thus, these proteins may serve antimicrobial purposes in the shark skin.In a bioinformatic report from 2016 in which mucin protein sequences in several species were predicted from genomic sequences, mucin-5, 2, and 6 were identified in elephant shark (Callorhinchus milii), although not verified experimentally [63].To the best of our knowledge, the present study is the first time these three mucins (mucin-5B, mucin-2, and mucin-2-like) are shown experimentally in shark skin mucus.
Jawed fish, including Osteichthyes (bony fish) and Chondrichthyes, are the most primitive animals that can make antibodies, however of different subclasses than mammals.Previously, several immunoglobulins (Ig) including IgM, IgH, IgD (IgW orthologue), and IgL were identified in bony fish, whereas three heavy chain isotopes including IgM, IgW, and immunoglobulin novel antigen receptor (IgNAR) were reported in Chondrichthyes [64].Due to its small size, shark IgNAR is often referred to as a nanobody and is the primary an-tibody of a shark's adaptive immune system with a serum concentration of 0.1-1.0mg/mL.Shark IgNAR may have developed from the IgW gene [65] and was previously identified in spiny dogfish serum [66].In spiny dogfish skin, we identified only the secreted IgW heavy chain (Table 1).IgW is believed to be the primordial antibody rather than IgM [67] and was first reported in the spleen of sandbar sharks (Carcharhinus plumbeus) in the early 1990s [41], and later in serum and lymphoid tissues of other sharks [68].Although discovered before in spiny dogfish serum, as well as well as in other shark species organs such as pancreas [69], herein we report for the first time that IgW is present in the shark skin mucin as well, where it may serve an antimicrobial role.
Furthermore, we discovered proteins and enzymes such as GDP-L-fucose synthase, fucolectin tachylectin-4 pentraxin-1 (FTP) domain-containing protein (fragment) and GDPmannose 4,6-dehydratase, which are involved in glycosylation, specifically fucosylation.Fucosylation is a glycan sugar protein modification essential to biological processes such as host-microbiota communication, viral infection or immunity [70].We have previously shown that fucosylated glycans are common on spiny dogfish, chain catshark, and little skate skin mucus proteins [26].Moreover, other proteins that are commonly posttranslationally modified by glycosylation including antithrombin, fibrinogen beta chain, transferrin and serotransferrin, hemoglobin subunit alpha, syndecan binding protein, and cystatin kininogen-type domain-containing protein were also identified in this study.Apart from their well-known role in hemostasis, these proteins have a role in the activation of the immune system [37,38].For instance, Raeder et al. [71] first identified a transferrin-like molecule in Atlantic salmon (Salmo salar) mucus infected with Vibrio salmonicida.The primary role of transferrin, which is a glycoprotein, is to sequester iron in a redox-inactive form making iron unavailable to pathogens, thus starving them [72].This is probably important in the sharks' skin antimicrobial defense, as iron is very limited in sea water [73].
The complement system, a network of more than 50 plasma and membrane-associated proteins, plays a vital role in vertebrate defense against pathogens in the blood as part of the innate and adaptive immune system [74].Upon activation, the intermediate key factor, complement component (C3), acts as a chemoattractant, phagocytotic agent and as agglutinin and initiates a cascade of events leading to bacterial lysis and also acts as an inflammation mediator.While most studies on the complement system have been carried out on blood and internal organs, it has been described to be active in the skin as well since human keratinocytes infected with intracellular Staphylococcus aureus can be attacked by the complement system [75].Notably, in the skin, complement dysregulation, deficiency, and genetic polymorphisms have been associated with a number of diseases such as psoriasis and recurrent cutaneous infection [76].In dogfish, we identified C3, as well as component 1Q (C1q) and complement protein 1S.In addition, sushi domaincontaining protein, which binds complement factors, was found both in spiny dogfish and chain catsharks.The presence of these proteins points to an active immune system, in general, and active complement cascade, specifically, in the skin mucus of dogfish sharks.In fact, a report published as early as 1907 suggested the presence of complement-like activity in dogfish serum [77].Sixty years later, Legler and Evans described the serum hemolytic complement activity in three elasmobranch species including sting ray (Dasyatis americana) and two species of shark, lemon shark (Nagaparion brevirostria) and nurse shark (Ginglyraostoma cirraium) [78].The C1q protein (complement system member) was first reported in skin mucus of European sea bass (Dicentrarchus labrax) [19].To our knowledge, our data are the first description of complement components being present in shark skin mucus where it may play an antibacterial role.
Lectins are proteins that bind to carbohydrates, for example, on bacteria, and serve multiple roles including antimicrobial and developmental [79].Lectins have been reported from various tissues of many fish species including skin mucus [28].We found four lectins in the shark skin mucus, including the following: (1) L-type lectin-containing protein (dogfish), which interacts with N-glycans (components of glycoproteins) in a Ca 2+dependent manner [80].( 2) Calreticulin (dogfish, catshark), which is important for the cell surface expression of MHC class I molecules and antigen recognition [81].(3) F-type lectins such as fucolectin tachylectin-4 pentraxin-1 (FTP) domain-containing protein (catshark), which is implicated in innate immunity (Table 5, detailed explanation).Of note, F-type lectin has been discovered in several fish species in the liver, intestines, and eggs [82][83][84] but to date not in the skin and not in sharks; however, C-type lectin has been found in the skin of Japanese bullhead shark (Heterodontus japonicus) [85].( 4) Calmodulin (dogfish), which also is involved in immune and inflammatory responses [86].Several proteasomes (protease complex, "genetic information processing" group, Tables 1 and 2) were found in both shark types, whereas cysteine proteases such as cathepsin L and B protein were identified in catsharks ("protein metabolism" group, Table 2).Proteases are essential for activation of both the innate and adaptive immune systems and perform complement activation, initiation of proinflammatory responses and the generation of peptides from foreign antigens that are then presented to the major histocompatibility complex in the adaptive immune response [36,95].Proteases have been detected in fish mucus of several cold water fish [96] as well as in fish preferring warmer waters such as the greater amberjack (Seriola dumerili), in which ectoparasite infection increases protease activity [97].Proteases have also been found in the gut of bonnethead sharks (Sphyrna tiburo) where these contribute to food digestion [98]; however, these have not been studied in shark skin.
Several annexins were found in both sharks and are known to regulate the activities of innate immune cells, in particular the generation of proinflammatory mediators, as was described in Atlantic cod [99].Although the role of annexins in the shark skin mucus has not been studied before, epigonal media derived from bonnethead sharks induced apoptosis in human cancer cells, possibly due to annexin as an apoptosis inducer [100], which highlights how sharks can be useful in human medicine.
Actin is one of the most prevalent proteins in eukaryote cells and has several roles including cell movement, cytoplasmic streaming, phagocytosis, and cytokinesis [101].Several reports suggested that the presence of actin and other cytoskeleton related proteins may not simply be due to contamination from ruptured cells but may have a separate role in mucus structure and immune system [102][103][104].In both shark types, we found cytoskeletonrelated molecules (actin, filamin, tubulin, gelsolin, tropomyosin, septin, and keratin), which is not surprising, as these proteins are common and were probably sloughed off.However, these proteins may have immune-relevant function in shark mucus, as shown in Atlantic salmon for actin [103], in rainbow trout (Salmo gairdneri) for keratin [105], and in zebrafish for septin [106].In addition, extracellular actin from insects can bind to bacteria and stimulate their killing by phagocytosis [104].Thus, an intracellular protein may change role when extracellularly located on the skin surface.Moreover, cytoskeletal-related proteins identified in spiny dogfish seemed to participate in shark osmoregulatory tissues [22].All together, these findings suggest that cytoskeletal proteins could be functionally active extracellularly in the shark skin mucus as well.
The 14-3-3 proteins are acidic proteins with several isoforms that are ubiquitously expressed, participate in regulatory processes, and are indirectly involved in immune response [28,107].Ras-related proteins are involved in signal transduction, the regulation of several biological processes, and, aptly, the immune system [108].In both shark types, we identified these proteins ("cell communication" group, Tables 1 and 2).14-3-3 was present in the skin mucus of several fish types [109], but its implication in fish skin (and shark skin) has yet to be determined.Ras proteins were shown to interact with parasite proteins in the skin mucus of common carp (Ichthyophthirius multifiliis) and thus might serve as a drug target [55].As the mucus layer is formed both by secretion and cellular sloughing, proteomics will naturally identify both secretory, membranous as well as intracellular proteins.Of note is that proteins may have dual functions; for example, ribosomal proteins with antimicrobial properties have been identified in rainbow trout and cod skin [56,110].

Protein Interaction
Protein-protein interaction network analysis may shed a light on the predicted function of the identified proteins by revealing their interaction, as well as reveal how similar these interactions are to other species.For that purpose, we used the STRING database [111], and created a proteome interaction network by merging all the proteins identified from the skin mucus of the sharks investigated and compared to published orthologues from other shark and fish species (Figures 2-5).The sources for the maps include interactions from the published literature describing experimentally studied interactions as well as databases.
A confidence score for every protein-protein interaction was assigned to the network in which a higher score is assigned when an association is supported by several types of evidence.To minimize false positives as well as false negatives, all interactions tagged as "low confidence" (<0.4) in the STRING database were eliminated.Thus, the networks are composed of a set number of nodes (proteins) and edges (interactions) (Tables 6 and 7).We found a much higher number of edges when comparing the spiny dogfish and chain catsharks to the phylogenetically close shark species cloudy catshark (Scyliorhinus torazame) and brownbanded bamboo shark (Chiloscyllium punctatum) as compared to the much more distant elephant shark (Callorhinchus milii) and zebrafish (Danio rerio) [112].There is also a similar pattern in number of nodes (proteins) albeit less significant.From the STRING analysis, examining the percentage of proteins that had orthologues with other species revealed that (1) cloudy catshark overlaps 88% with dogfish and 99% with chain catsharks, which suggests that the two catshark species are closely related; (2) brownbanded bamboo shark overlaps 88% with dogfish and 93% with chain catsharks; (3) elephant shark overlaps 81% with dogfish and 89% with catshark, somewhat counterintuitive, as elephant sharks are phylogenetically distant; and (4) zebrafish overlaps 82% with dogfish and 86% with catsharks.Furthermore, up to 19% of the proteins did not have orthologues in other shark species, which could mean that they are unique in the respective sharks or, alternatively, this could be due to methodological differences.These data indicate that most of the skin mucus proteome is conserved and shared among close (although separated by millions of years of evolution) shark species and also a bony fish, and, while speculative, this argues that these proteins may serve important physiological functions.To determine whether the skin proteomes of different species evolved independently (convergent evolution) or were already present earlier in evolution, one would need to sample common ancestors such as coelacanths.Table 6.Spiny dogfish protein interaction summary table using STRING analysis.Proteins identified in skin mucus of spiny dogfish were analyzed when employing orthologues from four different species.Number of nodes depicts the number of orthologues found out of 206 proteins.Number of edges depicts the number of protein-protein interactions found with medium (0.4) confidence.Interaction source is shown both for all active sources (all) and also limited to experiments and databases for more stringent analysis.

No. of Nodes
No  Protein interaction map of identified spiny dogfish (A) and chain catshark (B) skin proteins using cloudy catshark orthologues.A possible protein-protein interaction map with high edge confidence was generated using STRING.Ticker edges (line joining the nodes) represent a confidence of 0.4.Edges represent protein-protein association where association does not necessarily mean physical binding of the proteins and there could be involvement of several proteins to a shared function.Note that colored nodes represent different clusters of the query proteins, as employed by STRING software.Full protein names for the abbreviations are provided in Supplementary Files S1 and S2.Note that the larger number of proteins identified in dogfish relative to catsharks yields more interactions; for relative comparisons, see Tables 6 and 7.
Figure 3. Protein interaction map of identified spiny dogfish (A) and chain catshark (B) skin proteins using brownbanded bamboo (bbb) shark orthologues.A possible protein-protein interaction map with high edge confidence was generated using STRING.Ticker edges (line joining the nodes) represent a confidence of 0.4.Edges represent protein-protein association where association does not necessarily mean physical binding of the proteins and there could be involvement of several proteins to a shared function.Note that colored nodes represent different clusters of the query proteins, as employed by STRING software.Full protein names for the abbreviations are provided in Supplementary Files S1 and S2.Note that the larger number of proteins identified in dogfish relative to catsharks yields more interactions; for relative comparisons, see Tables 6 and 7.
Figure 4. Protein interaction map of identified spiny dogfish (A) and chain catshark (B) skin proteins using elephant shark orthologues.A possible protein-protein interaction map with high edge confidence was generated using STRING.Ticker edges (line joining the nodes) represent a confidence of 0.4.Edges represent protein-protein association where association does not necessarily mean physical binding of the proteins and there could be involvement of several proteins to a shared function.Note that colored nodes represent different clusters of the query proteins, as employed by STRING software.Full protein names for the abbreviations are provided in Supplementary Files S1 and S2.Note that the larger number of proteins identified in dogfish relative to catsharks yields more interactions; for relative comparisons, see Tables 6 and 7.  Protein interaction map of identified spiny dogfish (A) and chain catshark (B) skin proteins using zebrafish orthologues.A possible protein-protein interaction map with high edge confidence was generated using STRING.Ticker edges (line joining the nodes) represent a confidence of 0.4.Edges represent protein-protein association where association does not necessarily mean physical binding of the proteins and there could be involvement of several proteins to a shared function.
Note that colored nodes represent different clusters of the query proteins, as employed by STRING software.Full protein names for the abbreviations are provided in Supplementary Files S1 and S2.Note that the larger number of proteins identified in dogfish relative to catsharks yields more interactions; for relative comparisons, see Tables 5 and 6.

Therapeutic Implications and Human Relevance
Several studies have suggested that sharks may be relevant for human medicine.A recent comparison of gene transcripts between white shark (Carcharodon carcharias) and zebrafish revealed, surprisingly, that white shark gene products associated with metabolism, molecular functions, and the cellular locations of these functions were more similar to humans than to zebrafish [113].Moreover, squalamine, a compound with a broad-spectrum antifungal, antibacterial, and antitumor activity, that was isolated from spiny dogfish tissues [1] has resulted in a phase I and phase II human trials [114,115].Proteoglycans with anti-osteoarthritic properties isolated from the bramble shark (Echinorhinus brucus) cartilage showed significant improvement in disease parameters in an osteoarthritis rat model [116].Elasmobranchs immunoglobulins and nanobodies (small monoclonal antibodies) have raised a great attention from the scientific community, as they are the earliest jawed vertebrates to possess all the components necessary to perform responses associated with the adaptive immune system [117].The topic of sharks' usefulness in human medicine was elegantly reviewed by Luer and Walsh [117].

Study Limitations
Only female spiny dogfish were sampled in this study.The mucus harvest method may have missed some proteins and did not work well in chain catsharks in which longer absorption time or scraping may be needed.Furthermore, the mass spectrometry analysis used only shows already known proteins; thus, novel proteins unique to sharks may have been missed, and complementary methods for novel protein discovery will thus need to be used in the future.

Animals
Spiny dogfish caught using hook gear were purchased from a commercial fisherman in Chatham, MA in 2022.Only female spiny dogfish were available, likely due to commercial fishing often targeting female schools [118].Chain catsharks were collected from a National Oceanic and Atmospheric Administration survey vessel by dredging in the mid-north Atlantic between 2017 and 2019.All elasmobranchs were housed in tanks with natural sea water flow-through systems, maintained year-round at 14 • C at the Marine Resources Center (MRC) at the MBL.Elasmobranchs were housed in single-species groups and fed a diet of food-grade frozen capelin (Atlantic-Pacific North Kingstown, RI, USA) and fresh, frozen, locally caught squid three days per week.Photos were taken with an iPhone 13 Pro (Apple Inc., Cupertino, CA, USA)).
Experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the MBL (protocol no 22-22).

Skin Mucus Sampling
Skin mucus were sampled using the Kleenex tissue absorption method, previously developed for salmonoids [119].Briefly, housed elasmobranchs were caught gently with a net and a Kleenex tissue was placed on the skin for 10 s whereafter the tissue was put in Spin-X tubes (Sigma-Aldrich, St. Louis, MO, USA) on ice and later spun down at 700 g in a 4 • C cooled benchtop centrifuge.Tank water controls samples was also harvested by placing the Kleenex (Kimberly-Clark, Irving, TX, USA) briefly in the tank water.The liquid samples were transferred to plastic cryotubes, snap-frozen on dry ice, and stored at −80 • C.

Sample Preparation
Protein content was determined using a colorimetric assay (Bradford protein assay, Bio-Rad, Hercules, CA, USA).Aliquots corresponding to 20 µg protein were processed using a modified version of filter-aided sample preparation (FASP) method [120].The mucus samples were reduced in reduction buffer (6 M GuHCl (guanidinum hydrochloride (ultrapure, MP Biomedicals, Santa Ana, CA, USA), 0.1 M TEAB (triethyl ammonium buffer pH 9.5), 5 mM ethylenediaminetetraacetic acid, 0.1 M dithiothreitol) for 30 min at 37 • C. The samples were transferred to 10 kDa Microcon Centrifugal Filter Units (MPE030025, polyetylensulfon filter, Millipore, Burlington, MA, USA), and washed repeatedly with 6M GuHCl, followed by alkylation with 100 µL 0.05 M iodoacetamide in 50 mM TEAB buffer for 30 min.Digestion was performed in 0.1M TEAB with the addition of sequencinggrade modified trypsin (Promega, Madison, WI, USA) in an enzyme-to-protein ratio of 1:100 at 37 • C overnight.An additional portion of trypsin was added and incubated for 4 h.Peptides were collected by centrifugation, followed by further purification using High Protein and Peptide Recovery Detergent Removal Spin Column and Pierce peptide desalting spin columns (both Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

NanoLC/MS
NanoLC-MS/MS was performed on an Orbitrap Exploris 480 mass spectrometer interfaced with Easy-nLC1200 liquid chromatography system (both Thermo Fisher Scientific, Waltham, MA, USA).Peptides were trapped on an Acclaim Pepmap 100 C18 trap column (100 µm × 2 cm, particle size 5 µm, Thermo Fischer Scientific, Waltham, MA, USA) and separated on an in-house packed analytical column (75 µm × 35 cm, particle size 3 µm, Reprosil-Pur C18, Dr. Maisch) using a gradient from 5% to 35% ACN in 0.2% formic acid over 40 min at a flow of 300 nL/min.Each preparation was analyzed using MS1 scans settings, m/z 380-1500, at a resolution of 120 K. MS2 analysis was performed in a data-dependent mode at a resolution of 30K, using a cycle time of 2 s.The most abundant precursors with charges 2-6 were selected for fragmentation using HCD at collision energy settings of 30.The isolation window was set to 1.2 m/z and the dynamic exclusion was set to 10 ppm for 30 s.

Proteomic Data Analysis
The acquired data were analyzed using Proteome Discoverer 2.4 (Thermo Fisher Scientific, Waltham, MA, USA).The raw files were matched against the Swissprot human database (March 2021) and Uniprot Chondrichthyes database (142,499 entries, February 2023) using Mascot 2.5.1 (Matrix Science, London, UK) as a database search engine with peptide tolerance of 5 ppm and fragment ion tolerance of 30 mmu.Tryptic peptides were accepted with one missed cleavage, mono-oxidation on methionine was set as a variable modification, and carbamidomethylation on cysteine was set as a fixed modification.Target Decoy was used for PSM validation.Tables referring to secreted proteins are based on targeted literature searches and UniProt data (www.uniprot.org(accessed on 1 June 2023)).
The proteins identified were clustered into different categories based on Gene Ontology category, biological process.Further classification of protein type and functional hierarchies of biological entities were based on information on KEGG BRITE Database (kegg.jp/kegg/brite.html(accessed on 1 June 2023)) and UniProt (uniprot.org(accessed on 1 June 2023)) for individual proteins.As most of the proteins are not well annotated in teleost species, the Gene Ontology terms were retrieved from its human counterparts.

Protein-Protein Interaction Network Analysis
Protein interaction network maps for the sharks' skin mucus proteins was generated using STRING (https://version-12-0.string-db.org/(accessed on 1 September 2023), employing the following organism UniProt IDs: Cloudy catshark (Scyliorhinnus torazame), Brownbanded bamboo shark (Chiloscyllium punctatum), Elephant shark (Callorhinchus milii, also called Australian ghost shark), Zebrafish (Danio rerio).To achieve a more stringent analysis, the active interaction sources were limited to experiments and databases, and an interaction score >0.4 was applied to construct the protein-protein interaction network.

Chemicals
The chemicals were from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise.

Conclusions
This is the first study that describes the skin mucus proteome of sharks.These proteins represent several basic functional groups, and while most of them are cellular proteins, a substantial minority are secreted.We propose these skin proteomes to be relatively conserved between close shark species.Further research on elasmobranch skin is warranted, especially bioprospecting studies that aim to identify completely novel molecules using protein sequencing, decipher their functions experimentally, and, if possible, translate to human clinical use albeit with shark conservation in mind.

Figure 1 .
Figure 1.Experimental setup.(A) Shark species examined.The chain catshark scale is in inches and the spiny dogfish scale is in cm.(B) Sample harvest and analysis.Proteins were harvested by wrapping wet shark skin with a Kleenex tissue for 10 s, followed by centrifugation in SpinX tubes and analysis using mass spectrometry (NanoLC-MS/MS).N = 10 for spiny dogfish and N = 10 for chain catsharks.

Figure 2 .
Figure2.Protein interaction map of identified spiny dogfish (A) and chain catshark (B) skin proteins using cloudy catshark orthologues.A possible protein-protein interaction map with high edge confidence was generated using STRING.Ticker edges (line joining the nodes) represent a confidence of 0.4.Edges represent protein-protein association where association does not necessarily mean physical binding of the proteins and there could be involvement of several proteins to a shared function.Note that colored nodes represent different clusters of the query proteins, as employed by STRING software.Full protein names for the abbreviations are provided in Supplementary Files S1 and S2.Note that the larger number of proteins identified in dogfish relative to catsharks yields more interactions; for relative comparisons, see Tables6 and 7.

Figure 5 .
Figure5.Protein interaction map of identified spiny dogfish (A) and chain catshark (B) skin proteins using zebrafish orthologues.A possible protein-protein interaction map with high edge confidence was generated using STRING.Ticker edges (line joining the nodes) represent a confidence of 0.4.Edges represent protein-protein association where association does not necessarily mean physical binding of the proteins and there could be involvement of several proteins to a shared function.Note that colored nodes represent different clusters of the query proteins, as employed by STRING software.Full protein names for the abbreviations are provided in Supplementary Files S1 and S2.Note that the larger number of proteins identified in dogfish relative to catsharks yields more interactions; for relative comparisons, see Tables5 and 6.

Table 1 .
Identified proteins from spiny dogfish skin mucus grouped into biological groups.

Table 2 .
Identified proteins from chain catshark skin mucus grouped into biological groups.

Table 4 .
Secreted proteins identified in the mucus of spiny dogfish.A literature-based distinction of their immune potential.Organism represents the protein reference species.

Table 5 .
Secreted proteins identified in the mucus of chain catshark.A literature-based distinction of their immune potential.Organism represents the protein reference species.

Table 7 .
Chain catshark protein interaction summary table using STRING analysis.Proteins identified in skin mucus of chain catshark were analyzed when employing orthologues from four different species.Number of nodes depicts the number of orthologues found out of 72 proteins.Number of edges depicts the number of protein-protein interaction found with medium (0.4) confidence.Interaction source is shown both for all active sources (all) and also limited to experiments and databases for more stringent analysis.
Institutional Review Board Statement: The animal study protocol was approved by the Institutional animal care & use committee of the Marine Biological Laboratory, Protocol number: 20-22, date of approval-22 April 2022.Not applicable.