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. 2019 Dec 29;18(1):31. doi: 10.3390/md18010031

Characterization of the Jumbo Squid (Dosidicus gigas) Skin By-Product by Shotgun Proteomics and Protein-Based Bioinformatics

Mónica Carrera 1,*, Josafat Marina Ezquerra-Brauer 2, Santiago P Aubourg 1
PMCID: PMC7024357  PMID: 31905758

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).

Table 1.

Jumbo squid (Dosidicus gigas) skin proteome (FDR < 1%). See Supplementary Tables S1–S3 for complete information.

N Accession Description Gene Uni. Pep. PSM Cov. (%)
1 A0A1Y1DCG9 Paramyosin OS = Dosidicus gigas DgPm 17 46 22
2 A0A2Z5EQ31 Symplectin/biotinidase-like protein OS = Dosidicus gigas sympp 1 2 3
3 A0A0P0UX03 Hemocyanin subunit 1 OS = Todarodes pacificus Tphcy 116 3007 38
4 A0A077B1P8 Hemocyanin subunit 2 OS = Euprymna scolopes HCY2 10 1608 24
5 A0A077B6R8 Hemocyanin subunit 1 OS = Euprymna scolopes HCY1 13 1437 19
6 T2F8L5 Hemocyanin OS = Sepiella maindroni HCY1 8 1544 18
7 W6CNR9 Hemocyanin subunit 3 OS = Sepia officinalis HCY3 10 1035 13
8 A0A1Q2SJF4 Hemocyanin-like protein OS = Uroteuthis edulis hc 8 746 14
9 F1ADJ4 Myosin heavy chain OS = Todarodes pacificus MYH 16 456 15
10 I0JGT9 Actin I OS = Sepia officinalis ACTI 11 202 53
11 G4V4Y8 Myosin heavy chain isoform C OS = Doryteuthis pealeii MYH 3 411 12
12 A4D0I0 Hemocyanin subunit 1 OS = Todarodes pacificus Tphcy 6 174 50
13 A0A0P0UX01 Hemocyanin subunit2 OS = Todarodes pacificus Tphcy 4 171 51
14 A0A0L8G4B4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22000685mg 27 53 13
15 V6A729 Myosin heavy chain isoform A OS = Octopus bimaculoides MYH 2 348 8
16 Q2V0V2 Tropomyosin OS = Todarodes pacificus tp-tm 27 127 46
17 A0A0L8GFI1 Spectrin beta chain OS = Octopus bimaculoides OCBIM_22034275mg 24 72 12
18 I7H9I6 Haemocyanin OS = Nautilus pompilius hc 1 532 5
19 A0A075IT96 Heat shock protein 70 OS = Sepiella maindroni HSP70 3 59 23
20 A0A0L8HMH4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22011261mg 12 35 3
21 E7CLR5 Hemocyanin (Fragment) OS = Spirula spirula HCY1 1 315 12
22 A0A0L8IA52 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22026555mg 1 49 18
23 A0A0L8GPG8 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22030693mg 11 59 17
24 A0A0L8FFZ3 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22022789mg 2 394 30
25 A0A0L8H027 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22024964mg 8 48 5
26 A0A0L8G0V9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22003270mg 6 32 16
27 Q06270 Intermediate filament protein OS = Nototodarus sloanii OCBIM_22025455mg 9 38 18
28 Q76EJ2 Cathepsin D OS = Todarodes pacificus tpaD 9 49 22
29 P08052 Myosin regulatory light chain LC-2, mantle muscle OS = Todarodes pacificus MYL 8 23 50
30 A0A0L8HC80 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22017953mg 8 16 5
31 A0A0L8G3E9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22001601mg 1 31 11
32 P30842 Omega-crystallin OS = Nototodarus sloanii N/A 5 22 9
33 Q68LN1 Filamin OS = Euprymna scolopes OCBIM_22031719mg 4 20 34
34 A0A0L8FU30 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22007941mg 1 12 33
35 A0A0L8I9I4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22028792mg 1 18 22
36 A0A0L8FNC4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22013362mg 5 12 4
37 Q6E216 Tropomysin-like protein OS = Todarodes pacificus ATRP 5 9 26
38 A0A0L8HDP4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22016840mg 3 27 5
39 A0A0L8FVD0 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22007411mg 4 15 27
40 A0A0L8GWE3 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22026600mg 3 13 12
41 A0A0L8HKK9 Fructose-bisphosphate aldolase OS = Octopus bimaculoides OCBIM_22013272mg 3 21 7
42 A0A0L8FP56 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22013360mg 1 16 3
43 G1CW44 Triosephosphate isomerase OS = Enteroctopus dofleini OCBIM_22037419mg 1 27 11
44 G1CW45 Triosephosphate isomerase OS = Euprymna scolopes OCBIM_22037419mg 1 8 19
45 A0A0L8GN79 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22030767mg 2 11 9
46 A0A0L8FZT7 Protein disulfide-isomerase OS = Octopus bimaculoides OCBIM_22003356mg 3 17 8
47 A0A0L8H0K3 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22024969mg 3 8 7
48 A0A0L8GNQ0 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22030666mg 2 5 10
49 A0A0L8IA72 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025549mg 4 8 7
50 A0A0L8IAK7 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025100mg 5 9 1
51 A0A0L8HDG9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22017348mg 3 4 20
52 Q86DP6 Malate dehydrogenase (Fragment) OS = Sepia officinalis Mdh 3 7 11
53 P05945 Myosin catalytic light chain LC-1, mantle muscle OS = Todarodes pacificus MYL 2 6 19
54 A0A0L8GQL2 Tubulin beta chain OS = Octopus bimaculoides OCBIM_22029847mg 3 8 8
55 A0A0L8HMP5 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22011994mg 3 10 16
56 A0A0L8IAD9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025091mg 3 3 6
57 A0A0L8FJA0 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22017780mg 2 6 3
58 A0A0L8G425 Adenosylhomocysteinase OS = Octopus bimaculoides OCBIM_22000532mg 3 6 7
59 A0A0L8FXP2 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22004658mg 3 3 5
60 A0A0L8I198 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22039192mg 3 8 19
61 A0A0L8I871 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22028797mg 2 7 18
62 A0A2S1FRU3 Elongation factor 1-alpha OS = Callistoctopus minor EEF1A1 4 6 7
63 A0A0L8FFD9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22023810mg 2 3 2
64 A0A0L8I874 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22028979mg 2 6 16
65 A0A0L8FK19 Tubulin alpha chain OS = Octopus bimaculoides OCBIM_22016917mg 2 5 3
66 A0A0K0WTY3 Arginine kinase OS = Sepia pharaonis AK 4 7 7
67 A0A0L8GXA0 Glucosamine-6-phosphate isomerase OS = Octopus bimaculoides OCBIM_22026276mg 1 3 9
68 F8V2T7 Sodium/potassium-transporting ATPase subunit alpha OS = Bathypolypus arcticus OCBIM_22028074mg 2 4 2
69 A0A0L8H4W4 Proteasome subunit alpha type OS = Octopus bimaculoides OCBIM_22022293mg 2 3 10
70 A0A0L8GSZ5 Histone H4 OS = Octopus bimaculoides OCBIM_22029078mg 2 5 10
71 A0A0L8GDJ1 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22035502mg 2 4 6
72 A0A159BRC2 ColAa OS = Sepia pharaonis N/A 2 6 1
73 A0A0L8FIB5 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22020215mg 1 2 5
74 A0A0L8G4U5 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22000359mg 2 4 6
75 Q9NL93 G protein a subunit o class OS = Octopus vulgaris OvGao 2 5 6
76 A0A0L8IG11 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22004528mg 2 8 11
77 A0A0L8GG89 Proteasome subunit alpha OS = Octopus bimaculoides OCBIM_22033871mg 2 3 9
78 A0A0L8H716 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22020867mg 2 12 11
79 A0A0S1U346 Triosephosphate isomerase OS = Amphioctopus fangsiao OCBIM_22037419mg 1 3 18
80 A0A0L8H4E7 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22022663mg 2 4 6
81 A0A0L8I919 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22027793mg 1 7 5
82 A0A0L8HN83 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22010679mg 1 1 3
83 A0A0L8ICB5 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22019476mg 2 4 4
84 A0A0L8FMD3 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22014986mg 1 2 4
85 A0A0L8H0E1 Sorting nexin OS = Octopus bimaculoides OCBIM_22024936mg 1 5 3
86 A0A0L8IA39 Tubulin alpha chain OS = Octopus bimaculoides OCBIM_22026381mg 1 2 3
87 A0A0L8IG73 Malic enzyme OS = Octopus bimaculoides OCBIM_22004207mg 1 1 3
88 A0A0L8H635 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22021483mg 1 2 8
89 A0A0L8GYT6 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22026168mg 1 3 10
90 A0A0L8GFD5 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22034343mg 1 2 3
91 A0A0L8HKN4 Ornithine aminotransferase OS = Octopus bimaculoides OCBIM_22012517mg 1 4 3
92 A0A0L8G0I6 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22003454mg 2 2 4
93 A0A0L8HE61 AP complex subunit beta OS = Octopus bimaculoides OCBIM_22016805mg 1 1 1
94 A0A0L8HMS6 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22011048mg 1 3 3
95 A0A0L8FWD6 Calcium-transporting ATPase OS = Octopus bimaculoides OCBIM_22006279mg 2 6 2
96 A0A0L8GP54 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22030838mg 1 2 7
97 A0A0L8G9P1 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22037676mg 1 4 9
98 A0A0L8HTA6 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22007620mg 1 4 6
99 A0A0L8IAN9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025097mg 1 1 8
100 A0A0L8HCU8 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22018310mg 1 3 2
101 A0A0A7NZU2 Putative chitotriosidase OS = Euprymna scolopes Chia 1 1 4
102 A0A0L8G3Z0 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22000581mg 1 3 4
103 A0A0L8I836 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22028993mg 1 3 3
104 A0A0L8IDP3 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22014847mg 1 1 4
105 A0A0L8FZ08 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22004461mg 1 1 1
106 A0A0L8GZM9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025211mg 1 4 2
107 A0A193PD55 Chitinase OS = Todarodes pacificus TpChi 1 2 2
108 Q8IS80 60S acidic ribosomal protein OS = Euprymna scolopes OCBIM_22035130mg 1 3 19
109 A0A0L8FQ90 Serine/threonine-protein phosphatase OS = Octopus bimaculoides OCBIM_22011907mg 1 1 4
110 A0A0L8FIY8 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22018177mg 1 3 13
111 A0A0L8I107 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22039276mg 1 2 4
112 A0A0L8G4M6 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22000216mg 1 2 0
113 A0A0L8GLC5 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22031874mg 1 3 8
114 A0A0L8HDX1 Superoxide dismutase OS = Octopus bimaculoides OCBIM_22016770mg 1 2 6
115 A0A0L8HU31 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22005978mg 1 2 3
116 Q8SWQ7 Non-muscle myosin II heavy chain OS = Doryteuthis pealeii MYH 1 1 1
117 B8Q2 × 2 G alpha q subunit OS = Euprymna scolopes COI 1 1 5
118 A0A0L8G1S2 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22001882mg 1 1 3
119 A0A0L8HAV5 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22019117mg 1 1 7
120 A0A0L8IDX1 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22013485mg 1 4 4
121 A0A0L8GRX5 Histone H2B OS = Octopus bimaculoides OCBIM_22029075mg 1 1 6
122 A0A0L8FS75 Proteasome subunit alpha type OS = Octopus bimaculoides OCBIM_22010113mg 1 2 4
123 A0A0L8FRK2 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22010655mg 1 2 6
124 A0A0L8GZX1 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025682mg 1 5 1
125 A0A0L8G456 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22000796mg 1 1 6
126 A0A0L8FF63 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22024380mg 1 1 10
127 A0A0L8H8U9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22020735mg 1 1 5
128 A0A0L8I5N4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22033390mg 1 2 3
129 A0A0L8I398 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22037157mg 1 1 11
130 A0A0L8GP93 Nicotinamide-nucleotide adenylyltransferase OS = Octopus bimaculoides OCBIM_22030204mg 1 1 6
131 A0A0L8IIH3 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025740mg 1 3 0
132 A0A0L8GZD4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025455mg 1 1 1
133 A0A0L8HQW9 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22008430mg 1 4 2
134 A0A0L8G2Z7 Small ubiquitin-related modifier OS = Octopus bimaculoides OCBIM_22001102mg 1 1 11
135 A0A0L8G8L3 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22038063mg 1 2 2
136 O46345 S-syntaxin OS = Doryteuthis pealeii STX1 1 1 3
137 A0A0L8GDD2 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22036000mg 1 1 2
138 C4N147 Sodium/calcium exchanger regulatory protein 1 OS = Doryteuthis pealeii SLC8A1 1 4 7
139 A0A0L8FJE4 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22017696mg 1 2 2
140 A0A0L8I067 Kinesin-like protein OS = Octopus bimaculoides OCBIM_22000619mg 1 1 1
141 A0A0L8FYB6 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22005155mg 1 1 1
142 A0A0L8GUV0 Serine/threonine-protein phosphatase OS = Octopus bimaculoides OCBIM_22027338mg 1 1 2
143 A0A0L8GJ12 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22032700mg 1 2 1
144 A0A0L8GLG2 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22032112mg 1 1 1
145 A0A0L8GY97 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22026356mg 1 2 2
146 Q27Q56 Hemocyanin subunit 2 OS = Sepia officinalis HCY2 1 961 7
147 A0A161HPY5 Actin OS = Crassostrea brasiliana ACTI 3 96 38
148 D2YZ90 Beta actin OS = Idiosepius paradoxus ACTI 2 95 37
149 K1QFR9 Spectrin beta chain OS = Crassostrea gigas CGI_10013845 1 34 4
150 C1KC83 Heat shock cognate protein 70 OS = Haliotis diversicolor HSP70 1 37 16
151 A0A2C9K1T4 Uncharacterized protein OS = Biomphalaria glabrata 106078167 1 68 13
152 A0A0B7B7H2 Uncharacterized protein OS = Arion vulgaris ORF162822 1 40 11
153 A0A2T7NLR4 Uncharacterized protein OS = Pomacea canaliculata C0Q70_17913 1 18 5
154 K1RH58 Alpha-actinin, sarcomeric OS = Crassostrea gigas CGI_10003110 1 43 10
155 A0A2P1H676 Heat shock protein 70 OS = Diplodon chilensis HSP70 1 36 12
156 K1PMY9 Calmodulin OS = Crassostrea gigas CGI_10006482 1 22 13
157 A0A2T7NGU8 Uncharacterized protein OS = Pomacea canaliculata C0Q70_18553 5 12 29
158 Q564J1 Haemocyanin OS = Aplysia californica hc 2 927 2
159 A0A2T7NV41 Uncharacterized protein OS = Pomacea canaliculata C0Q70_15545 4 18 25
160 E7DS67 Actin (Fragment) OS = Gonospira metablata ACTI 1 38 18
161 K1RBG6 Actin-1/3 OS = Crassostrea gigas CGI_10017112 1 39 8
162 P02595 Calmodulin OS = Patinopecten sp. CAM 1 16 30
163 V6A758 Myosin heavy chain isoform C OS = Sepia officinalis MYH 1 17 16
164 A0A0B7BLG3 Uncharacterized protein OS = Arion vulgaris ORF192624 3 23 2
165 K1PPW8 Coatomer subunit beta OS = Crassostrea gigas CGI_10006442 2 8 7
166 A0A210R0F2 Fructose-bisphosphate aldolase OS = Mizuhopecten yessoensis KP79_PYT16607 2 8 6
167 A0A2T7PZW7 Uncharacterized protein OS = Pomacea canaliculata C0Q70_01565 1 6 1
168 A0A0B7B4N1 Uncharacterized protein OS = Arion vulgaris ORF158201 1 10 4
169 A0A210QY92 Coatomer subunit beta’ OS = Mizuhopecten yessoensis KP79_PYT21841 1 5 5
170 V3ZPS1 Uncharacterized protein OS = Lottia gigantea LOTGIDRAFT_222012 2 9 12
171 E3VWM3 Fructose-bisphosphate aldolase OS = Meretrix meretrix FBA 1 20 4
172 A0A2T7PSV4 Uncharacterized protein OS = Pomacea canaliculata C0Q70_03483 2 10 11
173 A0A0B7AZA8 Uncharacterized protein OS = Arion vulgaris ORF148015 2 10 19
174 K7WKX6 Fructose-bisphosphate aldolase OS = Haliotis rufescens FBA 1 3 9
175 A0A2T7NF32 Uncharacterized protein OS = Pomacea canaliculata C0Q70_20261 1 5 4
176 A0A2T7NMW4 Uncharacterized protein OS = Pomacea canaliculata C0Q70_18325 2 4 5
177 K1QZU8 Calcium-transporting ATPase OS = Crassostrea gigas CGI_10023684 1 2 1
178 A0A0L8IAE8 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22025089mg 1 2 8
179 A0A2C9KC89 Uncharacterized protein OS = Biomphalaria glabrata 106056965 2 5 3
180 A0A210R746 Ras-related protein Rab-6A OS = Mizuhopecten yessoensis KP79_PYT20147 1 9 11
181 A0A0B6Z4Q3 Uncharacterized protein OS = Arion vulgaris ORF48472 2 12 8
182 A0A2T7PZP4 Uncharacterized protein OS = Pomacea canaliculata C0Q70_01513 1 4 3
183 A0A2C9JIZ4 Uncharacterized protein OS = Biomphalaria glabrata 106056849 1 9 13
184 K1PTH4 ADP-ribosylation factor OS = Crassostrea gigas CGI_10020174 1 2 1
185 Q6PTL0 Triosephosphate isomerase OS = Nucula proxima OCBIM_22037419mg 1 5 6
186 A0A2C9JZR8 Uncharacterized protein OS = Biomphalaria glabrata 106074442 1 2 2
187 A0A2C9JIA9 Uncharacterized protein OS = Biomphalaria glabrata 106056539 1 6 4
188 A0A385NHM7 Glutathione S-transferase OS = Tegillarca granosa GST 1 8 5
189 A0A210QUP5 Malic enzyme OS = Mizuhopecten yessoensis KP79_PYT06884 1 1 3
190 V3YXF9 Adenosylhomocysteinase OS = Lottia gigantea LOTGIDRAFT_184532 1 2 3
191 A0A210QGP4 Chitotriosidase-1 OS = Mizuhopecten yessoensis KP79_PYT06201 1 1 3
192 A0A210QHE1 Adenosylhomocysteinase OS = Mizuhopecten yessoensis KP79_PYT14445 1 4 3
193 A0A210PIA6 Ornithine aminotransferase OS = Mizuhopecten yessoensis KP79_PYT16913 1 3 3
194 K1QQB6 40S ribosomal protein S14 OS = Crassostrea gigas CGI_10011151 1 4 9
195 A0A2C9KEN8 Tubulin alpha chain OS = Biomphalaria glabrata 106069694 1 2 3
196 A0A2T7PWT6 Serine/threonine-protein phosph OS = Pomacea canaliculata C0Q70_00460 1 1 3
197 A0A0B7AJW7 Fructose-bisphosphate aldolase OS = Arion vulgaris ORF124546 1 8 4
198 A0A2C9L7N6 Uncharacterized protein OS = Biomphalaria glabrata 106080319 1 49 4
199 A0A210QTZ1 Peptidyl-prolyl cis-trans OS = Mizuhopecten yessoensis KP79_PYT00632 1 2 6
200 A0A2I7M8C2 Go protein alpha subunit OS = Argopecten irradians N/A 1 4 3
201 K1R2G8 Titin OS = Crassostrea gigas CGI_10016808 1 2 0
202 K1QVD7 Neuronal acetylcholine receptor subunit non-alpha-2 OS = Crassostrea gigas CGI_10016138 1 2 1
203 K1Q7G5 Ficolin-2 OS = Crassostrea gigas CGI_10026202 1 2 3
204 A0A2C9K9W9 Uncharacterized protein OS = Biomphalaria glabrata 106068683 1 1 1
205 A0A0B6ZP87 Uncharacterized protein OS = Arion vulgaris ORF71130 1 3 4
206 V4AP92 Elongation factor 1-alpha OS = Lottia gigantea LOTGIDRAFT_239271 1 2 2
207 A0A2T7PU69 Uncharacterized protein OS = Pomacea canaliculata C0Q70_03920 1 4 4
208 V3ZN51 Staphylococcal nuclease domain-cont. OS = Lottia gigantea LOTGIDRAFT_235720 1 3 1
209 A0A2T7PSF5 Uncharacterized protein OS = Pomacea canaliculata C0Q70_03333 1 2 0
210 K1PQD4 Phosphoglucomutase-1 OS = Crassostrea gigas CGI_10011818 1 1 2
211 A0A0B7BF17 Uncharacterized protein OS = Arion vulgaris ORF179770 1 3 2
212 A0A2T7Q0W0 Uncharacterized protein OS = Pomacea canaliculata C0Q70_01928 1 1 3
213 A0A0L8I692 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22034637mg 1 4 19
214 K1PQ79 Copine-3 OS = Crassostrea gigas CGI_10011897 1 3 1
215 K1PWB9 EH domain-containing protein 1 OS = Crassostrea gigas CGI_10005813 1 1 4
216 A0A2T7Q016 Uncharacterized protein OS = Pomacea canaliculata C0Q70_01636 1 1 2
217 V4AKV4 Calcium-transporting ATPase OS = Lottia gigantea LOTGIDRAFT_208914 1 3 1
218 A0A2T7NL99 Proteasome subunit beta OS = Pomacea canaliculata C0Q70_17739 1 2 4
219 A0A0L8HWW8 Uncharacterized protein OS = Octopus bimaculoides OCBIM_22003772mg 1 2 2
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N (Identification Number); FDR (False Discovery Rate); Uni. Pep. (Unique Peptides); PSMs (Peptide Spectrum Matches); Cov. (Protein Coverage).

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.

Figure 1.

Figure 1

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SDS-PAGE 10% profiles of the extracted proteins of jumbo squid skin samples (A–D replicates). MW denotes molecular weight.

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].

Figure 2.

Figure 2

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Protein classes of the jumbo squid skin proteome identified by shotgun proteomics and categorized by PANTHER (http://pantherdb.org/).

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).

Table 2.

KEGG pathway analysis of the jumbo squid skin proteome by DAVID.

KEGG Pathway p-Value
Metabolic pathways (cysteine and methionine metabolism) 4.53 × 10−4
Endocytosis/phagosome 1.05 × 10−2
RNA transport 2.24 × 10−2
Protein methylation 3.46 × 10−2
Calcium homeostasis 1.00 × 10−1
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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.

Table 3.

Functional InterPro motifs by DAVID.

InterPro Motifs p-Value
Small GTP-binding protein domain 3.1 × 10−4
Heat shock protein 70, conserved site 8.5 × 10−4
Small GTPase superfamily 8.6 × 10−4
Proteasome, alpha-subunit, N-terminal domain 1.3 × 10−3
P-loop containing nucleoside triphosphate hydrolase 8.3 × 10−3
EF-hand-like domain 2.9 × 10−2
Ubiquitin 3.4 × 10−2
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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).

Figure 3.

Figure 3

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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.

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.

Table 4.

Selected potential bioactive peptides of the jumbo squid skin proteome predicted by in-silico digestions with pepsin.

Proteins Peptides PeptideRanker Score Anti-Microbial Peptide (AMP) Discriminant Score for AMP
ADP-ribosylation factor OS = Crassostrea gigas SPSPKQMVSCPVCGL 0.915222 Non-AMP 0.043
Collagen ColAa OS = Sepia pharaonis PGDPGPVGRTGPMGL 0.934847 Non-AMP 0.003
Collagen ColAa OS = Sepia pharaonis RGPPGPPGL 0.912657 Non-AMP 0.030
Heat shock protein 70 OS = Sepiella maindroni GGMPGGMPGGMPGGMPNF 0.92432 AMP 0.504
Hemocyanin OS = Sepiella maindroni KKPMMPF 0.932566 AMP 0.978
Hemocyanin OS = Sepiella maindroni PNQPMRPF 0.920777 AMP 0.983
Hemocyanin subunit 1 OS = Todarodes pacificus NDPMRPF 0.923312 AMP 0.795
Hemocyanin subunit 2 OS = Sepia officinalis SDPMRPF 0.938433 AMP 0.879
Uncharacterized protein OS = Octopus bimaculoides CPCMGRF 0.985441 AMP 0.622
Uncharacterized protein OS = Octopus bimaculoides GGPPGMPPF 0.973279 Non-AMP 0.208
Uncharacterized protein OS = Octopus bimaculoides GRCVMCNCNKHSSTCDPQTGKCVNCQHNTL 0.969319 Non-AMP 0.238
Uncharacterized protein OS = Octopus bimaculoides GSCVPCNCNGF 0.952459 AMP 0.745
Uncharacterized protein OS = Octopus bimaculoides QPPQCCPSKGGSF 0.943546 AMP 0.687
Uncharacterized protein OS = Octopus bimaculoides GSWGNGNRW 0.915802 Non-AMP 0.403
Uncharacterized protein OS = Octopus bimaculoides PPPSKRF 0.911736 AMP 0.983
Uncharacterized protein OS = Biomphalaria glabrata PPPPQPVGGGGGNRW 0.955862 Non-AMP 0.092
Uncharacterized protein OS = Biomphalaria glabrata SRSPPRPF 0.904351 AMP 0.993
Uncharacterized protein OS = Pomacea canaliculata HDGDGPRPCCF 0.93215 Non-AMP 0.031
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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.

Table 5.

Selected potential bioactive peptides of the jumbo squid skin proteome predicted by in-silico digestions with trypsin.

Proteins Peptides PeptideRanker Score Anti-Microbial Peptide (AMP) Discriminant Score for AMP
ADP-ribosylation factor OS = Crassostrea gigas CPICYDFMHTAMILPECSHTFCSFCIR 0.902646 Non-AMP 0.160
Calcium-transporting ATPase OS = Octopus bimaculoides FSDDYPGFF 0.970864 Non-AMP 0.006
Calcium-transporting ATPase OS = Crassostrea gigas FLQFQLTVNCVAVMVAFFGACIINDSPLK 0.979848 Non-AMP 0.281
Calcium-transporting ATPase OS=Lottia gigantea FADAPFMK 0.93747 Non-AMP 0.014
Calmodulin OS = Crassostrea gigas GAFFVFDR 0.915228 Non-AMP 0.003
Chitinase OS = Todarodes pacificus MLAVSLLFLLAIGGVSSAGHR 0.976725 AMP 0.746
Chitotriosidase OS = Euprymna scolopes MASTFATVFGVLSLCFLGLHLTNGEYK 0.984749 Non-AMP 0.106
Coatomer subunit beta’ OS = Mizuhopecten yessoensis YCLCLFR 0.924855 AMP 0.579
Collagen ColAa OS = Sepia pharaonis GPPGIPGLPGPK 0.93716 AMP 0.504
Collagen ColAa OS = Sepia pharaonis GPPGPPGLK 0.913133 Non-AMP 0.119
Collagen ColAa OS = Sepia pharaonis AGPPGFPGTPGPK 0.907398 AMP 0.682
Ficolin-2 OS = Crassostrea gigas DQDNDMYVSDNCGILFPSGWWHR 0.901865 Non-AMP 0.008
Fructose-bisphosphate aldolase OS = Mizuhopecten yessoensis KPWALTFSFGR 0.93422 Non-AMP 0.123
Hemocyanin OS = Aplysia californica MVGYLGQALMALLLLALSNAALVR 0.993669 Non-AMP 0.380
Hemocyanin OS = Aplysia californica FEPNPFFSGK 0.924588 Non-AMP 0.093
Hemocyanin OS = Aplysia californica VACCLHGMPVFPHWHR 0.903581 Non-AMP 0.106
Hemocyanin OS = Nautilus pompilius MATHWHSLLLFSLQLLVFTYATSDPTNIR 0.97599 Non-AMP 0.008
Hemocyanin OS = Sepiella maindroni GSPIGVPYWDWTKPMK 0.917605 Non-AMP 0.027
Hemocyanin-like protein OS = Uroteuthis edulis TNFFFLALIATVWLGNAETETETSK 0.90323 Non-AMP 0.062
Hemocyanin subunit 1 OS = Euprymna scolopes VFVGFLLHGFGSSAYATFDICNDAGECR 0.96087 Non-AMP 0.233
Hemocyanin subunit 1 OS = Euprymna scolopes LNHLPLLCLAVILTLWMSGSNTVNGNLVR 0.926117 Non-AMP 0.287
Hemocyanin subunit 1 OS = Euprymna scolopes VFAGFLFMGIK 0.904542 AMP 0.865
Hemocyanin subunit 2 OS = Euprymna scolopes VFAGFWFHGIK 0.943 AMP 0.506
Hemocyanin subunit 2 OS = Sepia officinalis VFGGFWLHGIK 0.907156 AMP 0.739
Hemocyanin subunit 3 OS = Sepia officinalis TSFLFLAFVATSWFVYAVTASK 0.905214 Non-AMP 0.136
Malate dehydrogenase OS = Sepia officinalis DLFNTNASIVANLADACAQYCPK 0.965037 Non-AMP 0.251
Myosin heavy chain isoform A OS = Octopus bimaculoides YQSGFIYTYSGLFCVAINPYR 0.956725 Non-AMP 0.024
Myosin heavy chain OS = Todarodes pacificus NWEWWR 0.951523 Non-AMP 0.478
Myosin II heavy chain OS = Doryteuthis pealeii NWQWWR 0.973264 AMP 0.959
Myosin II heavy chain OS = Doryteuthis pealeii YYSGLIYTYSGLFCVVVNPYK 0.939159 Non-AMP 0.032
Neuronal acetylcholine receptor subunit non-alpha-2 OS = Crassostrea gigas LLIDLCLSVLVTTLAIVSLYFYDMSDSR 0.904075 Non-AMP 0.015
Peptidyl-prolyl cis-trans isomerase OS = Mizuhopecten yessoensis MAGAGIGCVLLFLLPALLSAGK 0.996478 Non-AMP 0.159
Phosphoglucomutase-1 OS = Crassostrea gigas DGLWAVLAWLSVLANQNCSVEECIK 0.991266 AMP 0.904
Protein disulfide-isomerase OS = Octopus bimaculoides NVFIEFYAPWCGHCK 0.907443 Non-AMP 0.053
S-syntaxin OS = Doryteuthis pealeii IAILVCLVILVLVIVSTVGGVFGG 0.965343 Non-AMP 0.000
Titin OS = Crassostrea gigas DGSWQNLVTVLGCLKPQFVNLQR 0.974127 AMP 0.724
Titin OS = Crassostrea gigas GYPPPIISWYR 0.917986 Non-AMP 0.074
Tubulin alpha chain OS = Octopus bimaculoides FVDWCPTGFK 0.923256 Non-AMP 0.010
Uncharacterized protein OS = Arion vulgaris APDFIFYAPR 0.921198 Non-AMP 0.009
Uncharacterized protein OS = Octopus bimaculoides FLQFQLTVNVVAVLVAFFGACTINVSI 0.978717 AMP 0.916
Uncharacterized protein OS = Octopus bimaculoides YYTFFVTIFLFATTLCSTIPKPK 0.984914 Non-AMP 0.012
Uncharacterized protein OS = Octopus bimaculoides LFPAFGFGAR 0.94902 AMP 0.505
Uncharacterized protein OS = Octopus bimaculoides ATMLGAQGNIFFASLSCCCLILSCS 0.999233 AMP 0.879
Uncharacterized protein OS = Octopus bimaculoides SGPFYIFSGGMPR 0.939205 Non-AMP 0.089
Uncharacterized protein OS = Octopus bimaculoides EFSMMFR 0.931708 Non-AMP 0.001
Uncharacterized protein OS = Octopus bimaculoides YGSCVPCNCNGFSNDCDPVTGECIDCQR 0.980617 Non-AMP 0.243
Uncharacterized protein OS = Octopus bimaculoides HNPEGCISCFCMGVTEFCTSTSR 0.964134 Non-AMP 0.083
Uncharacterized protein OS = Octopus bimaculoides APMVELCECPQGYTGVSCQECSPGYSR 0.963828 Non-AMP 0.012
Uncharacterized protein OS = Octopus bimaculoides GCGCSAGQFECQNGLCINENK 0.930153 AMP 0.982
Uncharacterized protein OS = Octopus bimaculoides EECMSCFCFK 0.918951 AMP 0.982
Uncharacterized protein OS = Octopus bimaculoides NSEYGFACFCPQGFAGYQCDTVGER 0.906197 AMP 0.576
Uncharacterized protein OS = Octopus bimaculoides MIIYILSLAGVALGVYFLSCVR 0.995663 Non-AMP 0.008
Uncharacterized protein OS = Octopus bimaculoides MILTIFACLMALDIELNTSNSIQEE 0.968187 Non-AMP 0.026
Uncharacterized protein OS = Octopus bimaculoides AIGALVDACGPGLCPDWADWAPK 0.948884 AMP 0.774
Uncharacterized protein OS = Octopus bimaculoides QGDWTCPNPACGNNNFGWR 0.9572 Non-AMP 0.286
Uncharacterized protein OS = Octopus bimaculoides GGFGGGGGGGGGMGGDR 0.928063 Non-AMP 0.065
Uncharacterized protein OS = Octopus bimaculoides GFFEDDYDEYGGGYGGGMGFGGLNR 0.944869 Non-AMP 0.143
Uncharacterized protein OS = Octopus bimaculoides LDDGDACLLDMGTEYCCYASDITCSYPVNGK 0.968621 Non-AMP 0.056
Uncharacterized protein OS = Octopus bimaculoides MAFYTILNVVTIVLLIIVGQCR 0.998628 Non-AMP 0.031
Uncharacterized protein OS = Octopus bimaculoides GGSFGFNFR 0.969779 Non-AMP 0.355
Uncharacterized protein OS = Octopus bimaculoides NSTDVCNCSIYVGLFPCNECTK 0.994975 Non-AMP 0.462
Uncharacterized protein OS = Octopus bimaculoides PPSPPIYFR 0.946483 Non-AMP 0.226
Uncharacterized protein OS = Octopus bimaculoides CFLCATGTGTSIEVLALVTIGWCLLHATGTR 0.96344 AMP 0.768
Uncharacterized protein OS = Octopus bimaculoides FDFFYK 0.96245 Non-AMP 0.032
Uncharacterized protein OS = Octopus bimaculoides FSPIPFLFCTISGTCNFATR 0.95134 AMP 0.505
Uncharacterized protein OS = Octopus bimaculoides FWELTECCPHQCLEWLSNLVTR 0.933791 Non-AMP 0.106
Uncharacterized protein OS = Octopus bimaculoides DAFCSSPNFNSWLK 0.922125 Non-AMP 0.058
Uncharacterized protein OS = Octopus bimaculoides NGYEEDDALIGLLNLCTAILK 0.917521 Non-AMP 0.479
Uncharacterized protein OS = Octopus bimaculoides DYFWLVCR 0.911557 Non-AMP 0.001
Uncharacterized protein OS = Biomphalaria glabrata QGELGDCWLLAAVASLTCNPK 0.919385 AMP 0.783
Uncharacterized protein OS = Biomphalaria glabrata SPPRPFEWK 0.905581 Non-AMP 0.006
Uncharacterized protein OS = Pomacea canaliculata SVFNIPPNCFSEMM 0.908085 Non-AMP 0.003
Uncharacterized protein OS = Pomacea canaliculata SCLMGHGSLFGAGAGSLHLQAIAALK 0.919795 Non-AMP 0.315
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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.

Acknowledgments

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

Supplementary Materials

Click here for additional data file. (3.2MB, zip)

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).

Conflicts of Interest

The authors declare no conflict of interest.

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