Next Article in Journal
Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase
Previous Article in Journal
High-Throughput Identification of Putative Antimicrobial Peptides from Multi-Omics Data of the Lined Seahorse (Hippocampus erectus)

Mar. Drugs 2020, 18(1), 31; https://doi.org/10.3390/md18010031

Article
Characterization of the Jumbo Squid (Dosidicus gigas) Skin By-Product by Shotgun Proteomics and Protein-Based Bioinformatics
1
Department of Food Technology, Marine Research Institute (IIM), Spanish National Research Council (CSIC), 36208 Vigo, Pontevedra, Spain
2
Department of Food Research and Postgraduate, University of Sonora, 83100 Hermosillo, Sonora, Mexico
*
Author to whom correspondence should be addressed.
Received: 26 November 2019 / Accepted: 26 December 2019 / Published: 29 December 2019

Abstract

:
Jumbo squid (Dosidicus gigas) is one of the largest cephalopods, and represents an important economic fishery in several regions of the Pacific Ocean, from southern California in the United States to southern Chile. Large and considerable discards of this species, such as skin, have been reported to constitute an important source of potential by-products. In this paper, a shotgun proteomics approach was applied for the first time to the characterization of the jumbo squid (Dosidicus gigas) skin proteome. A total of 1004 different peptides belonging to 219 different proteins were identified. The final proteome compilation was investigated by integrated in-silico studies, including gene ontology (GO) term enrichment, pathways, and networks studies. Potential new valuable bioactive peptides such as antimicrobial, bioactive collagen peptides, antihypertensive and antitumoral peptides were predicted to be present in the jumbo squid skin proteome. The integration of the global proteomics results and the bioinformatics analysis of the jumbo squid skin proteome show a comprehensive knowledge of this fishery discard and provide potential bioactive peptides of this marine by-product.
Keywords:
Dosidicus gigas; squid; skin; by-product; shotgun proteomics; mass spectrometry; protein-based bioinformatics; bioactive peptides

1. Introduction

Marine by-products are the body parts of marine species that are removed before they reach the final consumer in order to improve their preservation, reduce the shipping weight, and increase the quality of the main product [1,2]. These organic materials are the main concern for current fishery management policies and legislation because they represent a significant source of valuable compounds such as proteins, minerals and lipids. In fact, from 2019 new regulations of fishery landing in the European Commission (EU) (European Commission Regulation (EU) No 1380/2013) oblige to keep and not discard all the species that are caught that are subjected to quota as well as underutilized commercial species [3]. For this reason, valorization solutions of marine discards biomasses have to be implemented. These new potential bioactive compounds could be used for human nutrition, as well as for their functional properties for nutraceutical, pharmaceutical, and cosmeceuticals industries [4,5,6,7].
Jumbo squid (Dosidicus gigas), also known as Humboldt squid, is one of the largest cephalopods and lives in the waters of the Humboldt Current in the eastern Pacific Ocean. It represents an important economic fishery resource in a wide number of countries such as Chile, Peru, Japan, and Mexico [8]. Nevertheless, only the jumbo squid mantle is marketed. During its processing, large amounts (up to 60% of whole weight) of squid off-products, such as skin, heads, fins, tentacles, and guts are generated and discarded [9].
By-products of the jumbo squid have recently attracted great attention due to the discovery of the presence of several relevant bioactive compounds. These include valuable and profitable bio-ingredients such as chitin, chitosan, collagen, gelatin, and pigments [10,11,12,13,14].
Particularly, the skin constitutes a significant sub-product in the jumbo squid fishery industry. Skin is actually a biological cooperative tissue formed by four different tissue types (epithelial, connective, muscle, and nerve tissues). Peptides derived from a tryptic hydrolysate of jumbo squid skin exhibited strong inhibition of lipid peroxidation that was much higher than the natural antioxidant α-tocopherol [15]. Skin molecules as xanthommatin also showed in vitro antioxidant effects [16]. Additionally, cytotoxic, antimicrobial, anti-biofilm, angiotensin converting enzyme (ACE)-inhibitory peptides, and anti-tumoral properties have been demonstrated for skin ink and the hydrolyzed skin of different squid species [14,17,18]. Recently, the inclusion on ice of a jumbo squid skin extract led to a remarkable microbial inhibition and a significant shelf life extension during fish chilled storage [19,20]. However, the global characterization of proteins and peptides from jumbo skin proteome has not been investigated to date.
Proteomics, as the discipline for the large-scale analysis of proteins of a particular biological system, has greatly contributed to the assessment of quality, safety, and bioactivity of seafood products [21,22,23,24]. In a shotgun proteomics approach, a mixture of proteins is digested with a protease (i.e., trypsin), and the resulting mixture of peptides is then analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) [25]. Using database searching programs, like SEQUEST [26] or Mascot [27], fragmentation spectra obtained are assigned to putative peptide sequences and the assignments are then validated with programs like PeptideProphet [28] or Percolator [29]. The identification of these peptides allows for the identification of proteins present in the complex mixture.
Additionally, potential bioactive proteins and peptides can be characterized by protein-based bioinformatics tools. Such software includes programs to simulate in-silico proteolysis and to predict the physicochemical properties of the released peptides (i.e., antihypertensive, antimicrobial, immunomodulatory). Several bioactive peptide databases are available online such as APD3 [30], BioPep [31], BioPD [32], BioPepDB [33], CAMP [34], PPIP [35], starPepDB [36] and StraPep [37].
Therefore, the present work focuses for the first time on the global characterization of the jumbo squid (Dosidicus gigas) skin proteome using a shotgun proteomic approach. Meanwhile, a combination of different protein-based bioinformatics programs is carried out to determine potential bioactive peptides of this marine discard.

2. Results and Discussion

2.1. Jumbo Squid (Dosidicus gigas) Skin Proteome

A shotgun proteomics analysis for the jumbo squid (Dosidicus gigas) skin proteome is presented in this work, to our knowledge, for the first time. This repository was created merging a total of 6559 identified spectra (PSMs) from 1004 different peptides belonging to 219 different non-redundant annotated proteins from the different sample replicates (n = 4) (Supplementary Tables S1–S3). Table 1 summarizes the list of the non-redundant annotated proteins of the jumbo squid skin proteome (n = 219). This discovery stage was based on the LC-MS/MS analysis and SEQUEST-HT search of the tryptic digestions for the global protein extracts from the skin of each jumbo squid specimens studied (A–D replicates).
Additionally, to visualize and corroborate the intact protein extraction of the jumbo squid skin fraction, complete protein extracts of the four replicates (A–D) were separated by SDS-PAGE 10% (Figure 1). This gel illustrates that all replicate extracts show the same protein weight distribution.
To our knowledge, this is the most comprehensive dataset of peptides and proteins for jumbo squid (D. gigas) skin identified to date. This valuable protein repository will add new and significant information to the universal public protein databases and could be very useful for new investigations of this marine by-product. Raw data and analyses outputs are publicly available in MassIVE data repository (https://massive.ucsd.edu/) (Reference: MSV000084702).
We need to take into account the difficulties and limitations of working with un-sequenced organisms as in the case of D. gigas. Thus, due to the fact that in the universal UniprotKB protein database only 40 different proteins for D. gigas are registered (Cytochrome c oxidase subunit 1, subunit 3; Cytochrome b; NADH-ubiquinone oxidoreductase chain 2, chain 4, chain 5; Cytochrome c oxidase subunit 2; ATP synthase subunit a; Histone H3; Chitin binding beak protein 1, 2, 3, 4; NADH dehydrogenase subunit 4L, subunit 2; ATP synthetase subunit 8; Paramyosin; Histidine rich beak protein 1, protein 2, protein 3; Suckerin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -12, -13, -14, -15, -16, -17, -18, -20, -21; Symplectin/biotinidase-like protein), we decided to perform the protein identification using Proteome Discoverer 2.2 using a global database according to phylogenetic similarity for the class “Cephalopoda”. This class presents 40,780 entries, these including the 40 different proteins for D. gigas in order to increase the number of protein identifications. In Table 1, assignments for D. gigas protein are indicated in the first lines (Paramyosin and Symplectin/biotinidase-like protein). Many of the protein assignments are uncharacterized proteins (n = 109 proteins; n = 1393 PSMs) that may change with future Cephalopoda and D. gigas specific databases updates.
Thus, the final global dataset of the jumbo squid skin proteome was subsequently investigated by protein-based bioinformatics, like gene ontologies, pathways, network analyses and by prediction of potential bioactive peptides to gather more functional insights.

2.2. Functional Analysis: Gene Ontologies and Pathways Analysis

PANTHER analysis revealed the presence of 11 different protein classes in the jumbo squid skin proteome (Figure 2). The most prominent classes were oxidoreductases (37.0%), nucleic acid binding proteins (12.1%), hydrolases (12.1%), calcium-binding proteins (12.1%), transferases (9.8%), and enzyme modulator (9.8%). Thus, in the jumbo squid skin, oxidoreductases are mainly involved in the energetic metabolism, antioxidant defense and cephalopod coloration [38]. Another significant protein class is that of calcium-binding proteins, which are involved in muscle relaxation and nervous transmission in the marine skin species [39,40].
KEGG pathway analysis was carried out by comparing the input data with the background of the Octopus bimaculoides genome by DAVID version 6.8 program (https://david.ncifcrf.gov/home.jsp); this cephalopod species is the most phylogenetically closest included in DAVID software. KEGG showed that most of the identified proteins were involved in metabolic pathways (cysteine and methionine metabolism), endocytosis/phagosome, RNA transport, protein methylation, and calcium homeostasis (Table 2).
The study of functional domains by InterPro performed by DAVID software revealed that the top protein motifs corresponded to small GTP-binding protein domains, heat shock protein 70, small GTPase superfamily, proteasome, P-loop containing nucleoside triphosphate hydrolase and EF-hand-like domains (Table 3). These EF-hand domains corresponded to calcium-binding domains in concordance with the calcium homeostasis pathway discovered for the calcium-binding proteins, which correspond to 12.1% of the total jumbo squid skin proteome.

2.3. Network Analysis

Network analysis was created merging all the proteins identified for the jumbo squid skin proteome using the STRING software (v.11.0) (https://string-db.org/). A specific organism was not selected (organism Auto-detect) because the genome of D. gigas is not available in the STRING software. According to MCL inflation clustering (MCL = 3), 21 nodes (proteins) and 61 edges (interactions) were obtained (Figure 3).
Physical direct interactions are represented with continuous lines and functional interactions with interrupted lines. The topological analysis of this network demonstrated mainly four different sub-networks. Two of them are relevant sub-networks implicated in metabolic and oxidative cellular respiration (Figure 3 in green and yellow).
Other relevant sub-network is composed of three nodes and is referred as calcium homeostasis (Figure 3 in blue). The results of this sub-network are in concordance with one of the top protein classes categorized previously by PANTHER and DAVID (Figure 2 and Table 2).
Other relevant sub-network is referred as transmembrane transport proteins (Figure 3, in red), as was obtained previously by PANTHER (Figure 2).
Finally, this network represents to date the first most comprehensive interactomic map for the jumbo squid skin proteome.

2.4. Putative Bioactive Peptides

Bioactive peptides are inactive when they are part of parent protein, but become active when released due to the action of enzymes. Thus, bioactive peptides encrypted in the parent jumbo squid skin proteome (n = 219) were predicted using different in-silico software. Thus, protein hydrolysates with pepsin and trypsin were performed in-silico using the MS-Digest program. No missed cleavages and a minimum of six residues per peptide were selected as parameters. Thus, the predicted peptides after every enzymatic digestion (pepsin and trypsin) are presented in Supplementary Table S4.
The first enzymatic digestion using pepsin released a total of 5077 different peptides (6–39 amino acid residues). This enzyme cleaves the proteins at Phe, Tyr, Trp, and Leu residues in positions P1 and P1’ [41]. Compared with the most used and conventional BIOPEP database, no bioactive peptides were identified probably because none squid bioactive peptide is included in the database. However, by using PeptideRanker (http://distilldeep.ucd.ie/PeptideRanker/), the complete list of potential bioactive peptides was ranked using the N-to-1 neural network probability [42], which predicts the peptides that may be more bioactive (Supplementary Table S4). Among them, 18 peptides with a PeptideRanker score higher than 0.9 (7–30 amino acid residues) were selected as potential bioactive peptides (Table 4). The majority of the results corresponded to collagen ColAa proteins, hemocyanin subunit proteins and different uncharacterized proteins.
Regarding tryptic digestion, this enzyme predicted the release of a total of 8042 different peptides (6–45 amino acid residues) (Supplementary Table S4). This enzyme preferentially cleaves the proteins at Lys and Arg residues in position P1 except for the case in which Pro is found in position P1’ [41]. Using a PeptideRanker score higher than 0.9, a total of 73 tryptic peptides (7–30 amino acid residues) were selected as potential bioactive peptides (Table 5). The majority of such peptides corresponded to calcium-transporting ATPase, collagen ColAa proteins, hemocyanin proteins, myosin heavy chain, titin and different uncharacterized proteins.
It is known that the employment of collagenous residues obtained from jumbo squid skin after hydrolysis with pepsin exhibit a good gelatin gel-forming ability including the absence of color, opacity and high-puncture deformation [43]. The collagen alpha chains proteins determined in this study were characterized as belonging to type-I. Additionally, jumbo squid skin collagen was explored to enhance the anti-damage and anti-osteoporosis activity in osteoblast cells [44,45]. Thus, potential pepsin (PGDPGPVGRTGPMGL, RGPPGPPGL) and tryptic (GPPGIPGLPGPK, GPPGPPGLK, AGPPGFPGTPGPK) bioactive collagen peptides determined in this study may be used to stimulate the regeneration of joint cartilages in patients with chronic joint symptoms (Table 4 and Table 5). GELITA® and CH-Alpha® are examples of commercial products containing collagen hydrolysates.
Hemocyanins are the oxygen transporters of cephalopods and mollusks. These proteins play important immune-related roles as antimicrobial, antiviral, agglutinative and antitumor proliferation of cancer cells [46]. In fact, hemocyanin of marine mollusks (Megathura crenulata and Concholepas concholepas) has showed significant antitumor effects of breast, pancreas and prostate cancer cells [47,48]. Although, no previous studies are available related to the use of jumbo squid hemocyanin from a bioactive and immunotherapeutic point of view, it can be considered that the potential pepsin (KKPMMPF, PNQPMRPF, NDPMRPF, SDPMRPF) and tryptic (MVGYLGQALMALLLLALSNAALVR, FEPNPFFSGK, VACCLHGMPVFPHWHR, MATHWHSLLLFSLQLLVFTYATSDPTNIR, GSPIGVPYWDWTKPMK, TNFFFLALIATVWLGNAETETETSK, VFVGFLLHGFGSSAYATFDICNDAGECR, LNHLPLLCLAVILTLWMSGSNTVNGNLVR, VFAGFLFMGIK, VFAGFWFHGIK, VFGGFWLHGIK, TSFLFLAFVATSWFVYAVTASK) bioactive hemocyanin peptides determined in this study may be used in the future as an antitumor therapy for cancer cells (Table 4 and Table 5).
Calcium-transporting ATPase protein is an important regulator of the Ca2+ concentration in the cells and extracellular space. It is necessary for the cell signaling and for the nerve transmission of the squid axons [49]. Potential tryptic (FSDDYPGFF, FLQFQLTVNCVAVMVAFFGACIINDSPLK, FADAPFMK) bioactive calcium-transporting ATPase peptides determined in this study may be used in a future to investigate the in vitro axon stimulation (Table 5).
Myosin heavy chain is one of the major components of the muscle that participates in the muscle contraction as well as in a wide variety of non-muscular cells movements. Previous studies identified different ACE-inhibitory peptides from alcalase hydrolysis of a protein concentrate recovered from a cuttlefish (Sepia officinalis) industrial manufacturing effluent [17]. In fact, several potential bioactive peptides had a proline residue in one of the last positions of C-terminal which promotes enzyme binding (YQSGFIYTYSGLFCVAINPYR, YYSGLIYTYSGLFCVVVNPYK) [50] (Table 5). However, these results need to be further investigated because this is neither sufficient nor essential to confer bioactivity.
Titin (also known as connectin) is a giant protein that works as a molecular spring for the passive elasticity of tissues. The degradation of this protein is one of the major reasons for quality changes in fresh raw squid tissues [51]. Potential tryptic (DGSWQNLVTVLGCLKPQFVNLQR, GYPPPIISWYR) bioactive titin peptides determined in this study may be used as potential biomarkers of quality changes or processing time in squid products (Table 5).
The antimicrobial activity of jumbo squid skin crude pigments extracts has been recently demonstrated [52]. In the present work, antimicrobial peptides (AMPs) were identified using the CAMP (Collection of Anti-Microbial Peptides) database (http://www.bicnirrh.res.in/antimicrobial/) and applying the DAC score (Discriminate Analysis Classifier score) [34]. Table 4 and Table 5 show the potential anti-microbial bioactive peptides. A total of 16 pepsin peptides and 20 tryptic peptides with anti-microbial peptides were predicted. Among them, seven anti-microbial peptides (four pepsin and three tryptic) were encrypted in the hemocyanin parent protein (KKPMMPF, PNQPMRPF, NDPMRPF, SDPMRPF, VFAGFLFMGIK, VFAGFWFHGIK, VFGGFWLHGIK), two anti-microbial tryptic peptides in the collagen parent protein (GPPGIPGLPGPK, AGPPGFPGTPGPK), one anti-microbial tryptic peptide in the myosin heavy chain protein (NWQWWR) and one anti-microbial tryptic peptide in the titin protein (DGSWQNLVTVLGCLKPQFVNLQR).
All these potential bioactive peptides need to be validated by further bioactivity assays using synthetic versions of the peptides. Nevertheless, compared with the classical approaches, the bioinformatics methods are faster and lower-cost alternatives that predict and reduce the number of potential targets to be investigated.

3. Materials and Methods

3.1. Chemicals and Reagents

Bicinchoninic acid (BCA), dithiothreitol (DTT), sodium dodecyl sulphate (SDS), Tris-HCl, and the protease inhibitor phenylmethylsulphonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO, USA). Ammonium persulphate (APS), bromophenol blue and N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased from GE Healthcare Science (Uppsala, Sweden). Acrylamide and bis N,N′-methylene-bis-acrylamide were obtained from Bio-rad (Hercules, CA, USA). Glycerol was obtained from Merck (Darmstadt, Germany). Sequencing grade porcine trypsin was purchased from Promega (Madison, WI, USA). All other chemicals were reagent/analytical grade and water was purified using a Milli-Q system (Millipore, Billerica, MA, USA).

3.2. Jumbo Squids

Jumbo squid (D. gigas) specimens were harvested off the coast of Kino Bay, Mexico. Specimens were degutted and major beheaded on site, and the skins bagged and placed in alternate layers of ice-squid-ice in a portable cooler, and transported to the laboratory. Time between capture and arrival at the laboratory did not exceed 12 h.

3.3. Skin Protein Samples

A total of 0.25 g of lyophilized jumbo squid skin were homogenized in 4 mL of lysis buffer (10 mM Tris-HCl buffer pH 7.2, 5 mM of PMSF) on ice for 6 cycles of 5 s pulses in a sonicator device (Werke, Germany). Samples were centrifuged at 40,000× g for 20 min at 4 °C in a J221-M centrifuge (Beckman, Palo Alto, CA, USA). The supernatant proteins were recovered and stored at −80 °C until used. Protein concentration in the protein extracts was determined by the bicinchoninic acid (BCA) method (Sigma Chemical Co., St. Louis, MI, USA).

3.4. SDS-Polyacrylamide Gel Electrophoresis

Squid skin proteins were separated on 10% (v/v) polyacrylamide gels (acrylamide/N,N′-ethylene-bis-acrylamide, 200:1) with a stacking gel of 4% polyacrylamide. A total of 25 µg of proteins in Laemmli buffer were boiled for 5 min at 100 °C and separated per well in a Mini-PROTEAN 3 cell (Bio-Rad, Hercules, CA, USA). The running buffer consisted of an aqueous solution, composed by 1.44% (w/v) glycine, 0.67% Tris-base, and 0.1% SDS. Running conditions were 80 V for the first 20 min and then 120 V until the end of the electrophoresis. PageRuler unstained protein ladder was also used as molecular weight (MW) indicator (Thermo Fisher Scientific, San Jose, CA, USA).
Gels were stained overnight with Coomassie dye PhastGel Blue R-350 (GE Healthcare, Uppsala, Sweden). Scanned Coomassie-stained gels were analysed by means of the 1-d gel electrophoresis analysis software LabImage 1D (Kapelan Bio-Imaging Solutions, Halle, Germany).

3.5. In-Solution Protein Digestion with Trypsin

A total of 100 μg of jumbo squid skin protein extract were denatured in 8 M urea and then reduced with 5 mM TCEP (Pierce, Thermo Fisher Scientific) for 30 min at 37 °C. After alkylation with 50 mM iodoacetamide (Pierce, Thermo Fisher Scientific) in 25 mM ammonium bicarbonate pH 8.25 for 60 min at room temperature in dark, samples being diluted 4-fold with 25 mM ammonium bicarbonate pH 8.25 to decrease the urea concentration. Proteins were digested with trypsin (Promega) (1:100 protease-to-protein ratio) overnight at 37 °C.

3.6. Shotgun LC-MS/MS Analysis

Peptides were acidified with formic acid, cleaned on a C18 MicroSpinTM column (The Nest Group, South-borough, MA) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Proxeon EASY-nLC II liquid chromatography system (Thermo Fisher Scientific, San Jose, CA, USA) coupled to a LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Peptide separation (1 µg) was done on a RP column (EASY-Spray column, 50 cm × 75 µm ID, PepMap C18, 2 µm particles, 100 Å pore size, Thermo Fisher Scientific) with a 10-mm pre-column (Accucore XL C18, Thermo Fisher Scientific) using 0.1% formic acid (mobile phase A) and 98% acetonitrile (98% ACN) with 0.1% formic acid (mobile phase B). A 120 min linear gradient from 5 to 35% B, at a flow rate of 300 nL min−1, was used. A spray voltage of 1.95 kV and a capillary temperature of 230 °C were used for ionization. The peptides were analyzed in positive mode (1 µscan; 400–1600 amu), followed by 10 data-dependent collision-induced dissociation (CID) MS/MS scans (1 µscans), using a normalized collision energy of 35% and an isolation width of 3 amu. Dynamic exclusion for 30 s after the second fragmentation event was applied and unassigned charged ions were excluded from the analysis.
A total of four replicates (n = 4) were analyzed independently.

3.7. Processing of the Mass Spectrometry Data

All the MS/MS spectra were analyzed using SEQUEST-HT (Proteome Discoverer 2.2 package, Thermo Fisher Scientific) against the Cephalopoda UniProt/TrEMBL database (release 2018_11; 40,780 entries). The following restrictions were used: tryptic cleavage with up to 2 missed cleavage sites and tolerances of 0.8 Da for parent ions and 0.6 Da for MS/MS fragment ions. Carbamidomethylation of Cys (C*) was considered as a fixed modification. The permissible variable modifications were: methionine oxidation (Mox) and acetylation of the N-terminus of the protein (N-Acyl). The results were subjected to statistical analysis with the Percolator algorithm to keep a false discovery rate (FDR) below 1%.

3.8. Functional Gene Ontologies and Pathways Analysis

The final list of non-redundant protein IDs was submitted to PANTHER program (http://www.pantherdb.org/), for the classification based on two main types of annotations: protein class and biological process. A statistical significance of representation for the analysis was also provided.
KEGG pathway analysis was performed by comparing the input data with the background of the Octopus bimaculoides genome by DAVID version 6.8 (https://david.ncifcrf.gov/home.jsp). Functional domains by InterPro Motifs were also obtained using DAVID version 6.8 software.

3.9. Network Analysis

Network analysis was performed submitting the protein dataset to the STRING (Search Tool for the Retrieval of Interacting Genes) software (v.11.0) (http://stringdb.org/) [53]. This is a large database of known and predicted protein interactions. Proteins were represented with nodes and the interactions with continuous lines to represent direct interactions (physical), while indirect ones (functional) were presented by interrupted lines. To minimize false positives as well as false negatives, all interactions tagged as “low-confidence” (<0.4) in STRING software have been eliminated from the analysis. Cluster networks were created using the MCL inflation algorithm which is included in the STRING website and a value of 3 was selected for all the analyses.

3.10. Bioactive Peptides Prediction

Bioactive peptides encrypted in the parent jumbo squid skin proteome were predicted combining different in-silico protein hydrolysates using pepsin and trypsin enzymes. For that, all the proteolytic digestions were performed in-silico using the MS-Digest software, which is included in ProteinProspector v.5.24.0 website (http://prospector.ucsf.edu/prospector/mshome.htm).
To evaluate the results, all the potential peptides were ranked using the PeptideRanker software (http://bioware.ucd.ie/~testing/biowareweb/) using the N-to-1 neural network probability to predict which peptides can be more bioactive [42]. In addition, all the potential peptides were compared with previous databases that included known bioactive peptides, such as BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep/) and CAMP (http://www.bicnirrh.res.in/antimicrobial/).

4. Conclusions

In this study, a shotgun proteomics strategy was applied for the first time for the characterization of the jumbo squid skin proteome. A total of 1004 different peptides belonging to 219 different proteins were identified. The final proteome compilation was investigated using different in-silico studies, including GO term enrichment, pathways and networks studies. The most prominent protein classes were oxidoreductases, calcium-binding proteins, hydrolases, nucleic acid binding, enzyme modulation, transferases involved in metabolic pathways (cysteine and methionine metabolism), endocytosis/phagosome, RNA transport, protein methylation, and calcium homeostasis. The first most comprehensive interactomic network map for the jumbo squid skin proteome was built up containing 21 nodes and 61 interactions. Most of the jumbo squid skin proteins were grouped under pathways and networks referring to metabolic and oxidative metabolism, calcium homeostasis, transmembrane transport and metabolic and cellular respiration. Moreover, potential valuable bioactive peptides were predicted after different in-silico digestions with pepsin and trysin. Antimicrobial, bioactive collagen peptides, antihypertensive, and antitumor properties were predicted to be present in the jumbo squid skin proteome. The integration of the global proteomics results and the bioinformatics analysis of the jumbo squid skin proteome show a comprehensive knowledge of this fishery discard and provide potential bioactive peptides of this marine by-product.

Supplementary Materials

The following are available online at https://www.mdpi.com/1660-3397/18/1/31/s1, Table S1: Peptide Spectrum Matches (PSMs), Table S2: Peptide Groups, Table S3: Proteins, Table S4: Potential bioactive peptides predicted after pepsin or trypsin digestion.

Author Contributions

M.C. and J.M.E.-B. performed experiments and analyzed data. M.C. wrote the manuscript. J.M.E.-B. and S.P.A. conceptualized, designed the research, revised and corrected the paper. All authors agreed with the final submitted version. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Ramón Areces Foundation (XVII National Grant), GAIN-Xunta de Galicia Project (IN607D 2017/01) and by CONACyT-Mexico under grant 2174. M.C. is supported by the Ramón y Cajal Contract (Ministry of Science, Innovation and Universities of Spain).

Acknowledgments

We are grateful to Lorena Barros (IIM-CSIC, Vigo, Spain) for her excellent technical assistance during the experiments.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Rustad, T.; Storrø, I.; Slizyte, R. Possibilities for the utilization of marine by-products. Int. J. Food Sci. Technol. 2011, 46, 2001–2014. [Google Scholar] [CrossRef]
  2. Blanco, M.; Vázquez, J.A.; Pérez-Martín, R.I.; Sotelo, C.G. Hydrolysates of fish skin collagen: An opportunity for valorizing fish industry byproducts. Mar. Drugs 2017, 15, 131. [Google Scholar] [CrossRef] [PubMed]
  3. European Commission. Regulation (EU) No 1380/2013 of the European Parliament and the Council of 11 December 2013 on the Common Fisheries Policy, Amending Council Regulations (EC) No 1954/2003 and (EC) No 1224/2009 and Repealing Council Regulations (EC) No 2371/2002 and (EC) No 639/2004 and Council Decision 2004/585/EC; European Commission: Brussels, Belgium, 2013. [Google Scholar]
  4. Carrera, M.; Cañas, B.; Gallardo, J.M. The sarcoplasmic fish proteome: Pathways, metabolic networks and potential bioactive peptides for nutritional inferences. J. Proteomics 2013, 78, 211–220. [Google Scholar] [CrossRef] [PubMed]
  5. Venkatesan, J.; Anil, S.; Kim, S.K.; Shim, M.S. Marine fish proteins and peptides for cosmeceuticals: A review. Mar. Drugs 2017, 15, 143. [Google Scholar] [CrossRef] [PubMed]
  6. Sanchez, A.; Blanco, M.; Correa, B.; Pérez-Martín, R.I.; Sotelo, C.G. Effect of fish collagen hydrolysates on type I collagen mRNA levels of human dermal fibroblast culture. Mar. Drugs 2018, 16, 144. [Google Scholar] [CrossRef]
  7. Vázquez, J.A.; Meduíña, A.; Durán, A.I.; Nogueira, M.; Fernández-Compás, A.; Pérez-Martín, R.I.; Rodríguez-Amado, I. Production of valuable compounds and bioactive metabolites from by-products of fish discards using chemical processing, enzymatic hydrolysis, and bacterial fermentation. Mar. Drugs 2019, 17, 139. [Google Scholar] [CrossRef]
  8. Food and Agriculture Organisation of the United Nations. Global Production Statistics-Fisheries and Aquaculture. Available online: http://www.fao.org/fishery/statistics/global-aqua (accessed on 27 May 2017).
  9. Ezquerra-Brauer, J.M.; Aubourg, S. Recent trends for the employment of jumbo squid (Dosidicus gigas) by products as a source of bioactive compounds with nutritional, functional and preservative applications: A review. Int. J. Food Sci. Technol. 2019, 54, 987–998. [Google Scholar] [CrossRef]
  10. Mäthger, L.M.; Denton, E.J.; Marshall, N.J.; Hanlon, R.T. Mechanisms and behavioral functions of structural coloration in cephalopods. J. R. Soc. Interface. 2009, 6, S149–S163. [Google Scholar] [CrossRef]
  11. Deravi, L.F.; Magyar, A.P.; Sheehy, S.P.; Bell, G.R.; Mäthger, L.M.; Senft, S.L.; Wardill, T.J.; Lane, W.S.; Kuzirian, A.M.; Hanlon, R.T.; et al. The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores. J. R. Soc. Interface 2014, 11, 20130942. [Google Scholar] [CrossRef]
  12. Aubourg, S.P.; Torres-Arreola, W.; Trigo, M.; Ezquerra-Brauer, J.M. Partial characterization of jumbo squid skin pigment extract and its antioxidant potential in a marine oil system. Eur. J. Lipid Sci. Technol. 2016, 118, 1293–1304. [Google Scholar] [CrossRef]
  13. Mosquera, M.; Giménez, B.; Montero, P.; Gómez-Guillén, M.C. Incorporation of liposomes containing squid tunic ACE-inhibitory peptides into fish gelatin. J. Sci. Food Agric. 2016, 96, 769–776. [Google Scholar] [CrossRef] [PubMed]
  14. Shahidi, S.; Jamili, S.; Ghavam Mostafavi, P.; Rezaie, S.; Khorramizadeh, M. Assessment of the inhibitory effects of ficin-hydrolyzed gelatin derived from squid (Uroteuthis duvauceli) on breast cancer cell lines and animal model. Iran. J. Allergy Asthma Immunol. 2018, 17, 436–452. [Google Scholar] [CrossRef] [PubMed]
  15. Mendis, E.; Rajapakse, N.; Byun, H.G.; Kim, S.K. Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects. Life Sci. 2005, 77, 2166–2178. [Google Scholar] [CrossRef] [PubMed]
  16. Chan-Higuera, J.E.; Santacruz-Ortega, H.D.C.; Carbonell-Barrachina, A.A.; Burgos-Hernández, A.; Robles-Sánchez, R.M.; Cruz-Ramírez, S.G.; Ezquerra-Brauer, J.M. Xanthommatin is behind the antioxidant activity of the skin of Dosidicus gigas. Molecules 2019, 24, 3420. [Google Scholar] [CrossRef] [PubMed]
  17. Amado, I.R.; Vázquez, J.A.; Gónzález, P.; Esteban-Fernández, D.; Carrera, M.; Piñeiro, C. Identification of the major ACE-inhibitory peptides produced by enzymatic hydrolysis of a protein concentrate from cuttlefish wastewater. Mar. Drugs 2014, 12, 1390–1405. [Google Scholar] [CrossRef]
  18. Kumar, P.; Kannan, M.; ArunPrasanna, V.; Vaseeharan, B.; Vijavakumar, S. Proteomic analysis of crude squid ink isolated from Sepia esculenta for their antimicrobial, antibiofilm and cytotoxic properties. Microb. Pathog. 2018, 116, 345–350. [Google Scholar] [CrossRef]
  19. Ezquerra-Brauer, J.M.; Miranda, J.M.; Cepeda, A.; Barros-Velázquez, J.; Aubourg, S.P. Effect of jumbo squid (Dosidicus gigas) skin extract on the microbial activity in chilled mackerel (Scomber scombrus). LWT-Food Sci. Technol. 2016, 72, 134–140. [Google Scholar] [CrossRef]
  20. Ezquerra-Brauer, J.M.; Miranda, J.M.; Chan-Higuera, J.E.; Barros-Velázquez, J.; Aubourg, S.P. New icing media for quality enhancement of chilled hake (Merluccius merlucius) using a jumbo squid (Dosidicus gigas) skin extract. J. Sci. Agric. 2017, 97, 3412–3419. [Google Scholar] [CrossRef]
  21. Carrera, M.; Cañas, B.; Gallardo, J.M. Proteomics for the assessment of quality and safety of fishery products. Food Res. Int. 2013, 54, 972–979. [Google Scholar] [CrossRef]
  22. Stryiński, R.; Mateos, J.; Pascual, S.; González, A.F.; Gallardo, J.M.; Łopieńska-Biernat, E.; Medina, I.; Carrera, M. Proteome profiling of L3 and L4 Anisakis simplex development stages by TMT-based quantitative proteomics. J. Proteomics 2019, 201, 1–11. [Google Scholar] [CrossRef]
  23. Gallardo, J.M.; Carrera, M.; Ortea, I. Proteomics in food science. In Foodomics: Advanced Mass Spectrometry in Modern Food Science and Nutrition; Cifuentes, A., Ed.; John Wiley & Sons Inc.: Hoboken, NJ, USA, 2013; pp. 125–165. [Google Scholar]
  24. Carrera, M.; Cañas, B.; Gallardo, J.M. Advanced proteomics and systems biology applied to study food allergy. Curr. Opin. Food Sci. 2018, 22, 9–16. [Google Scholar] [CrossRef]
  25. Carrera, M.; González-Fernández, A.; Magadán, S.; Mateos, J.; Pedrós, L.; Medina, I.; Gallardo, J.M. Molecular characterization of B-cell epitopes for the major fish allergen, parvalbumin, by shotgun proteomics, protein-based bioinformatics and IgE-reactive approaches. J. Proteomics 2019, 200, 123–133. [Google Scholar] [CrossRef] [PubMed]
  26. Eng, J.K.; McCormack, A.L.; Yates, J.R., III. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 1994, 5, 976–989. [Google Scholar] [CrossRef]
  27. Perkins, D.N.; Pappin, D.J.C.; Creasy, D.M.; Cottrell, J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551–3567. [Google Scholar] [CrossRef]
  28. Keller, A.; Nesvizhskii, A.I.; Kolker, E.; Aebersold, R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem. 2002, 74, 5383–5392. [Google Scholar] [CrossRef]
  29. Kall, L.; Canterbury, J.D.; Weston, J.; Noble, W.S.; MacCoss, M.J. Semi-supervised learning for peptide identification from shotgun proteomics datasets. Nat. Methods 2007, 4, 923–925. [Google Scholar] [CrossRef]
  30. Wang, G.; Li, X.; Wang, Z. APD3: The antimicrobial peptide database as a tool for research and education. Nucleic Acids Res. 2016, 4, D1087–D1093. [Google Scholar] [CrossRef]
  31. Iwaniak, A.; Dziuba, J.; Niklewicz, M. The BIOPEP database—A tool for the in silico method of classification of food proteins as the source of peptides with antihypertensive activity. Acta Aliment. Hung. 2005, 34, 417–425. [Google Scholar] [CrossRef]
  32. Shi, L.; Zhang, Q.; Rui, W.; Lu, M.; Jing, X.; Shang, T.; Tang, J. BioPD: A web-based information center for bioactive peptides. Regul. Pept. 2004, 120, 1–3. [Google Scholar] [CrossRef]
  33. Li, Q.; Zhang, C.; Chen, H.; Xue, J.; Guo, X.; Liang, M.; Chen, M. BioPepDB: An integrated data platform for food-derived bioactive peptides. Int. J. Food Sci. Nutr. 2018, 69, 963–968. [Google Scholar] [CrossRef]
  34. Thomas, S.; Karnik, S.; Barai, R.S.; Jayaraman, V.K.; Idicula-Thomas, S. CAMP: A useful resource for research on antimicrobial peptides. Nucleic Acids Res. 2010, 38, D774–D780. [Google Scholar] [CrossRef] [PubMed]
  35. Rong, M.; Zhou, B.; Zhou, R.; Liao, Q.; Zeng, Y.; Xu, S.; Liu, Z. PPIP: Automated software for identification of bioactive endogenous peptides. J. Proteome Res. 2019, 18, 721–727. [Google Scholar] [CrossRef] [PubMed]
  36. Aguilera-Mendoza, L.; Marrero-Ponce, Y.; Beltran, J.A.; Tellez Ibarra, R.; Guillen-Ramirez, H.A.; Brizuela, C.A. Graph-based data integration from bioactive peptide databases of pharmaceutical interest: Towards an organized collection enabling visual network analysis. Bioinformatics 2019, 35, 4739–4747. [Google Scholar] [CrossRef]
  37. Wang, J.; Yin, T.; Xiao, X.; He, D.; Xue, Z.; Jiang, X.; Wang, Y. StraPep: A structure database of bioactive peptides. Database (Oxford) 2018. [Google Scholar] [CrossRef] [PubMed]
  38. Kinoshita, Y.; Yoshioka, T.; Kato, S.; Konno, K. Color development of squid skin as affected by oxygen concentrations. J. Food Sci. 2009, 74, S142–S146. [Google Scholar] [CrossRef] [PubMed]
  39. Celio, M.R.; Heizmann, C.W. Calcium-binding protein parvalbumin is associated with fast contracting muscle fibres. Nature 1982, 297, 504–506. [Google Scholar] [CrossRef] [PubMed]
  40. Nelson, T.J.; Cavallaro, S.; Yi, C.L.; McPhie, D.; Schreurs, B.G.; Gusey, P.A.; Favit, A.; Zohar, O.; Kim, J.; Beushausen, S. Calexcitin: A signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability. Proc. Natl. Acad. Sci. USA 1996, 93, 13808–13813. [Google Scholar] [CrossRef]
  41. Keil, B. Specificity of proteolysis, 1st ed.; Springer-Verlag: Berlin/Heidelberg, Germany, 1992. [Google Scholar]
  42. Mooney, C.; Haslam, N.J.; Pollastri, G.; Shields, D.C. Towards the improved discovery and design of functional peptides: Common features of diverse classes permit generalized prediction of bioactivity. PLoS ONE 2012, 7, e45012. [Google Scholar] [CrossRef]
  43. Giménez, B.; Gómez-Estaca, J.; Alemán, A.; Gómez-Guillén, M.C.; Montero, P. Physico-chemical and film forming properties of giant squid (Dosidicus gigas) gelatin. Food Hydrocoll. 2009, 23, 585–592. [Google Scholar] [CrossRef]
  44. Cai, J.; Li, Y.; Zhang, Y.; Tong, Q.; Wang, F.; Su, X. Protective effects of collagen extracted from Dosidicus gigas skin on MC3T3-E1 cell induced by H2O2. J. Chin. Inst. Food Sci. Technol. 2015, 15, 6–12. [Google Scholar]
  45. Cai, J.; Li, Y.; Quan, J.; Lin, J.; Zhang, Y.; Wang, F.; Su, X. Effect of collagen peptide extracted from Dosidicus gigas skin on proliferation, differentiation and calcification of MC3T3-E1 cell induced by Cd. J. Chin. Inst. Food Sci. Technol. 2015, 15, 18–24. [Google Scholar]
  46. Liu, S.; Aweya, J.J.; Zheng, L.; Wang, F.; Zheng, Z.; Zhong, M.; Lun, J.; Zhang, Y. A Litopenaeus vannamei hemocyanin-derived antimicrobial peptide (peptide B11) attenuates cancer cells’ proliferation. Molecules 2018, 23, 3202. [Google Scholar] [CrossRef] [PubMed]
  47. Atala, A. This month in investigative urology. J. Urol. 2006, 176, 2335–2336. [Google Scholar] [CrossRef]
  48. McFadden, D.W.; Riggs, D.R.; Jackson, B.J.; Vona-Davis, L. Keyhole limpet hemocyanin, a novel immune stimulant with promising anticancer activity in Barrett’s esophageal adenocarcinoma. Am. J. Surg. 2003, 186, 552–555. [Google Scholar] [CrossRef]
  49. Dipolo, R. Ca pump driven by ATP in squid axons. Nature 1978, 274, 390–392. [Google Scholar] [CrossRef]
  50. Cheung, H.S.; Wang, F.L.; Ondetti, M.; Sabo, E.; Cushman, D. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme: Importance of the COOH-terminal dipeptide sequences. J. Biol. Chem. 1980, 255, 401–407. [Google Scholar]
  51. Kasamatsu, C.; Kimura, S.; Kagawa, M.; Hatae, K. Identification of high molecular weight proteins in squid muscle by western blotting analysis and postmortem rheological changes. Biosci. Biotechnol. Biochem. 2004, 68, 1119–1124. [Google Scholar] [CrossRef]
  52. Chan-Higuera, J.E.; Carbonell-Barrachina, A.A.; Cárdenas-López, J.L.; Kačániová, M.; Burgos-Hernández, A.; Ezquerra-Brauer, J.M. Jumbo squid (Dosidicus gigas) skin pigments: Chemical analysis and evaluation of antimicrobial and antimutagenic potential. J. Microbiol. Biotech. Food Sci. 2019, 9, 349–353. [Google Scholar] [CrossRef]
  53. Szklarczyk, D.; Franceschini, A.; Kuhn, M.; Simonovic, M.; Roth, A.; Minguez, P.; Doerks, T.; Stark, M.; Muller, J.; Bork, P.; et al. The STRING database in 2011: Functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011, 39, D561–D568. [Google Scholar] [CrossRef]
Figure 1. SDS-PAGE 10% profiles of the extracted proteins of jumbo squid skin samples (A–D replicates). MW denotes molecular weight.
Figure 1. SDS-PAGE 10% profiles of the extracted proteins of jumbo squid skin samples (A–D replicates). MW denotes molecular weight.
Marinedrugs 18 00031 g001
Figure 2. Protein classes of the jumbo squid skin proteome identified by shotgun proteomics and categorized by PANTHER (http://pantherdb.org/).
Figure 2. Protein classes of the jumbo squid skin proteome identified by shotgun proteomics and categorized by PANTHER (http://pantherdb.org/).
Marinedrugs 18 00031 g002
Figure 3. Protein network for the jumbo squid skin proteome using the STRING (v.11.0) software. Physical direct interactions are represented with continuous lines and functional interactions with interrupted lines.
Figure 3. Protein network for the jumbo squid skin proteome using the STRING (v.11.0) software. Physical direct interactions are represented with continuous lines and functional interactions with interrupted lines.
Marinedrugs 18 00031 g003
Table 1. Jumbo squid (Dosidicus gigas) skin proteome (FDR < 1%). See Supplementary Tables S1–S3 for complete information.
Table 1. Jumbo squid (Dosidicus gigas) skin proteome (FDR < 1%). See Supplementary Tables S1–S3 for complete information.
NAccessionDescriptionGeneUni. Pep.PSMCov. (%)
1A0A1Y1DCG9Paramyosin OS = Dosidicus gigasDgPm 174622
2A0A2Z5EQ31Symplectin/biotinidase-like protein OS = Dosidicus gigassympp123
3A0A0P0UX03Hemocyanin subunit 1 OS = Todarodes pacificusTphcy116300738
4A0A077B1P8Hemocyanin subunit 2 OS = Euprymna scolopesHCY2 10160824
5A0A077B6R8Hemocyanin subunit 1 OS = Euprymna scolopesHCY1 13143719
6T2F8L5Hemocyanin OS = Sepiella maindroniHCY1 8154418
7W6CNR9Hemocyanin subunit 3 OS = Sepia officinalisHCY310103513
8A0A1Q2SJF4Hemocyanin-like protein OS = Uroteuthis edulishc 874614
9F1ADJ4Myosin heavy chain OS = Todarodes pacificusMYH 1645615
10I0JGT9Actin I OS = Sepia officinalisACTI 1120253
11G4V4Y8Myosin heavy chain isoform C OS = Doryteuthis pealeiiMYH 341112
12A4D0I0Hemocyanin subunit 1 OS = Todarodes pacificusTphcy617450
13A0A0P0UX01Hemocyanin subunit2 OS = Todarodes pacificusTphcy 417151
14A0A0L8G4B4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22000685mg 275313
15V6A729Myosin heavy chain isoform A OS = Octopus bimaculoidesMYH 23488
16Q2V0V2Tropomyosin OS = Todarodes pacificustp-tm 2712746
17A0A0L8GFI1Spectrin beta chain OS = Octopus bimaculoidesOCBIM_22034275mg 247212
18I7H9I6Haemocyanin OS = Nautilus pompiliushc 15325
19A0A075IT96Heat shock protein 70 OS = Sepiella maindroniHSP70 35923
20A0A0L8HMH4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22011261mg 12353
21E7CLR5Hemocyanin (Fragment) OS = Spirula spirulaHCY1 131512
22A0A0L8IA52Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22026555mg 14918
23A0A0L8GPG8Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22030693mg 115917
24A0A0L8FFZ3Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22022789mg 239430
25A0A0L8H027Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22024964mg 8485
26A0A0L8G0V9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22003270mg 63216
27Q06270Intermediate filament protein OS = Nototodarus sloaniiOCBIM_22025455mg93818
28Q76EJ2Cathepsin D OS = Todarodes pacificustpaD 94922
29P08052Myosin regulatory light chain LC-2, mantle muscle OS = Todarodes pacificusMYL82350
30A0A0L8HC80Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22017953mg 8165
31A0A0L8G3E9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22001601mg 13111
32P30842Omega-crystallin OS = Nototodarus sloaniiN/A5229
33Q68LN1Filamin OS = Euprymna scolopesOCBIM_22031719mg42034
34A0A0L8FU30Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22007941mg 11233
35A0A0L8I9I4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22028792mg 11822
36A0A0L8FNC4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22013362mg 5124
37Q6E216Tropomysin-like protein OS = Todarodes pacificusATRP5926
38A0A0L8HDP4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22016840mg 3275
39A0A0L8FVD0Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22007411mg 41527
40A0A0L8GWE3Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22026600mg 31312
41A0A0L8HKK9Fructose-bisphosphate aldolase OS = Octopus bimaculoidesOCBIM_22013272mg 3217
42A0A0L8FP56Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22013360mg 1163
43G1CW44Triosephosphate isomerase OS = Enteroctopus dofleiniOCBIM_22037419mg12711
44G1CW45Triosephosphate isomerase OS = Euprymna scolopesOCBIM_22037419mg1819
45A0A0L8GN79Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22030767mg 2119
46A0A0L8FZT7Protein disulfide-isomerase OS = Octopus bimaculoidesOCBIM_22003356mg 3178
47A0A0L8H0K3Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22024969mg 387
48A0A0L8GNQ0Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22030666mg 2510
49A0A0L8IA72Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025549mg 487
50A0A0L8IAK7Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025100mg 591
51A0A0L8HDG9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22017348mg 3420
52Q86DP6Malate dehydrogenase (Fragment) OS = Sepia officinalisMdh 3711
53P05945Myosin catalytic light chain LC-1, mantle muscle OS = Todarodes pacificusMYL2619
54A0A0L8GQL2Tubulin beta chain OS = Octopus bimaculoidesOCBIM_22029847mg 388
55A0A0L8HMP5Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22011994mg 31016
56A0A0L8IAD9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025091mg 336
57A0A0L8FJA0Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22017780mg 263
58A0A0L8G425Adenosylhomocysteinase OS = Octopus bimaculoidesOCBIM_22000532mg 367
59A0A0L8FXP2Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22004658mg 335
60A0A0L8I198Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22039192mg 3819
61A0A0L8I871Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22028797mg 2718
62A0A2S1FRU3Elongation factor 1-alpha OS = Callistoctopus minorEEF1A1 467
63A0A0L8FFD9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22023810mg 232
64A0A0L8I874Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22028979mg 2616
65A0A0L8FK19Tubulin alpha chain OS = Octopus bimaculoidesOCBIM_22016917mg 253
66A0A0K0WTY3Arginine kinase OS = Sepia pharaonisAK477
67A0A0L8GXA0Glucosamine-6-phosphate isomerase OS = Octopus bimaculoidesOCBIM_22026276mg 139
68F8V2T7Sodium/potassium-transporting ATPase subunit alpha OS = Bathypolypus arcticusOCBIM_22028074mg242
69A0A0L8H4W4Proteasome subunit alpha type OS = Octopus bimaculoidesOCBIM_22022293mg 2310
70A0A0L8GSZ5Histone H4 OS = Octopus bimaculoidesOCBIM_22029078mg 2510
71A0A0L8GDJ1Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22035502mg 246
72A0A159BRC2ColAa OS = Sepia pharaonisN/A261
73A0A0L8FIB5Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22020215mg 125
74A0A0L8G4U5Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22000359mg 246
75Q9NL93G protein a subunit o class OS = Octopus vulgarisOvGao 256
76A0A0L8IG11Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22004528mg 2811
77A0A0L8GG89Proteasome subunit alpha OS = Octopus bimaculoidesOCBIM_22033871mg 239
78A0A0L8H716Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22020867mg 21211
79A0A0S1U346Triosephosphate isomerase OS = Amphioctopus fangsiaoOCBIM_22037419mg1318
80A0A0L8H4E7Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22022663mg 246
81A0A0L8I919Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22027793mg 175
82A0A0L8HN83Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22010679mg 113
83A0A0L8ICB5Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22019476mg 244
84A0A0L8FMD3Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22014986mg 124
85A0A0L8H0E1Sorting nexin OS = Octopus bimaculoidesOCBIM_22024936mg 153
86A0A0L8IA39Tubulin alpha chain OS = Octopus bimaculoidesOCBIM_22026381mg 123
87A0A0L8IG73Malic enzyme OS = Octopus bimaculoidesOCBIM_22004207mg 113
88A0A0L8H635Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22021483mg 128
89A0A0L8GYT6Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22026168mg 1310
90A0A0L8GFD5Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22034343mg 123
91A0A0L8HKN4Ornithine aminotransferase OS = Octopus bimaculoidesOCBIM_22012517mg 143
92A0A0L8G0I6Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22003454mg 224
93A0A0L8HE61AP complex subunit beta OS = Octopus bimaculoidesOCBIM_22016805mg 111
94A0A0L8HMS6Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22011048mg 133
95A0A0L8FWD6Calcium-transporting ATPase OS = Octopus bimaculoidesOCBIM_22006279mg 262
96A0A0L8GP54Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22030838mg 127
97A0A0L8G9P1Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22037676mg 149
98A0A0L8HTA6Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22007620mg 146
99A0A0L8IAN9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025097mg 118
100A0A0L8HCU8Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22018310mg 132
101A0A0A7NZU2Putative chitotriosidase OS = Euprymna scolopesChia114
102A0A0L8G3Z0Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22000581mg 134
103A0A0L8I836Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22028993mg 133
104A0A0L8IDP3Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22014847mg 114
105A0A0L8FZ08Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22004461mg 111
106A0A0L8GZM9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025211mg 142
107A0A193PD55Chitinase OS = Todarodes pacificusTpChi 122
108Q8IS8060S acidic ribosomal protein OS = Euprymna scolopesOCBIM_22035130mg1319
109A0A0L8FQ90Serine/threonine-protein phosphatase OS = Octopus bimaculoidesOCBIM_22011907mg 114
110A0A0L8FIY8Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22018177mg 1313
111A0A0L8I107Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22039276mg 124
112A0A0L8G4M6Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22000216mg 120
113A0A0L8GLC5Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22031874mg 138
114A0A0L8HDX1Superoxide dismutase OS = Octopus bimaculoidesOCBIM_22016770mg 126
115A0A0L8HU31Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22005978mg 123
116Q8SWQ7Non-muscle myosin II heavy chain OS = Doryteuthis pealeiiMYH 111
117B8Q2 × 2G alpha q subunit OS = Euprymna scolopesCOI115
118A0A0L8G1S2Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22001882mg 113
119A0A0L8HAV5Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22019117mg 117
120A0A0L8IDX1Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22013485mg 144
121A0A0L8GRX5Histone H2B OS = Octopus bimaculoidesOCBIM_22029075mg 116
122A0A0L8FS75Proteasome subunit alpha type OS = Octopus bimaculoidesOCBIM_22010113mg 124
123A0A0L8FRK2Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22010655mg 126
124A0A0L8GZX1Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025682mg 151
125A0A0L8G456Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22000796mg 116
126A0A0L8FF63Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22024380mg 1110
127A0A0L8H8U9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22020735mg 115
128A0A0L8I5N4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22033390mg 123
129A0A0L8I398Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22037157mg 1111
130A0A0L8GP93Nicotinamide-nucleotide adenylyltransferase OS = Octopus bimaculoidesOCBIM_22030204mg 116
131A0A0L8IIH3Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025740mg 130
132A0A0L8GZD4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025455mg 111
133A0A0L8HQW9Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22008430mg 142
134A0A0L8G2Z7Small ubiquitin-related modifier OS = Octopus bimaculoidesOCBIM_22001102mg 1111
135A0A0L8G8L3Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22038063mg 122
136O46345S-syntaxin OS = Doryteuthis pealeiiSTX1113
137A0A0L8GDD2Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22036000mg 112
138C4N147Sodium/calcium exchanger regulatory protein 1 OS = Doryteuthis pealeiiSLC8A1 147
139A0A0L8FJE4Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22017696mg 122
140A0A0L8I067Kinesin-like protein OS = Octopus bimaculoidesOCBIM_22000619mg 111
141A0A0L8FYB6Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22005155mg 111
142A0A0L8GUV0Serine/threonine-protein phosphatase OS = Octopus bimaculoidesOCBIM_22027338mg 112
143A0A0L8GJ12Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22032700mg 121
144A0A0L8GLG2Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22032112mg 111
145A0A0L8GY97Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22026356mg 122
146Q27Q56Hemocyanin subunit 2 OS = Sepia officinalisHCY2 19617
147A0A161HPY5Actin OS = Crassostrea brasilianaACTI 39638
148D2YZ90Beta actin OS = Idiosepius paradoxusACTI 29537
149K1QFR9Spectrin beta chain OS = Crassostrea gigasCGI_10013845 1344
150C1KC83Heat shock cognate protein 70 OS = Haliotis diversicolorHSP70 13716
151A0A2C9K1T4Uncharacterized protein OS = Biomphalaria glabrata10607816716813
152A0A0B7B7H2Uncharacterized protein OS = Arion vulgarisORF162822 14011
153A0A2T7NLR4Uncharacterized protein OS = Pomacea canaliculataC0Q70_17913 1185
154K1RH58Alpha-actinin, sarcomeric OS = Crassostrea gigasCGI_10003110 14310
155A0A2P1H676Heat shock protein 70 OS = Diplodon chilensisHSP70 13612
156K1PMY9Calmodulin OS = Crassostrea gigasCGI_10006482 12213
157A0A2T7NGU8Uncharacterized protein OS = Pomacea canaliculataC0Q70_18553 51229
158Q564J1Haemocyanin OS = Aplysia californicahc 29272
159A0A2T7NV41Uncharacterized protein OS = Pomacea canaliculataC0Q70_15545 41825
160E7DS67Actin (Fragment) OS = Gonospira metablataACTI 13818
161K1RBG6Actin-1/3 OS = Crassostrea gigasCGI_10017112 1398
162P02595Calmodulin OS = Patinopecten sp.CAM11630
163V6A758Myosin heavy chain isoform C OS = Sepia officinalisMYH 11716
164A0A0B7BLG3Uncharacterized protein OS = Arion vulgarisORF192624 3232
165K1PPW8Coatomer subunit beta OS = Crassostrea gigasCGI_10006442 287
166A0A210R0F2Fructose-bisphosphate aldolase OS = Mizuhopecten yessoensisKP79_PYT16607 286
167A0A2T7PZW7Uncharacterized protein OS = Pomacea canaliculataC0Q70_01565 161
168A0A0B7B4N1Uncharacterized protein OS = Arion vulgarisORF158201 1104
169A0A210QY92Coatomer subunit beta’ OS = Mizuhopecten yessoensisKP79_PYT21841 155
170V3ZPS1Uncharacterized protein OS = Lottia giganteaLOTGIDRAFT_222012 2912
171E3VWM3Fructose-bisphosphate aldolase OS = Meretrix meretrixFBA 1204
172A0A2T7PSV4Uncharacterized protein OS = Pomacea canaliculataC0Q70_03483 21011
173A0A0B7AZA8Uncharacterized protein OS = Arion vulgarisORF148015 21019
174K7WKX6Fructose-bisphosphate aldolase OS = Haliotis rufescensFBA 139
175A0A2T7NF32Uncharacterized protein OS = Pomacea canaliculataC0Q70_20261 154
176A0A2T7NMW4Uncharacterized protein OS = Pomacea canaliculataC0Q70_18325 245
177K1QZU8Calcium-transporting ATPase OS = Crassostrea gigasCGI_10023684 121
178A0A0L8IAE8Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22025089mg 128
179A0A2C9KC89Uncharacterized protein OS = Biomphalaria glabrata106056965253
180A0A210R746Ras-related protein Rab-6A OS = Mizuhopecten yessoensis KP79_PYT20147 1911
181A0A0B6Z4Q3Uncharacterized protein OS = Arion vulgarisORF48472 2128
182A0A2T7PZP4Uncharacterized protein OS = Pomacea canaliculataC0Q70_01513 143
183A0A2C9JIZ4Uncharacterized protein OS = Biomphalaria glabrata1060568491913
184K1PTH4ADP-ribosylation factor OS = Crassostrea gigasCGI_10020174 121
185Q6PTL0Triosephosphate isomerase OS = Nucula proximaOCBIM_22037419mg156
186A0A2C9JZR8Uncharacterized protein OS = Biomphalaria glabrata106074442122
187A0A2C9JIA9Uncharacterized protein OS = Biomphalaria glabrata106056539164
188A0A385NHM7Glutathione S-transferase OS = Tegillarca granosaGST 185
189A0A210QUP5Malic enzyme OS = Mizuhopecten yessoensisKP79_PYT06884 113
190V3YXF9Adenosylhomocysteinase OS = Lottia giganteaLOTGIDRAFT_184532 123
191A0A210QGP4Chitotriosidase-1 OS = Mizuhopecten yessoensisKP79_PYT06201 113
192A0A210QHE1Adenosylhomocysteinase OS = Mizuhopecten yessoensisKP79_PYT14445 143
193A0A210PIA6Ornithine aminotransferase OS = Mizuhopecten yessoensis KP79_PYT16913 133
194K1QQB640S ribosomal protein S14 OS = Crassostrea gigasCGI_10011151 149
195A0A2C9KEN8Tubulin alpha chain OS = Biomphalaria glabrata106069694123
196A0A2T7PWT6Serine/threonine-protein phosph OS = Pomacea canaliculataC0Q70_00460 113
197A0A0B7AJW7Fructose-bisphosphate aldolase OS = Arion vulgarisORF124546 184
198A0A2C9L7N6Uncharacterized protein OS = Biomphalaria glabrata1060803191494
199A0A210QTZ1Peptidyl-prolyl cis-trans OS = Mizuhopecten yessoensisKP79_PYT00632 126
200A0A2I7M8C2Go protein alpha subunit OS = Argopecten irradiansN/A143
201K1R2G8Titin OS = Crassostrea gigasCGI_10016808 120
202K1QVD7Neuronal acetylcholine receptor subunit non-alpha-2 OS = Crassostrea gigasCGI_10016138 121
203K1Q7G5Ficolin-2 OS = Crassostrea gigasCGI_10026202 123
204A0A2C9K9W9Uncharacterized protein OS = Biomphalaria glabrata106068683111
205A0A0B6ZP87Uncharacterized protein OS = Arion vulgarisORF71130 134
206V4AP92Elongation factor 1-alpha OS = Lottia giganteaLOTGIDRAFT_239271 122
207A0A2T7PU69Uncharacterized protein OS = Pomacea canaliculataC0Q70_03920 144
208V3ZN51Staphylococcal nuclease domain-cont. OS = Lottia giganteaLOTGIDRAFT_235720 131
209A0A2T7PSF5Uncharacterized protein OS = Pomacea canaliculataC0Q70_03333 120
210K1PQD4Phosphoglucomutase-1 OS = Crassostrea gigasCGI_10011818 112
211A0A0B7BF17Uncharacterized protein OS = Arion vulgarisORF179770 132
212A0A2T7Q0W0Uncharacterized protein OS = Pomacea canaliculataC0Q70_01928 113
213A0A0L8I692Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22034637mg 1419
214K1PQ79Copine-3 OS = Crassostrea gigasCGI_10011897 131
215K1PWB9EH domain-containing protein 1 OS = Crassostrea gigasCGI_10005813 114
216A0A2T7Q016Uncharacterized protein OS = Pomacea canaliculataC0Q70_01636 112
217V4AKV4Calcium-transporting ATPase OS = Lottia giganteaLOTGIDRAFT_208914 131
218A0A2T7NL99Proteasome subunit beta OS = Pomacea canaliculataC0Q70_17739 124
219A0A0L8HWW8Uncharacterized protein OS = Octopus bimaculoidesOCBIM_22003772mg 122
N (Identification Number); FDR (False Discovery Rate); Uni. Pep. (Unique Peptides); PSMs (Peptide Spectrum Matches); Cov. (Protein Coverage).
Table 2. KEGG pathway analysis of the jumbo squid skin proteome by DAVID.
Table 2. KEGG pathway analysis of the jumbo squid skin proteome by DAVID.
KEGG Pathwayp-Value
Metabolic pathways (cysteine and methionine metabolism)4.53 × 10−4
Endocytosis/phagosome1.05 × 10−2
RNA transport2.24 × 10−2
Protein methylation3.46 × 10−2
Calcium homeostasis1.00 × 10−1
Table 3. Functional InterPro motifs by DAVID.
Table 3. Functional InterPro motifs by DAVID.
InterPro Motifsp-Value
Small GTP-binding protein domain 3.1 × 10−4
Heat shock protein 70, conserved site8.5 × 10−4
Small GTPase superfamily8.6 × 10−4
Proteasome, alpha-subunit, N-terminal domain1.3 × 10−3
P-loop containing nucleoside triphosphate hydrolase8.3 × 10−3
EF-hand-like domain2.9 × 10−2
Ubiquitin3.4 × 10−2
Table 4. Selected potential bioactive peptides of the jumbo squid skin proteome predicted by in-silico digestions with pepsin.
Table 4. Selected potential bioactive peptides of the jumbo squid skin proteome predicted by in-silico digestions with pepsin.
ProteinsPeptidesPeptideRanker ScoreAnti-Microbial Peptide (AMP)Discriminant Score for AMP
ADP-ribosylation factor OS = Crassostrea gigasSPSPKQMVSCPVCGL0.915222Non-AMP0.043
Collagen ColAa OS = Sepia pharaonisPGDPGPVGRTGPMGL0.934847Non-AMP0.003
Collagen ColAa OS = Sepia pharaonisRGPPGPPGL0.912657Non-AMP0.030
Heat shock protein 70 OS = Sepiella maindroniGGMPGGMPGGMPGGMPNF0.92432AMP0.504
Hemocyanin OS = Sepiella maindroniKKPMMPF0.932566AMP0.978
Hemocyanin OS = Sepiella maindroniPNQPMRPF0.920777AMP0.983
Hemocyanin subunit 1 OS = Todarodes pacificusNDPMRPF0.923312AMP0.795
Hemocyanin subunit 2 OS = Sepia officinalisSDPMRPF0.938433AMP0.879
Uncharacterized protein OS = Octopus bimaculoidesCPCMGRF0.985441AMP0.622
Uncharacterized protein OS = Octopus bimaculoidesGGPPGMPPF0.973279Non-AMP0.208
Uncharacterized protein OS = Octopus bimaculoidesGRCVMCNCNKHSSTCDPQTGKCVNCQHNTL0.969319Non-AMP0.238
Uncharacterized protein OS = Octopus bimaculoidesGSCVPCNCNGF0.952459AMP0.745
Uncharacterized protein OS = Octopus bimaculoidesQPPQCCPSKGGSF0.943546AMP0.687
Uncharacterized protein OS = Octopus bimaculoidesGSWGNGNRW0.915802Non-AMP0.403
Uncharacterized protein OS = Octopus bimaculoidesPPPSKRF0.911736AMP0.983
Uncharacterized protein OS = Biomphalaria glabrataPPPPQPVGGGGGNRW0.955862Non-AMP0.092
Uncharacterized protein OS = Biomphalaria glabrataSRSPPRPF0.904351AMP0.993
Uncharacterized protein OS = Pomacea canaliculataHDGDGPRPCCF0.93215Non-AMP0.031
Table 5. Selected potential bioactive peptides of the jumbo squid skin proteome predicted by in-silico digestions with trypsin.
Table 5. Selected potential bioactive peptides of the jumbo squid skin proteome predicted by in-silico digestions with trypsin.
ProteinsPeptidesPeptideRanker ScoreAnti-Microbial Peptide (AMP)Discriminant Score for AMP
ADP-ribosylation factor OS = Crassostrea gigasCPICYDFMHTAMILPECSHTFCSFCIR0.902646Non-AMP0.160
Calcium-transporting ATPase OS = Octopus bimaculoidesFSDDYPGFF0.970864Non-AMP0.006
Calcium-transporting ATPase OS = Crassostrea gigasFLQFQLTVNCVAVMVAFFGACIINDSPLK0.979848Non-AMP0.281
Calcium-transporting ATPase OS=Lottia giganteaFADAPFMK0.93747Non-AMP0.014
Calmodulin OS = Crassostrea gigasGAFFVFDR0.915228Non-AMP0.003
Chitinase OS = Todarodes pacificusMLAVSLLFLLAIGGVSSAGHR0.976725AMP0.746
Chitotriosidase OS = Euprymna scolopesMASTFATVFGVLSLCFLGLHLTNGEYK0.984749Non-AMP0.106
Coatomer subunit beta’ OS = Mizuhopecten yessoensisYCLCLFR0.924855AMP0.579
Collagen ColAa OS = Sepia pharaonisGPPGIPGLPGPK0.93716AMP0.504
Collagen ColAa OS = Sepia pharaonisGPPGPPGLK0.913133Non-AMP0.119
Collagen ColAa OS = Sepia pharaonisAGPPGFPGTPGPK0.907398AMP0.682
Ficolin-2 OS = Crassostrea gigasDQDNDMYVSDNCGILFPSGWWHR0.901865Non-AMP0.008
Fructose-bisphosphate aldolase OS = Mizuhopecten yessoensisKPWALTFSFGR0.93422Non-AMP0.123
Hemocyanin OS = Aplysia californicaMVGYLGQALMALLLLALSNAALVR0.993669Non-AMP0.380
Hemocyanin OS = Aplysia californicaFEPNPFFSGK0.924588Non-AMP0.093
Hemocyanin OS = Aplysia californicaVACCLHGMPVFPHWHR0.903581Non-AMP0.106
Hemocyanin OS = Nautilus pompiliusMATHWHSLLLFSLQLLVFTYATSDPTNIR0.97599Non-AMP0.008
Hemocyanin OS = Sepiella maindroniGSPIGVPYWDWTKPMK0.917605Non-AMP0.027
Hemocyanin-like protein OS = Uroteuthis edulisTNFFFLALIATVWLGNAETETETSK0.90323Non-AMP0.062
Hemocyanin subunit 1 OS = Euprymna scolopesVFVGFLLHGFGSSAYATFDICNDAGECR0.96087Non-AMP0.233
Hemocyanin subunit 1 OS = Euprymna scolopesLNHLPLLCLAVILTLWMSGSNTVNGNLVR0.926117Non-AMP0.287
Hemocyanin subunit 1 OS = Euprymna scolopesVFAGFLFMGIK0.904542AMP0.865
Hemocyanin subunit 2 OS = Euprymna scolopesVFAGFWFHGIK0.943AMP0.506
Hemocyanin subunit 2 OS = Sepia officinalisVFGGFWLHGIK0.907156AMP0.739
Hemocyanin subunit 3 OS = Sepia officinalisTSFLFLAFVATSWFVYAVTASK0.905214Non-AMP0.136
Malate dehydrogenase OS = Sepia officinalisDLFNTNASIVANLADACAQYCPK0.965037Non-AMP0.251
Myosin heavy chain isoform A OS = Octopus bimaculoidesYQSGFIYTYSGLFCVAINPYR0.956725Non-AMP0.024
Myosin heavy chain OS = Todarodes pacificusNWEWWR0.951523Non-AMP0.478
Myosin II heavy chain OS = Doryteuthis pealeiiNWQWWR0.973264AMP0.959
Myosin II heavy chain OS = Doryteuthis pealeiiYYSGLIYTYSGLFCVVVNPYK0.939159Non-AMP0.032
Neuronal acetylcholine receptor subunit non-alpha-2 OS = Crassostrea gigasLLIDLCLSVLVTTLAIVSLYFYDMSDSR0.904075Non-AMP0.015
Peptidyl-prolyl cis-trans isomerase OS = Mizuhopecten yessoensisMAGAGIGCVLLFLLPALLSAGK0.996478Non-AMP0.159
Phosphoglucomutase-1 OS = Crassostrea gigasDGLWAVLAWLSVLANQNCSVEECIK0.991266AMP0.904
Protein disulfide-isomerase OS = Octopus bimaculoidesNVFIEFYAPWCGHCK0.907443Non-AMP0.053
S-syntaxin OS = Doryteuthis pealeiiIAILVCLVILVLVIVSTVGGVFGG0.965343Non-AMP0.000
Titin OS = Crassostrea gigasDGSWQNLVTVLGCLKPQFVNLQR0.974127AMP0.724
Titin OS = Crassostrea gigasGYPPPIISWYR0.917986Non-AMP0.074
Tubulin alpha chain OS = Octopus bimaculoidesFVDWCPTGFK0.923256Non-AMP0.010
Uncharacterized protein OS = Arion vulgarisAPDFIFYAPR0.921198Non-AMP0.009
Uncharacterized protein OS = Octopus bimaculoidesFLQFQLTVNVVAVLVAFFGACTINVSI0.978717AMP0.916
Uncharacterized protein OS = Octopus bimaculoidesYYTFFVTIFLFATTLCSTIPKPK0.984914Non-AMP0.012
Uncharacterized protein OS = Octopus bimaculoidesLFPAFGFGAR0.94902AMP0.505
Uncharacterized protein OS = Octopus bimaculoidesATMLGAQGNIFFASLSCCCLILSCS0.999233AMP0.879
Uncharacterized protein OS = Octopus bimaculoidesSGPFYIFSGGMPR0.939205Non-AMP0.089
Uncharacterized protein OS = Octopus bimaculoidesEFSMMFR0.931708Non-AMP0.001
Uncharacterized protein OS = Octopus bimaculoidesYGSCVPCNCNGFSNDCDPVTGECIDCQR0.980617Non-AMP0.243
Uncharacterized protein OS = Octopus bimaculoidesHNPEGCISCFCMGVTEFCTSTSR0.964134Non-AMP0.083
Uncharacterized protein OS = Octopus bimaculoidesAPMVELCECPQGYTGVSCQECSPGYSR0.963828Non-AMP0.012
Uncharacterized protein OS = Octopus bimaculoidesGCGCSAGQFECQNGLCINENK0.930153AMP0.982
Uncharacterized protein OS = Octopus bimaculoidesEECMSCFCFK0.918951AMP0.982
Uncharacterized protein OS = Octopus bimaculoidesNSEYGFACFCPQGFAGYQCDTVGER0.906197AMP0.576
Uncharacterized protein OS = Octopus bimaculoidesMIIYILSLAGVALGVYFLSCVR0.995663Non-AMP0.008
Uncharacterized protein OS = Octopus bimaculoidesMILTIFACLMALDIELNTSNSIQEE0.968187Non-AMP0.026
Uncharacterized protein OS = Octopus bimaculoidesAIGALVDACGPGLCPDWADWAPK0.948884AMP0.774
Uncharacterized protein OS = Octopus bimaculoidesQGDWTCPNPACGNNNFGWR0.9572Non-AMP0.286
Uncharacterized protein OS = Octopus bimaculoidesGGFGGGGGGGGGMGGDR0.928063Non-AMP0.065
Uncharacterized protein OS = Octopus bimaculoidesGFFEDDYDEYGGGYGGGMGFGGLNR0.944869Non-AMP0.143
Uncharacterized protein OS = Octopus bimaculoidesLDDGDACLLDMGTEYCCYASDITCSYPVNGK0.968621Non-AMP0.056
Uncharacterized protein OS = Octopus bimaculoidesMAFYTILNVVTIVLLIIVGQCR0.998628Non-AMP0.031
Uncharacterized protein OS = Octopus bimaculoidesGGSFGFNFR0.969779Non-AMP0.355
Uncharacterized protein OS = Octopus bimaculoidesNSTDVCNCSIYVGLFPCNECTK0.994975Non-AMP0.462
Uncharacterized protein OS = Octopus bimaculoidesPPSPPIYFR0.946483Non-AMP0.226
Uncharacterized protein OS = Octopus bimaculoidesCFLCATGTGTSIEVLALVTIGWCLLHATGTR0.96344AMP0.768
Uncharacterized protein OS = Octopus bimaculoidesFDFFYK0.96245Non-AMP0.032
Uncharacterized protein OS = Octopus bimaculoidesFSPIPFLFCTISGTCNFATR0.95134AMP0.505
Uncharacterized protein OS = Octopus bimaculoidesFWELTECCPHQCLEWLSNLVTR0.933791Non-AMP0.106
Uncharacterized protein OS = Octopus bimaculoidesDAFCSSPNFNSWLK0.922125Non-AMP0.058
Uncharacterized protein OS = Octopus bimaculoidesNGYEEDDALIGLLNLCTAILK0.917521Non-AMP0.479
Uncharacterized protein OS = Octopus bimaculoidesDYFWLVCR0.911557Non-AMP0.001
Uncharacterized protein OS = Biomphalaria glabrataQGELGDCWLLAAVASLTCNPK0.919385AMP0.783
Uncharacterized protein OS = Biomphalaria glabrataSPPRPFEWK0.905581Non-AMP0.006
Uncharacterized protein OS = Pomacea canaliculataSVFNIPPNCFSEMM0.908085Non-AMP0.003
Uncharacterized protein OS = Pomacea canaliculataSCLMGHGSLFGAGAGSLHLQAIAALK0.919795Non-AMP0.315
Back to TopTop