Proteomic Identification of Stonefish Synanceja verrucosa Venom

Tai-Yuan Chen, Yu-Huai Chang, Hsi-Pin Lin, Shui-Tein Chen, Deng-Fwu Hwang

Journal of Food and Nutrition Research

Proteomic Identification of Stonefish Synanceja verrucosa Venom

Tai-Yuan Chen1, Yu-Huai Chang1, Hsi-Pin Lin2, Shui-Tein Chen3, Deng-Fwu Hwang1, 4,

1Department of Food Science and Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, Taiwan, ROC

2Department of Food Science, Yuanpei University, Hsinchu, Taiwan, ROC

3Institute of Biological Chemistry and Genomic Research Center, Academia Sinica, Taipei, Taiwan, ROC

4Department of Health and Nutrition, Asia University, Taichung, Taiwan, ROC

Abstract

Three venom toxins, neoverrucotoxin (neoVTX) α-subunit and β-subunit as well as verrucotoxin (VTX) β-subunit, were identified in the stonefish Synanceja verrucosa by SDS-PAGE, Native-PAGE and two-dimensional electrophoresis (2-DE) coupled with Matrix Assisted Laser Desorption Ionization-Quadrupole-Time-of-Flight (MALDI-Q-TOF). The venom estimated by Native-PAGE were 471, 358, 260 and 166 kDa. The predominate protein bands of crude venom were 84 and 75 kDa by SDS-PAGE. The crude venom protein fell in the region with pI values of 7-9 and molecular weights of 75-90 kDa by 2-DE. Peptide mass fingerprints (PMF) and MS/MS ions originated from MALDI-Q-TOF were used to identify the protein. Our results showed that the complete components of neoverrucotoxin (neoVTX) α-subunit and β-subunit as well as verrucotoxin (VTX) β-subunit were identified from SDS-PAGE and 2-DE patterns. Native-PAGE did not yield protein identifications but revealed the presence of protein complexes.

Cite this article:

  • Tai-Yuan Chen, Yu-Huai Chang, Hsi-Pin Lin, Shui-Tein Chen, Deng-Fwu Hwang. Proteomic Identification of Stonefish Synanceja verrucosa Venom. Journal of Food and Nutrition Research. Vol. 3, No. 8, 2015, pp 526-539. http://pubs.sciepub.com/jfnr/3/8/8
  • Chen, Tai-Yuan, et al. "Proteomic Identification of Stonefish Synanceja verrucosa Venom." Journal of Food and Nutrition Research 3.8 (2015): 526-539.
  • Chen, T. , Chang, Y. , Lin, H. , Chen, S. , & Hwang, D. (2015). Proteomic Identification of Stonefish Synanceja verrucosa Venom. Journal of Food and Nutrition Research, 3(8), 526-539.
  • Chen, Tai-Yuan, Yu-Huai Chang, Hsi-Pin Lin, Shui-Tein Chen, and Deng-Fwu Hwang. "Proteomic Identification of Stonefish Synanceja verrucosa Venom." Journal of Food and Nutrition Research 3, no. 8 (2015): 526-539.

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At a glance: Figures

1. Introduction

Stonefish of the genus Synanceja have caused a number of human deaths through stings by their extremely venomous spines. These fish inhabit shallow waters over wide areas of the Pacific and Indian oceans. S. verrucosa, belonging to this group, can be encountered in Taiwan. Intense pain is produced immediately after the sting and radiates from the wound through all the extremities. This is soon followed by cardiovascular effects, hemolytic activity, platelet aggregation and edema. It may take a few days or several weeks for recovery from the local effects. In severe cases, patients may experience weakness, sweating, respiratory distress, convulsions and coma; death may occur within a few hours [1].

S. verrucosa is equipped with 11–17 dorsal spines, and each spine is connected to a pair of protein venom sacs. The venom is collected from the sacs with a syringe, either by squeezing the sacs with a pair of tweezers or by sucking on them with an aspirator. S. verrucosa provides about of venom per sac [2, 3].

The venom showed activity of hyaluronidase, esterase and aminopeptidase, but not phospholipase A2 activity. From the venom, a toxic component called verrucotoxin (VTX) from the crude venom of S. verrucosa, was isolated [4]. The molecular weight of VTX was 322 kDa, and the VTX was a tetramer of glycoprotein and consisted of two subunits of 78 kDa and 83 kDa. The value of LD50 was 125 μg/kg in mice [4]. Furthermore, the complete amino acid sequence (708 amino acids) of the β-subunit of VTX from the venom of the stonefish S. verrucosa was reported, and the partial amino acid sequence of the VTX α-subunit was submitted to the GeneBank (CAA69254.1) [5]. VTX has been identified as a neurotoxin, inducing clinical symptoms including convulsions, epilepsy and spasms [6]. NeoVTX, a more recently identified component of the crude venom, showed hemolytic and lethal activity. The LD50 value was determined as 47 μg/kg in mice [3]. NeoVTX is a dimmer of 166 kDa, consisting of an α-subunit of 75 kDa and a β-subunit of 80 kDa. The LD50 value was 47 μg/kg. Furthermore, a hyaluronidase was purified from the venom of S. verrucosa with a molecular weight of 59 kDa. Its optimal pH and temperature were 6.6 and , respectively [7]. The reverse-transcription polymerase chain reaction (RT-PCR) method was used to obtain the sequences of gene transcripts for the α- and β-subunits of neoVTX as 3,675 bp and 3,510 bp, respectively; the responding amino acid numbers were 702 (79,540 Da) and 699 (79,370 Da), respectively [3]. In addition to proteinaceous toxin, the venom of S. verrucosa was shown to contain noradrenaline, dopamine, tryptophan and catecholamines [8, 9].

The protein profiles of the venom have not been clearly established due to the venom’s extreme lability. However, several electrophoretic techniques were validated for use in protein analysis [10, 11]. Peptide mass fingerprinting (PMF) using the matrix assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF) is a useful method for elucidating the toxin components [12, 13, 14, 15, 16]. Stinging incidents of S. verrucosa have been reported in Taiwan [17, 18]. To elucidate the toxin components, native polyacrylamide gel electrophoresis (Native-PAGE), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) were employed in this study to establish the protein pattern profiles of crude venom from S. verrucosa in Taiwan. After in-gel digestion, a matrix-assisted laser desorption/ionization-quadrupole time of flight mass spectrometer (MALDI-Q-TOF) was used to identify protein samples. MALDI-Q-TOF can analyze PMF and further provide product ion spectra (MS/MS ion search) for protein identification [19, 20].

Few reports were performed on the feasibility of the combination of the non-denatured electrophoresis (Native-PAGE) and the denatured electrophoresis (2-DE and SDS-PAGE) and MALDI-Q-TOF in identifying fish spine venom [3, 21, 22, 23]. Thus, the objective of this study was to determine the efficacy of current bottom-up proteomic and bioinformatic approaches in identifying proteinaceous venom toxins from S. verrucosa spines.

2. Materials and Methods

2.1. Venoms

Venom samples were taken from 5 live stonefish S. verrucosa (675 ± 124 g) captured along the northeast Taiwanese coast. The fish were put into an ice bath and the crude venom was collected using a 1 ml syringe from a pair of venom sacs in each dorsal spine. The crude venoms were stored in a liquid nitrogen bath until use.

2.2. Protein Determination

Protein concentrations were determined using the Bradford Protein Assay Kit (Bio-Rad, CA, USA) and bovine serum albumin (Bio-Rad) as a standard, according to manufacturer’s instructions.

2.3. Native-PAGE

A venom sample dissolved in distilled water was mixed with an equal volume of loading buffer without sodium dodecyl sulfate (SDS) and β-mercaptoethanol. The preparation was subjected to Native–PAGE in an 8% polyacrylamide gel using the Mini PROTEAN 3 Cell and BasicTM PowerPac device (Bio-Rad). The separated proteins were stained with Coomassie Brilliant Blue R250 dye (Aldrich-Sigma, MO, USA). The standard proteins were purchased from SERVA electrophoresis Co. (HDB, DE). The gel image was obtained using a Gel Doc photosystem (Bio-Rad) and calculated by Quantity One Software (Bio-Rad).

2.4. SDS-PAGE

This procedure was modified from our previous studies (Chen et al., 2002) [24]. The standard proteins were purchased from Bio-Rad (CA, USA). The gel image was obtained using a Gel Doc photosystem (Bio-Rad) and calculated by Quantity One Software (Bio-Rad).

2.5. Two-dimensional Gel Electrophoresis (2-DE)

The venom sample was treated using a 2-DE clean-up kit (GE Healthcare, NJ, USA) to free it from salt, detergent and lipids. Then the venom proteins were precipitated and dissolved with a rehydration buffer in a 200 μg/350 μl solution. For the first dimension-electrophoresis, each IPG strip (18 cm, pH 3-10) (GE Healthcare) was placed into the Ettan IPGphor 3 Isoelectric Focusing System (GE Healthcare) and the IPG strip was allowed to rehydrate at 20°C for 12 h in the DeStreak TM Rehydration Solution. Electrophoresis was performed at 250 V for 30 min, 500 V for 30 min, 1,000 V for 30 min, 2,000 V for 30 min, 4,000 V for 30 min, 8,000 V for 30 min, 10,000 V to 50,000 Vh (about 6-8 h) and 500 V for 30 min. For the second dimension-electrophoresis, the IPG strip focused on was equilibrated in a reduction buffer [50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% bromophenol blue and 1% (w/v) dithiothreitol] at 25°C for 15 min. The strip was then equilibrated in an alkylation buffer [50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002% bromophenol blue and 2.5% (w/v) iodoacetamide] at 25°C for 15 min. SDS-PAGE was carried out in a 10% polyacrylamide gel cast in the PROTEAN II xi 2-D Cell (Bio-Rad) at 10 mA/gel during the first 15 min and at 30 mA/gel with protein markers (Bioman, Taipei, ROC) until the tracking dye reached the lower edge of the gel. Then, the gel was stained with SYPRO ruby stain (Invitrogen, NY, USA) for the proteomic study. The 2-DE gel images were scanned using a Typhoon 9200 Laser Scanner (GE Healthcare) and exported to the image analysis software program using PDQuest Software Package Version 7.1.1 (Bio-Rad).

2.6. In-gel Digestion

An in-gel digestion procedure was processed according to a previously described method [25]. Briefly, the gel pieces were washed and rehydrated with trypsin (Promega, Madison, WI, USA) at 37°C for at least 16 h. Finally, the extracted proteins were eluted with 5 μl of 75% acetonithrile/0.1% formic acid for further analysis.

2.7. MALDI-Q-TOF Mass Spectrometry

The trypsinized samples were premixed at a ratio of 1:1 (v/v) with the matrix solution [5 mg/ml α-cyano-4-hydroxycinnamic acid (CHCA) in 50% acetonitrile, 0.1% (v/v) trifluoroacetic acid and 2% (w/v) ammonium citrate] and spotted onto the 96-well MALDI sample stage. The samples were analyzed by the Q-TOF UltimaTM MALDI instrument (MALDITM; Micromass, Manchester, UK), as described previously [26]. The MASCOT search engine (http://www.matrixscience.com) was applied in the analysis on the PMF and MS/MS ions searching programs. The product ion spectra generated by Q-TOF MS/MS were searched within the NCBInr and Swiss-PROT databases for exact matches using the in-house MASCOT software ver 2.2.04 (Matrix Science). The parameter settings for the MASCOT server were as follows: there were no fixed modifications; variable modifications were carbamidomethyl and oxidation (M); there was one missed cleavage site; peptide tolerance was 50 ppm; MS/MS tolerance was 0.25 Da.

3. Results and Discussion

3.1. Native-PAGE Coupled with MALDI-Q-TOF to Identify S. verrucosa spine Venom

The Native-PAGE pattern of crude spine venom in S. verrucosa was shown in Figure 1A. Four clearly visible protein bands were estimated at 471, 358, 260 and 166 kDa. Each protein band was treated with in-gel trypsin digestion and analyzed by MALDI-Q-TOF mass spectrometry. The peptide mass fingerprints (PMF) and MS/MS ion search obtained from each protein band of Native-PAGE were analyzed using the Mascot Search Program, and no matched protein was found. The proteins were not denatured during the Native-PAGE process. The complicated compositions in a native protein band might not perfectly be digested by trypsin. Hence, we could not match any protein from the Native-PAGE of crude venom in S. verrucosa using MALDI-Q-TOF analysis. The previous approaches to achieve protein identification were to excise the native gel band and re-analyze by denaturing SDS-PAGE [27] or LC-MS/MS [28]. In the Native-PAGE, both bands (358 kDa and 166 kDa) might be suspended to correspond to VTX (a tetramer glycoprotein of 322 kDa including α-subunit 83 kDa and β-subunit 78 kDa) and neoVTX (a dimer protein of 166 kDa including α-subunit 75 kDa and β-subunit 80 kDa), respectively [3, 4]. The 471 kDa venom protein would be a conjugate of different toxin subunits or of other nonprotein portions needing to be studied further for elucidation.

Figure 1. Electrophoretic patterns obtained from spine venom of S. verrucosa. (A) Native polyacrylamide gel electrophoresis (Native-PAGE) of 8% gel and CBB staining. The molecular mass markers were indicated at the left. (B) SDS-PAGE of 10% gel and CBB staining. The molecular mass markers were indicated at the left. (C) 2-DE was first separated by a pH 3-10 non-linear immobilized pH gradient (IPG) dry strip, followed by 10% SDS-PAGE and SYPRO ruby staining
3.2. SDS-PAGE Coupled with MALDI-Q-TOF to Identify S. verrucosa Spine Venom

In the SDS-PAGE pattern, the crude spine venom of S. verrucosa showed 6 estimated protein bands of 94, 84, 75, 71, 39 and 25 kDa (Figure 1B). Among them, both protein bands of 84 and 75 kDa (svs001 and 002), indicated in Figure 1B, exhibited much higher amounts than the other 4 protein bands. Regarding the protein band of 94 kDa, one research group in 1993 reported that one of lethal factors from S. verrucosa was related to the protein band of 90 kDa identified by SDS electrophoresis and HPLC methods [29], as well as protein bands of 71, 39 and 25 kDa were exhibited in the crude venom fluid of S. verrucosa [3]. Compared with data from PMF, svs001 was identified as a neoVTX β-subunit with 30% sequence coverage (SC) (Figure 2) and/or a VTX β-subunit with a lesser SC at 19% (shown in Supplementary information); svs002 was identified as a neoVTX α-subunit with 24% SC (Figure 3). Furthermore, according to the analysis of the MS/MS ion search, svs001 was identified as the VTX β-subunit (Table 1, Figure 2), and svs002 was identified as the neoVTX α-subunit (Table 1, Figure 3). A research group in Singapore applied 8% SDS-PAGE to obtain stonustoxin (SNTX) α-subunit (71 kDa) and β-subunit (79 kDa) from the sting toxin of S. horrida [21]. In Japan, an 8%–25% gradient SDS-PAGE was applied to obtain major components distributed between 75-150 kDa and 10-15 kDa from the crude venom of S. verrucosa [3]. In this study, we applied 10% SDS-PAGE to obtain 6 bands from the crude venom of S. verrucosa. The svs001 band (84 kDa) was matched to the VTX β-subunit and/or neoVTX β-subunit [3, 4]. The svs002 band (75 kDa) was matched to the neoVTX α-subunit [3]. The results obtained by MALDI-Q-TOF in the present study were in good agreement with the above observation.

Figure 2. Sequence coverage of the observed protein band svs001 peptides mapped to the known VTX β-subunit sequence. PMF matched peptides were shown with underlining. Further sequencing analysis was carried out by MS/MS, outlined in boxing. The B30.2/SPRY domain was outlined in shaded lettering
Figure 3. Sequence coverage of the observed protein band svs002 peptides mapped to the known neoVTX α-subunit sequence. PMF matched peptides were shown with underlining. Further sequencing analysis was carried out by MS/MS, outlined in boxing. The B30.2/SPRY domain was outlined in shaded lettering

Table 1. Identification of S. verrucosa spine venom by MALDI-Q-TOF mass spectrometry and MS/MS ion search

3.3. 2-DE Coupled with MALDI-Q-TOF to Identify S. verrucosa Spine Venom

The 2-DE pattern of crude spine venom in S. verrucosa was shown in Figure 1C. There were 6 major protein spots (svi001-006) distributed between 75-90 kDa and pI 7-9, as indicated in Figure 1C. Based on PDQuest Software analysis, the estimated molecular weights of svi001-003 were 88 kDa, and their respective pI values were 7.00, 7.17 and 7.35. The other 3 protein spots (svi004-006) showed the same molecular weight of 79 kDa, and their respective pI values were 8.09, 8.30 and 8.50. The posttranslational modifications or different isoforms with slight differences in amino acid composition may be present in the very same venom account for a shift in pI of venomous proteins [3, 22, 23].

Figure 4. Sequence coverage of the observed protein spot svi002 peptides mapped to the known neoVTX β-subunit sequence. PMF matched peptides were shown with underlining. Further sequencing analysis was carried out by MS/MS, outlined in boxing. The B30.2/SPRY domain was outlined in shaded lettering

Compared with data from PMF, 3 protein spots (svi001-003) were matched to the protein, neoVTX β-subunit. The matched peptides of svi002 to neoVTX β-subunit with 25% SC were shown in Figure 4. Two protein spots (svi001, 003) were also matched to the protein, VTX β-subunit. The other 3 protein spots (svi004-006) were matched to the protein neoVTX α-subunit. The peptides of svi006 matched to neoVTX α-subunit with 44% SC were shown in Figure 5. According to the analysis of the MS/MS ion search, 3 protein spots (svi001-003) were matched to the same protein, neoVTX β-subunit (Table 1, Figure 4), and another 2 protein spots (svi005-006) were matched to the other same protein, neoVTX α-subunit (Table 1, Figure 5). The representative MS and MS/MS spectrums of svi002 were shown in the supplementary information. On the other hand, based on the data from PMF, svi005 was also matched to the other protein, stonustoxin (SNTX) α-subunit (shown in Supplementary information). However, the matched peptides shared between SNTX and neoVTX α-subunits. SNTX was isolated from other stonefish S. horrida [9, 30]. These results show that three components, including α- and β-subunits of neoVTX and the β-subunit of VTX, were identified from SDS-PAGE and 2-DE patterns. Judging from the above data, at least neoVTX is a complete component of venom in Taiwanese S. verrucosa.

Figure 5. Sequence coverage of the observed protein spot svi006 peptides mapped to the known neoVTX α-subunit sequence. PMF matched peptides were shown with underlining. Further sequencing analysis was carried out by MS/MS, outlined in boxing. The B30.2/SPRY domain was outlined in shaded lettering

Specific proteins in the crude venom of Scorpaena plumier appeared between pI 4-7 and MW 6-120 kDa [11]. In this study, the major proteins appeared between pI 7-9 and MW 75-90 kDa. The major 6 protein spots (svs001-006) were the toxin components (VTX β-subunit, neoVTX α-subunit and neoVTX β-subunit) according to the molecular weight data. On the other hand, the pI values of the VTX β-subunit, the neoVTX α-subunit and the neoVTX β-subunit were 5.85, 6.49 and 5.93, respectively, calculated from amino acid sequences through the ExPASy database. However, the pI value of the VTX α-subunit is unknown due to the lack of complete amino acid sequences [3]. Furthermore, the amino acid sequence of the venom in S. verrunosa collected from different countries showed a slight difference, indicating that the molecular weights and pI values may have slight variations [3].

From the first report on the stonefish toxin [31], the spine venom of 3 species in the genus Synanceja has not been characterized until now. The complete amino acid sequences of these proteinaceous venom toxins were mainly achieved by the cDNA cloning approach [3, 5, 21, 22]. This study was to utilize proteomic platform to conduct prompt identification of these venom toxins. The identified toxin SNTX 148 kDa was isolated from S. horrida, containing α-subunit and β-subunit, which revealed 50% homology with each other [21, 22]. Then VTX and neoVTX, originated from S. verrucosa venom, were characterized as a glycoprotein 322 kDa and a dimer protein 166 kDa, respectively [3, 5]. The species S. trachynis was found to contain trachynilysin [32]. However, the amino acid sequence of the VTX α-subunit and trachynilysin remains unknown to date.

Alignment of the amino acid sequences revealed that the VTX β-subunit shared 86% identity with the SNTX β-subunit. The sequences of the neoVTX α-subunit and β-subunit exhibited 87% and 95% homology with the SNTX α-subunit and β-subunit, respectively. Besides, the sequences of the neoVTX β-subunit revealed 90% identity with the VTX β-subunit [33]. Furthermore, the stonefish-like toxins showed close similarity in properties and primary structures to stonefish toxins. Of these toxins, the toxin from devil stinger (Inimicus japonicus) venom even reached 90% amino acid sequence identity with the neoVTX α- and β-subunit individually [23]. The PMF and MS/MS ions achieved from one MALDI-Q-TOF analysis might match different proteins with excellent identity of amino acid sequences from the database. For example, svs001 was identified as the neoVTX β-subunit and VTX β-subunit by using the PMF and MS/MS ion search; svi005 was matched to the neoVTX α-subunit and the SNTX α-subunit by using the PMF search in this study. Hence, the PMF searching strategy is not recommended in fish sting toxin protein identification due to the close similarity of the primary structures among them. Instead, unique amino acid sequences for individual venom species identified by tandem MS or MSn would be more suitable. The complementary proteomic approaches such as the combination of both the MALDI and ESI ionization methods, digestion by Glu-C or Lys-C instead of trypsin, or the “top-down” proteomics that deal with intact proteins will lead to further understanding of the primary structures. The glycosylation status of VTX or other PTMs might be also responsible for the biological or pharmacological properties of these marine protein toxins.

Chemical modification of protein in solution could provide important structural and functional information. Several amino acids including cationic lysine and arginine residues, free thiols, tryptophan, and histidine have been characterized in the SNTX as playing important roles in lethal and hemolytic activities [9, 34, 35, 36, 37]. Modification of cationic amino acids in the SNTX by succinylation and 5,5′-Dithiobis (2-nitrobenzoic acid; DTNB) leads to lethality loss. Although hemolytic activity of neoVTX and SNTX was inhibited by an anionic lipid such as cardiolipin, it is not contained in the erythrocyte membrane. It is therefore likely that the hemolytic activity of neoVTX and SNTX is triggered by the electrostatic interaction of cationic residues of the toxins with anionic phospholipids (typically phosphatidylserine) abundant in the erythrocyte membrane [3, 9, 35].

The neoVTX and SNTX are very similar to the primary structures. A distinct difference between neoVTX and SNTX is recognized in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX) by DTNB titration measurement [3, 36]. Of these Cys residues, some are involved in the intrachain disulfide bridges of each subunit. The others are in the free thiol form, but two subunits are not linked by disulfide bridges [3, 21]. The neoVTX α-subunit has the extra three Cys residues at positions 567, 616 and 689 in addition to the seven Cys residues located at the same position as the SNTX α-subunit. On the other hand, the neoVTX β-subunit contains eight Cys residues at the same positions as the SNTX β-subunit. Only the VTX β-subunit has the additional three Cys residues. The essentiality of tryptophan residues in the activity of a lytic and lethal factor of SNTX was validated [37].

The scorpaeniform fish toxins contain a B30.2/SPRY domain (approximately 200 amino acid residues) in the C-terminal region of each subunit [23]. This domain is contained at positions 508–703 in the α-subunit of neoVTX, 506–700 in the β-subunit of neoVTX, and 506–696 in the β-subunit of VTX. The B30.2/SPRY domains are considered to function through protein-protein interaction [38]. The amino acid sequences are highly variable among the B30.2/SPRY domain in different proteins; therefore, the domains possibly recognize a specific individual protein ligand, namely xanthine oxidase [39]. The B30.2/SPRY domain is present in diverse proteins such as those belonging to the TRIM or RBCC (Ring-finger, B-box and coiled-coil domain), BTN (butyrophilin) and SPSB (cytokine signaling box protein) families [40]. Three highly conserved motifs (LDP, WEVE and LDYE) have been detected in the B30.2/SPRY domain, although their functions are still unknown [36]. Only the LDYE motif is not observable in the scorpaeniform fish toxins [22, 23].

4. Conclusion

These results showed that SDS-PAGE and 2-DE coupled with MALDI-Q-TOF could identify the neoVTX α- and β-subunits, and the VTX β-subunit in S. verrucosa venom. This study revealed the presence of protein isoforms and protein complexes as well as possible PTMs in the S. verrucosa venom proteins. Meanwhile, the VTX α-subunit and other venom components remain unclear and require further study. These techniques could be used in the future to identify venom proteins in other fish spine venoms.

Conflicts of Interest

The authors have declared no conflicts of interest.

Acknowledgements

This study was supported by funds from the Ministry of Science and Technology, Taiwan, ROC and the Center of Excellence for the Oceans, National Taiwan Ocean University.

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Supplement

Figure 2. MS/MS spectrum for the matched peptide sequence of FYEAVYPAFLIGDAQQK of protein spot svi002
Figure 3. MS/MS spectrum for the matched peptide sequence of HYWELEWSGHVSAGVTYK of protein spot svi002
Figure 4. MS/MS spectrum for the matched peptide sequence of NDDFLTVLNDFLDSPQSRPK of protein spot svi002

Table 1-1. Selected bands from SDS-PAGE of S. verrucosa spine venom identified by MALDI-Q-TOF mass spectrometry and SwissProt database search

Table 1-2. The peptide mass fingerprint (PMF) identification of protein band svs001 from SDS-PAGE of spine venom

Table 1-3. The peptide mass fingerprint (PMF) identification of protein band svs001 from SDS-PAGE of spine venom

Table 1-4. The peptide mass fingerprint (PMF) identification of protein band svs002 from SDS-PAGE of spine venom

Table 2-1. Selected protein spots from 2-DE of S. verrucosa spine venom identified by MALDI-Q-TOF mass spectrometry and SwissProt database search

Table 2-2. The peptide mass fingerprint (PMF) identification of protein spot svi001 from 2-DE of spine venom

Table 2-3. The peptide mass fingerprint (PMF) identification of protein spot svi001 from 2-DE of spine venom

Table 2-4. The peptide mass fingerprint (PMF) identification of protein spot svi002 from 2-DE of spine venom

Table 2-5. The peptide mass fingerprint (PMF) identification of protein spot svi003 from 2-DE of spine venom

Table 2-6. The peptide mass fingerprint (PMF) identification of protein spot svi003 from 2-DE of spine venom

Table 2-7. The peptide mass fingerprint (PMF) identification of protein spot svi004 from 2-DE of spine venom

Table 2-8. The peptide mass fingerprint (PMF) identification of protein spot svi005 from 2-DE of spine venom

Table 2-9. The peptide mass fingerprint (PMF) identification of protein spot svi005 from 2-DE of spine venom

Table 2-10. The peptide mass fingerprint (PMF) identification of protein spot svi006 from 2-DE of spine venom

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