Proteomics and immunocharacterization of Asian mountain pit viper (Ovophis monticola) venom.

The venomic profile of Asian mountain pit viper Ovophis monticola is clarified in the present study. Using mass spectrometry-based proteomics, 247 different proteins were identified in crude venom of O. monticola found in Thailand. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant proteins included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%), C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). The immunoreactivity of this viper's venom to a monovalent antivenom against green pit viper Trimeresurus albolabris, or to a polyvalent antivenom against hemotoxic venom was investigated by indirect ELISA and two-dimensional (2D) immunoblotting. Polyvalent antivenom showed substantially greater reactivity levels than monovalent antivenom. A titer for the monovalent antivenom was over 1:1.28x107 dilution while that of polyvalent antivenom was 1:5.12x107. Of a total of 89 spots comprising 173 proteins, 40 spots of predominantly SVMP, SVSP and PLA2 were specific antigens for antivenoms. The 49 unrecognized spots containing 72 proteins were characterized as non-reactive proteins, and included certain types of CTLs and CRISPs. These neglected venom constituents could limit the effectiveness of antivenom-based therapy currently available for victims of pit viper envenomation.

Introduction

Envenomation from snakebites affects over 2.7 million people in tropical and subtropical countries each year, leading to more than 130,000 deaths among victims [1]. Severe injuries and complications from bites also can lead to permanent disabilities and long-term health problems in survivors. In addition, the majority of snakebite victims have been reported within the productive age of the workforce [2]. This creates socio-ecomomic loss as seen in developing countries of Asia and Africa [3].

Currently, there are at least six known species of the venomous Ovophis genus (Family Viperidae, Subfamily Crotalinae) existed according to the phylogenetic and morphological analyses. Five closely related species are distributed across several geographical areas of the Asian mainland [4]. Ovophis tonkinensis occurs in northern Vietnam and southern China; O. zayuensis in southern China (Yunnan), northeastern India and Myanmar; O. makazayazaya in southern China (Sichuan, Yunnan), Taiwan and northern Vietnam; O. convictus is restricted to western Malaysia; and O. monticola is found in Nepal, northeastern India [5], southern China, Myanmar, southern Laos, central Vietnam and northern Thailand [4, 6]. The other member of this genus, O. okinavensis, inhabits Ryukyu Island of Japan [7].

In Thailand, the Asian mountain pit viper O. monticola is found in high-altitude mountains, particularly in the northern province of Chiang Mai [6]. It has a stout body with a short snout. Its triangular head is covered by small, smooth scales rather than large shields. These vipers also exhibit sexual dimorphism in body size, with an average male length of 49 cm and female length of 110 cm. These montane, terrestrial, nocturnal vipers generally live under the forest litter and prey on small mammals [8].

The medical significance of pit viper envenomation primarily relates to the hematotoxic activity on human victims. Severe clinical manifestation includes local damage (e.g., painful oedema, tissue necrosis) and systemic injuries, including haemorrhage, coagulopathy and thrombocytopenia, critically resulting in high mortality and morbidity [5, 9].With advanced proteomic technology, the heterogeneity of snake venoms has progressively been elucidated. For the Ovophis spp., venomic profiles of O. convictus from western Malaysia, O. tonkinensis from northern Vietnam and southern China and Japanese hime habu O. okinavensis from Okinawa, Japan were recently reported. The abundance of four major enzymes namely snake venom serine proteinase (SVSP), phospholipases A2 (PLA2), L-amino acid oxidases (LAAO) and snake venom metalloproteases (SVMP) were dominant within all venoms. Among these enzymatic proteins, SVSP was found in the greatest proportion, accounting for 35–53% of all constituents. The second most abundant enzyme was PLA2 ranging from 19–26%. In addition, various non-enzymatic proteins and peptides including cysteine-rich secretory proteins (CRISP), venom nerve growth factor (VNGF), venom endothelial growth factor (VEGF), kunitz peptides (KUN) and C-type lectins/snaclecs (CTL) were recorded, in varying amounts [10]. However, variation in snake venom composition occurs not only among distinct species but also among different population of the same species, due to ecological niches as well as availability of preys [11].

The present study aims to investigate the protein constituents of venom from the Asian mountain pit viper O. monticola found in Thailand. In addition, since there is no homospecific antivenom to Ovophis spp. venoms currently available, the therapeutic regime for bite victims depends largely on two types of antivenom: pit viper monovalent antivenom, raised against white-lipped green pit viper (Trimeresurus albolabris) venom; and polyvalent antivenom, produced against hematotoxic venom of Calloselasma rhodostoma (Malayan pit viper), Daboia siamensis (Russell’s viper) and T. albolabris. Cross reactivity of O. monticola venom to these readily available antivenoms was therefore evaluated. Compositional profiles of immunoreactive versus non-reactive proteins in O. monticola venom were also clarified. Knowledge gained from this study not only extends the Ovophis spp. venomic database, but also can lead to better management and therapeutic approaches for mountain pit viper envenomation.

Materials and methods

Snakes, venom and antivenoms

All O. monticola pit vipers (Fig 1) were captured in the wild and transferred to Snake Farm, Queen Saovabha Memorial Institute (QSMI) before being quarantined. All procedures were performed following the safety protocol for working with venomous snakes (No. SN 001/2016). Routine snake care and the venom collection was conducted according to the specific protocol. All protocols were approved by the Ethic Committee of the Queen Saovabha Memorial Institute Animal Care and Use (No. QSMI-ACUC-02-2018) in accordance with the guideline of the National Research Council of Thailand. Information about individual snakes used in this study is shown in Table 1.

Fig 1

A wild juvenile Asian mountain pit viper (O. monticola) found in Northern Thailand.

The venom of O. monticola was extracted and kept in individual 1.5 ml microcentrifuge tubes. After weighing, the fresh (liquid) venom was immediately frozen at -20°C and lyophilized. The lyophilized venom was then pooled and stored at -20°C until use.

Table 1

Biological and geographical data for all snakes used in the study.

Speciesa	Voucher no.b	Sexc	Snout-Vent Length (cm)	Total Length (cm)	Localityd
Ovophis monticola	QSMI 1441	F	32.0	36.5	Omkoi-Chiang Mai
Ovophis monticola	QSMI1443	F	32.0	36.5	Doi Pui-Chiang Mai
Ovophis monticola	QSMI 1449	M	34.0	40.0	Omkoi-Chiang Mai
Ovophis monticola	QSMI 1469	M	31.0	38.0	Omkoi-Chiang Mai
Ovophis monticola	QSMI 1559	M	35.5	43.0	Omkoi-Chiang Mai

a The identification of Ovophis monticola was made by specialized veterinarians according to the identification key [8]. Key characters are body coloration and pattern: predominantly tan or reddish-grey with irregular short, black-edged crossbars or blotches along the vertebral ridge, including smaller irregular dark blotches on both sides of the body along the edges of the dorsal scales (Fig 1).

b Voucher no. was attached to each preserved snake after it died.

c Sex: F: Female; M: Male.

d District or subdistrict-province in Thailand where snakes were captured.

Monovalent antivenom against the green pit viper T. albolabris venom (batch no. TA00219; expiry date 08/10/2024) and hematotoxic polyvalent antivenoms (against the venom of C. rhodostoma, D. siamensis and T. albolabris) (batch no. HP 00118; expiry date 16/ 01/2023) produced by QSMI available as a freeze-dried F(ab’)2 form, isolated from horse immunoglobulins were used within their shelf-life. Following reconstitution, each milliliter of monovalent antivenom neutralized 0.7 mg of T. albolabris venom; one milliliter of hematotoxic polyvalent antivenom neutralized 0.7 mg of T. albolabris venom, 1.6 mg of C. rhodostoma venom and 0.6 mg of D. siamensis venom [12].

O. monticola venom preparation and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Crude venom of O. monticola was mixed with lysis buffer (containing of 1% Triton X-100 (Merck, Germany), 1% sodium dodecyl sulfate (SDS) (Merck, Germany), and 1% NaCl (Merck, Germany). The venom was estimated for protein concentration by Quick Start™ Bradford Protein Assay (Bio-Rad, USA). A 30 μg sample of O. monticola venom was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, USA) and stained by Coomassie R-250 solution (Bio-Rad, USA) as previously described [13]. The whole lane of venom was excised into 10 pieces and further subjected to in-gel digestion.

Two-dimensional polyacrylamide gel electrophoresis (2DE)

A 100 μg protein was mixed with IPG sample buffer containing 8 M urea, 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 15 mM dithiothreitol (DTT), and 0.5% IPG sample buffer [14]. Afterwards, the protein solution was rehydrated overnight into a non-linear immobilized pH gradient (IPG) strip (pH 3–10; Amersham Bioscience, USA). Isoelectric focusing (pI) was done using an Ettan IPGphorII instrument (Amersham Bioscience, USA) with the following settings: 30 V for 14 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 3500 V for 1 h, and 8000 V for 18 h. The IPG strips were equilibrated with DTT for 15 min and with iodoacetamine for 15 min. After incubation, the strips were placed onto a 12% SDS-PAGE gel. All three 2DE gels were stained with silver stain and the immunoreactive spots in these gels were excised and pooled for mass spectrometric analysis. Other two 2DE gels were used for immunoblotting.

In-gel digestion

A mixture of 50% acetonitrile (ACN) in 50 mM ammonium bicarbonate was used for de-staining the blue color from gel slides [13]. Venom proteins were reduced by 4mM DTT and incubated at 60°C for 15 min. The reduced proteins were further alkylated by 250 mM iodoacetamine (IAA) (Sigma-Aldrich, USA) and incubated at room temperature for 30 min in dark. The gel pieces were dehydrated by removing all solution and adding 100% ACN (Thermo Scientific, USA). For tryptic digestion, trypsin (Sigma-Aldrich, USA, T6567) in 50 mM ammonium bicarbonate (Sigma-Aldrich, USA) was added to rehydrate the gels, which were then incubated overnight at 37 ˚C. Peptide extraction was performed by adding 100% ACN and incubating for 15 min. The resulting solution was transferred into a new microcentrifuge tube and dried using a centrifugal concentrator (TOMY, Japan). The peptide mixtures were stored at -20°C prior to mass spectrometric analysis.

Mass spectrometric analysis

Venom peptides were dissolved in 0.1% formic acid (Sigma-Aldrich, USA) and subjected to an Ultimate® 3000 Nano-LC systems (Thermo Scientific, USA). The peptides were eluted and infused to a microTOF-Q II (Bruker, Germany). The acquisition was operated by HyStar™ version 3.2 (Bruker, Germany), and the resulting data were processed and converted to mascot generics format (.mgf) files using Compass DataAnalysis™ software version 3.4 (Bruker, Germany). A database search was performed using Mascot Daemon software (Matrix Science, USA) against the NCBI snake database with the following parameters: one missed cleavage site, variable modifications of carbamidomethyl (C) and oxidation (M), 0.8 Da for MS peptide tolerance and 0.8 Da for MS/MS tolerance. The significance threshold was set at 95%. Three biological replications were performed for protein identification.

Indirect enzyme-linked immunosorbent assay (ELISA)

Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenom was assessed by indirect enzyme-linked immunosorbent assay (ELISA) modified from Gawtham and colleagues [15]. Each well of a 96-well Maxisorp Nunc immune plate (Thermo Fisher Scientific, Denmark) was coated with 5 ng of O. monticola venom in 0.05 M carbonate/bicarbonate buffer pH 9.6 (50 μl/well) and kept at 4°C overnight. Plates were washed three times with phosphate-buffered saline (PBS) pH 7.2, blocked by adding 200 μL of PBS containing 2% (w/v) bovine serum albumin (BSA) (Capricorn Scientific GmBH, Ebsdorfergrund, Germany) and incubated for 1.5 h at 37°C. The plates were then washed three times with PBS-0.05% Tween (PBST). They were incubated again for 1 h at 37°C with 50 μL of the serial dilution of either monovalent or polyvalent antivenom (1:105−1:5x107 in 0.2% BSA-PBS). After washing the plate three times with PBST, 50 μL of horseradish peroxidase-conjugated goat anti-horse-IgG (Abcam, Cambridge, UK) in PBST (1:1000) was added into each well and further incubated for another hour at 37°C. Plates were then washed three times with PBST. Fifty microliters of substrate solution (SureBlue TMB microwell peroxidase, Seracare Life Sciences, Milford, MA) was subsequently added to each well, and the plate was kept in the dark for 10 min at room temperature for the reaction to occur. The absorbance at 630 nm was read using a microplate reader (TECAN InfinitePro 200, Switzerland).

Immunoblot analysis

The separated polypeptide spots from 2DE gels were transferred to nitrocellulose membrane for 90 min at 18 V on a Trans-blot semi-dry Transfer CellTM (Biorad) in semi-dry transfer buffer (48 mM Tris and 2.93 g glycine) pH 9.2 containing 20% methanol. The membranes were blocked using 5% (w/v) non-fat milk in PBS for 2 h at room temperature. The membranes were rinsed twice with PBS-T buffer pH 7.4 (8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM NaCl, 2.7 mM KCl and 0.5% v/v Tween) for 30 s each. The blotted membranes were incubated with either monovalent or polyvalent antivenom (1:1000 in 0.2% BSA-PBS). After washing the membrane three times with PBS-T, 50 μL of horseradish peroxidase-conjugated goat anti-horse-IgG (Abcam, Cambridge, UK) in PBS-T (1:2000) was added, and the mixture was incubated for 1 h at ambient temperature under constant agitation. Membranes were washed three times with PBS-T buffer and one time with PBS. Immunogen spots were visualized by detection of peroxidase activity using Ultra TMB-Blotting Solution (ThermoFisher Scientific, UK).

Statistical analysis

Quantitative data are presented as mean ± SEM. Statistical significance between groups was analyzed using standard t-tests or two-way ANOVA followed by the Bonferroni test. Significant p-values are indicated within the figure panels. Error bars indicate SEM.

Results

Proteomic analysis of O. monticola venom

Detectable proteins in venom of O. monticola were between 10–95 kDa (Fig 2A). Intense protein bands at 10, 15, 50 and 72 kDa and faint bands at 26, 28, 30, 34 and 95 kDa were recorded. There were 247 proteins found in O. monticola venom (a list of all proteins is shown in S1 Table). A classification of constituent proteins based on their biological properties is presented in Fig 2B. The most abundant proteins were snake venom metalloproteases (SVMP) (36.8%), snake venom serine proteases (SVSP) (31.1%), and phospholipases A2 (PLA2) (12.1%). Less abundant groups included L-amino acid oxidase (LAAO) (5.7%), venom nerve growth factor (3.6%), nucleic acid degrading enzymes (3.2%) C-type lectins (CTL) (1.6%), cysteine-rich secretory proteins (CRISP) (1.2%) and disintegrin (1.2%). Toxin biosynthesis and other proteins comprised 0.4%. The top 15 unique proteins identified in O. monticola venom are shown in Table 2.

Fig 2

Venomics of Asian mountain pit viper O. monticola from Thailand.

(A) Coomassie blue-stained 12% SDS-PAGE of O. monticola venom (30 μg) under reducing conditions. (B) Proteome classification of O. monticola venom; percentages indicate relative abundance (% of total venom proteins) of protein family in snake venom.

Table 2

Fifteen most abundant unique proteins identified in Ovophis monticola venom.

No.	Accession no.	Protein	Score	MWa (Da)	No. of peptide	% Sequence coverage	pIb	emPAIc
1	sp|P0C2D5.2|OXLAPROFL	L-amino-acid oxidase	534	3601	2	80	4.44	14.55
2	sp|O93517.1|VM3S4GLOBR	Zinc metalloproteinase/disintegrin	1293	11254	5	48.6	4.42	5.18
3	sp|Q9PRP4.1|VSPFLACMR	Thrombin-like enzyme LMR-47	481	3168	2	100	4.31	3.82
4	sp|P0C590.1|VSP2GLOUS	Thrombin-like enzyme calobin-2	481	2159	2	100	4.65	3.81
5	sp|C0HLA2.1|VSP3LACMR	Thrombin-like enzyme LmrSP-3	335	2942	1	50	4.1	2.22
6	sp|P81478.1|PA2A2TRIGA	Acidic phospholipase A2 2	621	13784	4	33.6	4.95	1.73
7	sp|C0HLA3.1|VSP4LACMR	Snake venom serine protease LmrSP-4	1090	5841	2	62.3	4.28	1.60
8	sp|Q90W54.1|OXLA_GLOBL	L-amino-acid oxidase	2334	57056	19	32.7	6.52	1.37
9	BAA01566.1	Phospholipase A2	621	15697	3	40.6	4.99	1.35
10	pdb|1WVR|A	Chain A, Triflin	1099	24782	3	13.6	7.03	1.15
11	sp|Q7ZT99.1|CRVPCROAT	Cysteine-rich venom protein catrin	1099	26629	3	19.2	8.42	1.04
12	sp|Q7ZTA0.1|CRVPAGKPI	Cysteine-rich venom protein piscivorin	1099	26664	3	26.3	7.83	1.04
13	sp|E5L0E5.1|VSPPAAGKPL	Venom plasminogen activator	558	28060	5	12.8	5.78	1.02
14	AAM80563.1	Acidic phospholipase A2	372	15403	3	18.1	5.65	0.95
15	sp|P82896.1|PA2A5TRIST	Acidic phospholipase A2 5	427	13870	2	32.8	4.72	0.93

aMW: Molecular weight (Dalton).

bpI: isoelectric point.

cemPAI: exponentially modified protein abundance index.

The information of identified proteins including NCBI accession number (Accession no.), protein name (Protein), protein score (Score), molecular weight of protein in Dalton unit (Da), Number of identified peptides (No. of peptide), % sequence coverage of the identified peptides (%Sequence coverage), isoelectric point of protein (pI) and exponentially modified protein abundance index for semi-quantification (emPAI) are demonstrated.

Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenoms by indirect ELISA

Since there is no homospecific antivenom to Ovophis spp. venoms currently available, all pit viper envenoming victims are recommended to receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) to alleviate symptoms [16]. Indirect ELISA was used to determine the cross-reactivity of these antivenoms to O. monticola venom. Hematotoxic polyvalent antivenom exhibited a significantly greater level of immunoreactivity than the monovalent antivenom by 30–50% (up to the dilution 1: 1.6x106), P<0.001 (Fig 3). A titer for the monovalent antivenom was over 1:1.28x107 dilution, while that of hematotoxic polyvalent antivenom was 1:5.12x107 (Fig 3).

Fig 3

Immunoreactivity of O. monticola venom to antivenoms.

Cross-reactivity of crude O. monticola venom to monovalent antivenom raised against green pit viper venom and polyvalent antivenom against snake hemotoxins. Data represent the mean ± SEM from two independent experiments; * P < 0.05, ** P < 0.01 and *** P < 0.001.

O. monticola venom protein analysis by two-dimensional electrophoresis (2DE)

In order to explore the protein antigens present in O. monticola venom, crude venom was subjected to 2DE gel electrophoresis. There were 89 spots detected, with pI values ranging from 3 to 10 and MW from 10 to 95 kDa. Within the particular MW regions of 10–15, 30–34, 50 and 72–90 lies the greatest abundance of protein spots (Fig 4A). Using MALDI-TOF/TOF-MS/MS, all protein spots in 2DE gels were identified, and are listed in S2 Table. There were 461 different sequences, which correspond to 173 peptide accession identities.

Fig 4

The 2DE separations and immunoblot analysis of O. monticola venom.

(A) 2DE gels stained with silver stain; (B) 2D immunoblot of O. monticola proteins probed with monovalent antivenom and (C) polyvalent antivenom. Matched spots selected for subsequent LC-MS/MS analysis are marked and numbered.

Immunoreactive proteins in O. monticola venom by immunoblot analysis

The immunoblot analysis was performed with either monovalent or polyvalent antivenom to characterize specific protein antigens within the O. monticola venom. Twenty-six immunoreactive spots were detected with monovalent antivenom, with pI values ranging from 3 to 6, and MW ranging from 17 to 95 kDa. Most of these spots were observed at MW 50 to 95 kDa and pI between 3 to 5 (Fig 4B). When probed with hematotoxic polyvalent antivenom, 40 immunoreactive spots were recorded with a broader range of pI values from 3 to 8, and MW ranging from 17 to 95 kDa. A high number of the spots were detected within a MW range of 40–55 kDa (Fig 4C). Comparing the immunoreactive spots obtained from polyvalent antivenom with all protein spots visualized by silver staining (Fig 4A), 49 spots (numbered 24–25, 31–32, 38–40, 42–45, 48–51, 54–73 and 76–89) were not immunologically recognized. These non-reactive spots were grouped according to their MW and pI values into three clusters. Cluster 1 appeared in the MW range from 26 to 43 kDa, with high pI values (7–8); cluster 2 included those with MW ranging from 26 to 43 and with low pI values (3–5); and cluster 3 contained those with low MW from 10 to 26 kDa and with low pI values (3–5).

Identification of immunoreactive and non-reactive peptides in O. monticola venom by LC-MS/MS

LC-MS/MS analysis revealed a total of 202 distinct sequences in O. monticola venom identified within 101 protein types that were immunologically reactive with a polyvenom. All immunoreactive proteins recognized by monovalent and polyvalent antivenom are listed in Table 3. However, from all 173 protein types appearing by silver staining, 72 proteins were left unrecognized by both antivenoms, and are shown in Table 4. Interestingly, the majority of these neglected peptides are well-known members of CTLs (e.g., C-type lectin, snaclec and galactose-binding lectin) and different CRISPs including okinavin, catrin and piscivorin.

Table 3

List of identified proteins in Ovophis monticola venom immunologically reactive with monovalent and polyvalent antivenoms.

Spot no.	Protein/peptide accession	Description [Organisms]	MW (Da)	Monovalent	Polyvalent
1	XP_029142019.1	Zinc metalloproteinase-disintegrin-like atrolysin-A, partial [Protobothrops mucrosquamatus]	60272	√	√
	JAS04843.1	Metalloproteinase type III 2b [Crotalus horridus]	68297
	JAS04684.1	Metalloproteinase type III 1b [Crotalus adamanteus]	67284
	AAA03326.1	Hemorrhagic toxin a (partial)[Crotalus atrox]	46848
	GBP06242.1	Disintegrin and metalloproteinase domain-containing protein 12 [Eumeta japonica]	199170
2	XP_029142019.1	Zinc metalloproteinase-disintegrin-like atrolysin-A, partial [Protobothrops mucrosquamatus]	60272	√	√
	JAS04843.1	Metalloproteinase type III 2b [Crotalus horridus]	68297
	JAS04684.1	Metalloproteinase type III 1b [Crotalus adamanteus]	67284
3	sp|Q4VM07.1|VM3VB_MACLB	Zinc metalloproteinase-disintegrin-like VLAIP-B (Snake venom metalloproteinase)	68798	√	√
	JAS04447.1	Metalloproteinase type III 7 [Agkistrodon piscivorus conanti]	68638
	sp|P0DM87.1|VM2_TRIST	Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase)	54401
4	JAS04447.1	Metalloproteinase type III 7 [Agkistrodon piscivorus conanti]	68638	√	√
	sp|Q4VM07.1|VM3VB_MACLB	Zinc metalloproteinase-disintegrin-like VLAIP-B	68798
5	sp|Q4VM07.1|VM3VB_MACLB	Zinc metalloproteinase-disintegrin-like VLAIP-B (Snake venom metalloproteinase)	68798	√	√
	sp|P0DM87.1|VM2_TRIST	Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase)	54401
	JAS04675.1	Metalloproteinase type III 5 [Crotalus adamanteus]	69463
	JAS04447.1	Metalloproteinase type III 7 [Agkistrodon piscivorus conanti]	68638
	XP_023086434.2	disintegrin and metalloproteinase domain-containing protein 20-like [Piliocolobus tephrosceles]	84212
6	sp|Q4VM07.1|VM3VB_MACLB	Zinc metalloproteinase-disintegrin-like VLAIP-B (Snake venom metalloproteinase)	68798	√	√
	JAS04447.1	Metalloproteinase type III 7 [Agkistrodon piscivorus conanti]	68638
	sp|P0DM87.1|VM2_TRIST	Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase)	54401
7	-	Not identified		√	√
8	pdb|1REO|A	Chain A, Ahplaao	55097	√	√
	AAQ16182.1	L-amino acid oxidase [Trimeresurus stejnegeri]	58607
9	pdb|1REO|A	Chain A, Ahplaao	55097	√	√
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
10	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341	√	√
11	pdb|1REO|A	Chain A, Ahplaao	55097	-	√
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
	sp|P0C2D5.2|OXLA_PROFL	L-amino-acid oxidase (Okinawa Habu apoxin protein-1)	3601
	sp|P0C2D6.1|OXLA_PROMU	L-amino-acid oxidase	2929
12	pdb|1REO|A	Chain A, Ahplaao	55097	-	√
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
	BAP39915.1	L-amino acid oxidase [Protobothrops elegans]	57339
	sp|P0DI84.1|OXLA_VIPAA	L-amino-acid oxidase	54714
	sp|C0HJE7.2|OXLA_CRODU	L-amino acid oxidase bordonein-L	58882
	sp|Q4F867.2|OXLA_DABSI	L-amino-acid oxidase	46343
	sp|X2JCV5.1|OXLAA_CERCE	L-amino acid oxidase	58520
	sp|A8QL51.1|OXLA_BUNMU	L-amino-acid oxidase	58774
	sp|P0C2D5.2|OXLA_PROFL	L-amino-acid oxidase (Okinawa Habu apoxin protein-1)	3601
	sp|A0A2U8QPE6.1|OXLA_MICMP	L-amino acid oxidase	57079
	XP_026523888.1	titin isoform X41 [Notechis scutatus]	3637718
13	pdb|1REO|A	Chain A, Ahplaao	55097	-	√
	JAS04783.1	L-amino acid oxidase 1b [Crotalus horridus]	58587
	sp|P0DI84.1|OXLA_VIPAA	L-amino-acid oxidase	54714
	BAP39915.1	L-amino acid oxidase [Protobothrops elegans]	57339
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
	sp|C0HJE7.2|OXLA_CRODU	L-amino acid oxidase bordonein-L	58882
	JAV01888.1	BATXLAAO1 [Bothrops atrox]	56625
	sp|Q4F867.2|OXLA_DABSI	L-amino-acid oxidase	46343
	sp|P0C2D5.2|OXLA_PROFL	L-amino-acid oxidase (Okinawa Habu apoxin protein-1)	3601
	sp|X2JCV5.1|OXLAA_CERCE	L-amino acid oxidase	58520
	sp|A0A2U8QPE6.1|OXLA_MICMP	L-amino acid oxidase	57079
14	pdb|1REO|A	Chain A, Ahplaao	55097	-	√
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
	sp|P0DI84.1|OXLA_VIPAA	L-amino-acid oxidase	54714
	sp|C0HJE7.2|OXLA_CRODU	L-amino acid oxidase bordonein-L	58882
	sp|P0C2D5.2|OXLA_PROFL	L-amino-acid oxidase (Okinawa Habu apoxin protein-1)	3601
	JAV01888.1	BATXLAAO1 [Bothrops atrox]	56625
	sp|A0A2U8QPE6.1|OXLA_MICMP	L-amino acid oxidase	57079
15	pdb|1REO|A	Chain A, Ahplaao	55097	-	√
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
	sp|A0A2U8QPE6.1|OXLA_MICMP	L-amino acid oxidase	57079
	XP_026523846.1	Titin isoform X1 [Notechis scutatus]	3675875
16	pdb|1REO|A	Chain A, Ahplaao	55097	-	√
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
17	pdb|1REO|A	Chain A, Ahplaao	55097	√	√
	JAV01888.1	BATXLAAO1 [Bothrops atrox]	56625
18	pdb|1REO|A	Chain A, Ahplaao	55097	√	√
	AAQ16182.1	L-amino acid oxidase [Trimeresurus stejnegeri]	58607
	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341
	sp|P0DI84.1|OXLA_VIPAA	L-amino-acid oxidase	54714
	JAV01888.1	BATXLAAO1 [Bothrops atrox]	56625
	sp|A0A2U8QPE6.1|OXLA_MICMP	L-amino acid oxidase	57079
	sp|A8QL51.1|OXLA_BUNMU	L-amino-acid oxidase	58774
	sp|P0C2D5.2|OXLA_PROFL	L-amino-acid oxidase (Okinawa Habu apoxin protein-1)	3601
	XP_026523888.1	Titin isoform X41 [Notechis scutatus]	3637718
19	sp|A0A024BTN9.1|OXLA_BOTSC	L-amino acid oxidase Bs29	56341	√	√
20	BAN82126.1	Serine protease, partial [Ovophis okinavensis]	9035	-	√
	JAV51428.1	Serine proteinase 12a [Agkistrodon contortrix contortrix]	28885
	XP_026529526.1	Microtubule-actin cross-linking factor 1 isoform X1 [Notechis scutatus]	838459
21	sp|P0C578.1|VSP2_OVOOK	Thrombin-like enzyme okinaxobin-2 (Fibrinogen-clotting enzyme)	2310	-	√
	JAV51428.1	Serine proteinase 12a [Agkistrodon contortrix contortrix]	28885
	sp|I2C090.1|VCO3_OPHHA	Ophiophagus venom factor (Complement C3 homolog)	183812
	XP_026526061.1	ALK and LTK ligand 1 [Notechis scutatus]	21543
	sp|P85109.1|VSP1_GLOBR	Thrombin-like enzyme kangshuanmei (Fibrinogen-clotting enzyme)	26415
	JAG68112.1	Dynamin-binding protein [Boiga irregularis]	90258
22	JAV51428.1	Serine proteinase 12a [Agkistrodon contortrix contortrix]	28885	-	√
	BAN82126.1	serine protease, partial [Ovophis okinavensis]	9035
	sp|E5L0E5.1|VSPPA_AGKPL	Venom plasminogen activator	28060
	sp|Q5W958.1|VSP20_BOTJA	Venom serine proteinase-like HS120	27797
23	BAN82126.1	Serine protease, partial [Ovophis okinavensis]	9035	√	√
	sp|Q9PSN3.1|VSP2_AGKBI	Thrombin-like enzyme bilineobin (Fibrinogen-clotting enzyme/Snake venom serine protease)	26461
	BAN82122.1	Serine protease, partial [Ovophis okinavensis]	8080
	pdb|2AIP|A	Chain A, Protein C activator	25090
	sp|C0HLA2.1|VSP3_LACMR	Thrombin-like enzyme LmrSP-3	2942
	ADI47563.1	Serine protease, partial [Echis ocellatus]	27233
	sp|P0C5B4.2|VSPGL_GLOSH	Thrombin-like enzyme gloshedobin(Fibrinogen-clotting enzyme/Snake venom serine protease)	28597
	sp|Q9DF66.1|VSP3_PROJR	Snake venom serine protease 3	28007
	pdb|1OP0|A	Chain A, Venom serine proteinase	25318
26	JAV51428.1	Serine proteinase 12a [Agkistrodon contortrix contortrix]	28885	√	√
27	XP_029142018.1	Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus]	58843	√	√
	TSK34762.1	Disintegrin and metalloproteinase domain-containing protein 12 [Bagarius yarrelli]	146595
	XP_032089254.1	ras GTPase-activating-like protein IQGAP1 [Thamnophis elegans]	189690
28	XP_029142018.1	Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus]	58843	√	√
	sp|P0DM87.1|VM2_TRIST	Zinc metalloproteinase-disintegrin stejnitin	54401
	TSK34762.1	Disintegrin and metalloproteinase domain-containing protein 12 [Bagarius yarrelli]	146595
29	XP_029142018.1	Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus]	58843
	sp|P0DM87.1|VM2_TRIST	Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase)	54401	√	√
	TSK34762.1	Disintegrin and metalloproteinase domain-containing protein 12 [Bagarius yarrelli]	215963
	ETE65365.1	putative helicase senataxin, partial [Ophiophagus hannah]	146595
30	XP_029142018.1	Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus]	58843	√	√
	sp|P0DM87.1|VM2_TRIST	Zinc metalloproteinase-disintegrin stejnitin (Snake venom metalloproteinase)	54401
31	XP_029142018.1	Zinc metalloproteinase-disintegrin jerdonitin [Protobothrops mucrosquamatus]	58843	-	√
34	JAS05371.1	Serine proteinase 9d [Sistrurus miliarius barbouri]	28266	√	√
	sp|P0DMH6.1|VSP_BOTFO	Snake venom serine protease	1729
	sp|Q8AY78.1|VSP5M_TRIST	Snake venom serine protease 5	28117
	sp|Q8AY79.1|VSPS2_TRIST	Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease)	28010
	sp|Q5W958.1|VSP20_BOTJA	Venom serine proteinase-like HS120	27797
	sp|Q71QH7.1|VSPP_TRIST	Snake venom serine protease PA	27933
	XP_026540424.1	Inositol hexakisphosphate and diphosphoinositol-pentakisphosphate kinase 1 isoform X1 [Notechis scutatus]	135476
35	sp|P0DMH6.1|VSP_BOTFO	Snake venom serine protease	1729	√	√
	sp|E5L0E5.1|VSPPA_AGKPL	Venom plasminogen activator	28060
	sp|Q8AY78.1|VSP5M_TRIST	Snake venom serine protease 5	28117
	sp|Q5W958.1|VSP20_BOTJA	Venom serine proteinase-like HS120	27797
	sp|Q71QH7.1|VSPP_TRIST	Snake venom serine protease PA	27933
	sp|Q8AY79.1|VSPS2_TRIST	Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease)	28010
36	JAS05372.1	Serine proteinase 9c [Sistrurus miliarius barbouri]	28221	√	√
	JAS05371.1	Serine proteinase 9d [Sistrurus miliarius barbouri]	28266
	JAV51414.1	Serine proteinase 8 [Agkistrodon contortrix contortrix]	28242
	sp|P0DMH6.1|VSP_BOTFO	Snake venom serine protease	1729
	sp|P0C5B4.2|VSPGL_GLOSH	Thrombin-like enzyme gloshedobin (Fibrinogen-clotting enzyme/Snake venom serine protease)	28597
	ADI47574.1	Serine protease, partial [Echis coloratus]	28437
	sp|Q8AY78.1|VSP5M_TRIST	Snake venom serine protease 5	28117
	sp|Q5W958.1|VSP20_BOTJA	Venom serine proteinase-like HS120	27797
	sp|Q8AY79.1|VSPS2_TRIST	Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease)	28010
	sp|Q8UUJ2.2|VSPUI_GLOUS	Snake venom serine protease ussurin;	26184
	sp|Q71QH7.1|VSPP_TRIST	Snake venom serine protease PA	27933
	XP_032092228.1	Vitelline membrane outer layer protein 1 homolog isoform X1 [Thamnophis elegans]	21236
	JAI10638.1	Vacuolar protein sorting-associated protein 18 homolog [Crotalus adamanteus]	111967
37	JAS05372.1	Serine proteinase 9c [Sistrurus miliarius barbouri]	28221	-	√
	JAS05371.1	Serine proteinase 9d [Sistrurus miliarius barbouri]	28266
	sp|P0DMH6.1|VSP_BOTFO	Snake venom serine protease	1729
	JAV51414.1	Serine proteinase 8 [Agkistrodon contortrix contortrix]	28242
	sp|Q9PT41.1|VSPF5_MACLB	Factor V activator (Lebetina viper venom FV activator/Snake venom serine protease	28577
	ADI47574.1	Serine protease, partial [Echis coloratus]	28437
	sp|Q8AY78.1|VSP5M_TRIST	Snake venom serine protease 5;	28117
	XP_023086434.2	Disintegrin and metalloproteinase domain-containing protein 20-like [Piliocolobus tephrosceles]	84212
	sp|Q8AY79.1|VSPS2_TRIST	Beta-fibrinogenase stejnefibrase-2 (Snake venom serine protease)	28010
41	-	Not identified		-	√
46	pdb|1BQY|A	Chain A, Plasminogen Activator	25590	√	√
	JAS04757.1	Serine proteinase 1f [Crotalus horridus]	28133
	JAS04429.1	Serine proteinase 13e [Agkistrodon piscivorus conanti]	27985
	JAS04417.1	Serine proteinase 18b [Agkistrodon piscivorus conanti]	27728
	JAS04415.1	Serine proteinase 19b [Agkistrodon piscivorus conanti]	27782
	sp|Q072L7.1|VSP_LACST	Snake venom serine protease	27796
	sp|O13069.1|VSP2_BOTJA	Thrombin-like enzyme KN-BJ 2 (Kinin-releasing and fibrinogen-clotting serine protease 2)	26399
	pdb|4E7N|A	Chain A, Snake-venom Thrombin-like Enzyme	28333
	XP_032089049.1	Spectrin alpha chain, non-erythrocytic 1 [Thamnophis elegans]	263010
	XP_032064352.1	Zinc finger protein 347-like [Thamnophis elegans]	169270
	sp|Q9PT41.1|VSPF5_MACLB	Factor V activator/Lebetina viper venom FV activatorSnake venom serine protease	28577
	ADI47574.1	Serine protease, partial [Echis coloratus]	28437
47	JAS04757.1	Serine proteinase 1f [Crotalus horridus]	28133	√	√
	pdb|1BQY|A	Chain A, Plasminogen Activator	25590
	JAS04417.1	Serine proteinase 18b [Agkistrodon piscivorus conanti]	27728
	JAS04429.1	Serine proteinase 13e [Agkistrodon piscivorus conanti]	27985
	JAS04415.1	Serine proteinase 19b [Agkistrodon piscivorus conanti]	27782
	JAV01826.1	BATXSVSP10 [Bothrops atrox]	28606
	pdb|4E7N|A	Chain A, Snake-venom Thrombin-like Enzyme	26370
	sp|Q6T5L0.2|VSPSH_GLOSH	Alpha-fibrinogenase shedaoenase (Snake venom serine protease)	26399
	sp|O13069.1|VSP2_BOTJA	Thrombin-like enzyme KN-BJ 2 (Kinin-releasing and fibrinogen-clotting serine protease 2)	27876
	sp|Q71QI0.1|VSP07_TRIST	Snake venom serine protease homolog KN7	28703
	XP_015671556.1	Snake venom serine protease [Protobothrops mucrosquamatus]	28023
	JAS04671.1	Serine proteinase 3b [Crotalus adamanteus]	28890
	QHR82809.1	Serine protease 2 [Vipera anatolica senliki]	28084
	sp|A8QL53.1|VSP1_NAJAT	Snake venom serine protease NaSP	31117
	XP_026523831.1	Integrin alpha-4 [Notechis scutatus]	114850
52	JAS05359.1	Cysteine-rich secretory protein 1c [Sistrurus tergeminus]	26787	-	√
53	ETE67131.1	Keratin, type II cytoskeletal 1, partial [Ophiophagus hannah]	240496	-	√
74	sp|A8E2V8.1|PA2A_TRIGS	Acidic phospholipase A2 Tgc-E6	15678	√	√
	sp|P0DJJ7.1|PA2A_OVOMO	Acidic phospholipase A2 Omo-E6	3261
	JAV51451.1	Phospholipase A2 1a [Agkistrodon contortrix contortrix]	15952
	sp|Q6EAN6.1|PA2A_SISTE	Acidic phospholipase A2 homolog sistruxin APrecursor	15419
	XP_032088152.1	Group IIE secretory phospholipase A2-like [Thamnophis elegans]	17310
	sp|Q7ZTA6.1|PA2AB_CROVV	Acidic phospholipase A2 Cvv-E6b	15429
	AAB28455.1	Phospholipase A2 isozyme III, PLA2-III [Trimeresurus gramineus]	13716
	JAV01879.1	BATXPLA5 [Bothrops atrox]	15504
	sp|P06860.1|PA2BX_PROFL	Basic phospholipase A2 PL-X	13971
	AAB28454.1	Phospholipase A2 isozyme IV, PLA2-IV [1] [Trimeresurus gramineus]	13783
	sp|C0HJC1.1|PA2_BOTLA	Acidic phospholipase A2 BlatPLA2	13881
75	sp|A8E2V8.1|PA2A_TRIGS	Acidic phospholipase A2 Tgc-E6	15678	√	√
	sp|P0DJJ7.1|PA2A_OVOMO	Acidic phospholipase A2 Omo-E6	3261
	pdb|1C1J|A	Chain A, Basic phospholipase A2	13888
	JAS04499.1	Phospholipase A2 1s [Agkistrodon piscivorus conanti]	15776
	sp|P82896.1|PA2A5_TRIST	Acidic phospholipase A2 5	13870
	sp|D6MKR0.1|PA2A6_CROHD	Acidic phospholipase A2 CH-E6	15498
	sp|Q7ZTA6.1|PA2AB_CROVV	Acidic phospholipase A2 Cvv-E6b	15429
	JAV51451.1	Phospholipase A2 1a [Agkistrodon contortrix contortrix]	15952
	sp|P86907.1|PA2A_BOTAM	Acidic phospholipase A2	13858
	sp|C9DPL5.1|PA2A1_BOTPI	Acidic phospholipase A2 BpirPLA2-I	13627
	sp|C0HLF0.1|PA2_POROP	Basic phospholipase A2	14042
	sp|C0HJC1.1|PA2_BOTLA	Acidic phospholipase A2 BlatPLA2	13881
	sp|P86456.1|PA2A4_BOTAL	Acidic phospholipase A2 SpII RP4	13733
	QHR82796.1	Phospholipase A2 3 [Vipera anatolica senliki]	17437

Table 4

List of non-immunoreactive proteins/peptides in Ovophis monticola venom.

	Protein/peptide accession	Description [Organisms]	MW (Da)	Spot no.
1	AAZ75628.1	Kallikrein-Phi4, partial [Philodryas olfersii]	26827	45, 49, 50
2	BAN82001.1	Galactose binding lectin, partial [Protobothrops flavoviridis]	17654	80, 82, 84, 85
3	BAN82034.1	Serine protease, partial [Protobothrops flavoviridis]	22377	55
4	BAN82147.1	Cysteine rich secretory protein [Ovophis okinavensis]	26920	59, 60, 61, 62
5	BAN82148.1	Galactose binding lectin [Ovophis okinavensis]	18480	80, 82
6	BAN82149.1	C-type lectin alpha subunit [Ovophis okinavensis]	17686	87, 88
7	ETE59238.1	Fascin-3, partial [Ophiophagus hannah]	14941	48
8	ETE60526.1	Trichohyalin, partial [Ophiophagus hannah]	80507	51
9	ETE61374.1	Dynein heavy chain 8, axonemal [Ophiophagus hannah]	284552	72
10	ETE64295.1	Glycerol-3-phosphate acyltransferase 4 [Ophiophagus hannah]	50772	86
11	ETE66458.1	Helicase SRCAP, partial [Ophiophagus hannah]	494261	87
12	ETE70787.1	N6-adenosine-methyltransferase 70 kDa subunit, partial [Ophiophagus hannah]	59797	89
13	JAI12774.1	Leucine-rich repeat-containing protein 7-like [Crotalus adamanteus]	163679	45
14	JAS04407.1	Serine proteinase 6 [Agkistrodon piscivorus conanti]	28115	43,44, 48, 76
15	JAS04411.1	Serine proteinase 2 [Agkistrodon piscivorus conanti]	28333	49, 50, 55, 57,
16	JAS04568.1	Phospholipase A2 1b [Boiga irregularis]	16906	67, 72, 73, 78
17	JAS04670.1	Serine proteinase 3c [Crotalus adamanteus]	28849	45, 49, 50, 54, 55, 56, 57, 58
18	JAS04734.1	Cysteine-rich secretory protein [Crotalus adamanteus]	26612	59, 60, 61, 62
19	JAS04742.1	Serine proteinase 9d [Crotalus horridus]	28299	49
20	JAS04748.1	Serine proteinase 6 [Crotalus horridus]	28594	54, 55, 56, 57
21	JAS05249.1	Serine proteinase 2 [Sistrurus tergeminus]	28326	49
22	JAS05472.1	C-type lectin 2 [Sistrurus miliarius barbouri]	18147	87
23	JAS05484.1	Cysteine-rich secretory protein 1b [Sistrurus miliarius barbouri]	26772	61
24	JAV51425.1	Serine proteinase 15a [Agkistrodon contortrix contortrix]	28940	54, 55, 56, 57
25	JAV51455.1	C-type lectin 9a [Agkistrodon contortrix contortrix]	18657	80
26	pdb|1BK9|A	Chain A, Phospholipase A2	13964	72
27	pdb|1GMZ|A	Chain A, Phospholipase A2	13850	67
28	pdb|1JZN|A	Chain A, Galactose-specific lectin	16281	80, 82
29	pdb|1WVR|A	Chain A, Triflin	24782	59, 60, 61, 62
30	pdb|3JR8|A	Chain A, Phospholipase A2 bothropstoxin-2	13985	67
31	sp|A0A1I9KNP0.1|VSPH1_VIPAA	Vaa serine proteinase homolog 1	28909	49
32	sp|A8QL56.1|VSP1_OPHHA	Alpha- and beta-fibrinogenase OhS1	28637	45, 49, 50
33	sp|B0VXW0.1|OXLA_SISCA	L-amino-acid oxidase	58532	77
34	sp|B0ZT25.1|VSPH_PROJR	Snake venom serine protease homolog	28776	58
35	sp|C0HLA1.1|VSP2_LACMR	Thrombin-like enzyme LmrSP-2 (Snake venom serine protease)	3271	25, 32
36	sp|E5AJX2.1|VSP_VIPBN	Snake venom serine protease nikobin	28197	45, 49
37	sp|J3S832.1|VSPB_CROAD	Snake venom serine proteinase 11	28033	58
38	sp|J3S833.1|VSP2_CROAD	Snake venom serine proteinase 2	28298	49
39	sp|K4LLQ2.1|VSP_BOTBA	Thrombin-like enzyme barnettobin (Snake venom serine protease)	27567	38, 39
40	sp|O13057.1|VSP2_PROFL	Snake venom serine protease 2	28623	56
41	sp|O93421.2|VSPPE_GLOHA	Snake venom serine protease pallase	26031	49
42	sp|P0DJG8.1|CRVP_HELAG	Helicopsin	2618	61
43	sp|P0DL18.1|CRVP_OVOOK	Cysteine-rich venom protein okinavin	3496	59, 60, 61
44	sp|P0DM36.1|LECG_AGKPI	C-type lectin APL	16195	80, 82, 84, 85
45	sp|P81114.1|SLA4_TRIAB	Snaclec alboaggregin-A subunit beta	14357	81, 83, 86
46	sp|P81176.1|VSP1_GLOBL	Thrombin-like enzyme halystase (Snake venom serine protease)	26466	49
47	sp|P82981.1|VSP2_AGKCO	Thrombin-like enzyme contortrixobin/Fibrinogen-clotting enzyme (Snake venom serine protease)	25396	54, 55, 56, 57, 58
48	sp|Q27J47.1|VSPPA_LACMU	Venom plasminogen activator LV-PA	28044	44
49	sp|Q71QJ4.1|VSP04_TRIST	Snake venom serine protease homolog KN4	28685	49, 50, 55, 56, 57
50	sp|Q7SZE2.1|VSPD_GLOUS	Bradykinin-releasing enzyme KR-E-1 (Snake venom serine protease)	25335	49
51	sp|Q7T229.1|VSPH_BOTJR	Snake venom serine protease homolog	28636	54, 55, 56, 57
52	sp|Q7ZT99.1|CRVP_CROAT	Cysteine-rich venom protein catrin	26629	59
53	sp|Q7ZTA0.1|CRVP_AGKPI	Cysteine-rich venom protein piscivorin	26664	60, 61, 62
54	sp|Q8AY81.1|VSPST_TRIST	Thrombin-like enzyme stejnobin (Fibrinogen-clotting enzyme/Snake venom serine protease)	29309	25, 32
55	sp|Q91053.1|VSP1_GLOUS	Thrombin-like enzyme calobin-1 (Snake venom serine protease)	28889	49
56	sp|Q9YGJ2.1|VSP1_GLOHA	Snake venom serine protease pallabin	28662	49
57	XP_015671564.1	Snake venom serine protease serpentokallikrein-1 [Protobothrops mucrosquamatus]	88822	56
58	XP_023418723.1	Disintegrin and metalloproteinase domain-containing protein 17 [Cavia porcellus]	92703	64
59	XP_024069019.3	Disintegrin and metalloproteinase domain-containing protein 17 [Terrapene carolina triunguis]	99769	64
60	XP_025414344.1	Disintegrin and metalloproteinase domain-containing protein 9 [Sipha flava]	138126	54
61	XP_026527653.1	Laminin subunit alpha-1 [Notechis scutatus]	331363	80
62	XP_026535629.1	Dynein heavy chain 8, axonemal [Notechis scutatus]	511281	66
63	XP_026540213.1	Regulatory solute carrier protein family 1 member 1 [Notechis scutatus]	37705	80
64	XP_026541175.1	N-acetylated-alpha-linked acidic dipeptidase-like protein [Notechis scutatus]	81911	50
65	XP_028906446.1	Disintegrin and metalloproteinase domain-containing protein 17 [Ornithorhynchus anatinus]	94942	63
66	XP_032078796.1	Laminin subunit alpha-1 [Thamnophis elegans]	339446	85
67	XP_032080246.1	Centromere-associated protein E [Thamnophis elegans]	308791	59
68	XP_032085798.1	60S ribosomal protein L6 isoform X1 [Thamnophis elegans]	30312	42, 81
69	XP_032088226.1	Forkhead-associated domain-containing protein 1 [Thamnophis elegans]	137802	81
70	XP_032091805.1	Glial fibrillary acidic protein [Thamnophis elegans]	52228	88
71	XP_039181676.1	Snake venom serine protease-like isoform X1 [Crotalus tigris]	25811	49
72	XP_039181680.1	Snake venom serine proteinase 12-like [Crotalus tigris]	24539	56

Discussion

The protein constituents within the venom of Asian mountain pit viper O. monticola from northern Thailand were investigated in the present study. SDS-PAGE revealed a protein band pattern ranging between MW 10–90 kDa with intense bands representing low MW proteins (10–15 kDa), those of 50 and 72 kDa, and faint bands between 30–50 kDa. This corresponded well to the spot pattern obtained from 2DE, where the clouds of protein spots were observed within MW regions of 10–15, 30–34, 50 and 72–90 kDa. The overall MW range of proteins in O. monticola venom was comparable to those from three other Ovophis species, namely O. convictus, O. tonkinensis and O. okinavensis. The pattern of dominant bands was most similar to the venom of O. tonkinensis found in China [10].

The proteomic profile showed that enzymatic components which are SVMP (36.8%), SVSP (31.1%), PLA2 (12.1%) and LAAO (5.7%) mainly make up the venom of Asian mountain pit viper O. monticola. The overall composition of these major enzymes was comparable to those recently reported from venoms of O. convictus, O. tonkinensis and O. okinavensis, only content proportion seemed to be different. Among these three Ovophis spp., the most abundant proteins were of SVSP (35–53%), followed by PLA2 (19–25%) and LAAO (5–17%). SVMP (11–19%) was detected at a lower percentage than in our venom [10]. Such venom variation in venom composition could would not only be attributed by speciation but also other factors including prey diversity reflecting different ecological habitats [11], snake sex, [17] and age [18, 19]. In addition, the variation in quantity of identified toxin types within snake venom might be resulted from the different quantitative approaches and accompanying calculation methods as well as proteomic database availability [20]. The 2DE indicated that O. monticola venom contained more acidic than basic protein spots. This finding was confirmed by our list of all identified peptides obtained from LC-MS/MS showing that the majority possessed pI values lower than 7. Our spot pattern also confirmed previous 2DE analysis of Trimeresurus sumatranus (another Viperid) venom, in which more proteins were identified in the acidic range than in Elapid venom [21]. The overall acidic properties of 4 main protein groups of vipers greatly contribute to the hemorrhagic and coagulopathic effect on victims [22, 23].

Our results revealed SVMPs as representing more than one-third of the entire O. monticola venom. The greatest amount found in O. monticola was sp|O93517.1|VM3S4_GLOBR Zinc metalloproteinase/disintegrin or disintegrin-like salmosin-4, first identified within Korean Agkistrodon halys brevicaudus snake venom [24]. SVMPs potentially inhibit platelet aggregation and integrin-dependent cell adhesion via interrupting glycoprotein IIb-IIIa/fibrinogen interaction and fibrinogenolysis [25, 26]. Additionally, SVMPs interact with the various types of cellular matrix and exerts the most haemorrhagic effect on hosts [27]. SVSPs were found to be second-most abundant in O. monticola venom. The majority are thrombin-like enzymes including sp|Q9PRP4.1|VSPF_LACMR thrombin-like enzyme LMR-47 and sp|Q9PRP4.1|VSPFLACMR thrombin-like enzyme calobin-2. Known as fibrinogen-clotting enzymes, they are common, and found in large amounts in the venoms of the genera Agkistrodon, Bothrops, Lachesis and Trimeresurus [28]. Thrombin-like enzymes demonstrate strong hydrolytic activity, primarily against triad residues of His57, Asp102 and Ser195 of fibrinogen [29]. Resembling thrombin, they act on blood plasma by forming friable and translucent clots which later are degraded due to non-functional cross-linked structures, leading to coagulopathy and hypofibrinogenemia [30, 31]. However, in terms of enzyme proportion, our findings contrast with previous reports on the transcriptomic analysis of O. okinavensis venom glands, which mainly contained SVSP (93.1%) and relatively little SVMP (4.2%) [32]. It remains unclear whether the microenvironment within the venom gland might preferentially activate or interfere with the functioning of newly synthesized enzymes.

Compared with SVMP and SVSP, we detected smaller amounts of PLA2 and LAAO in O. monticola venom. PLA2 hydrolyzes phospholipids at the sn-2 position, generating fatty acids and lysophospholipids [33]. Group II PLA2 is expressed exclusively in the venoms of the Viperidae [34]. We found both acidic and basic PLA2 subtypes in O. monticola venom. It is noteworthy that the acidic PLA2 (sp|P81478.1|PA2A2TRIGA and sp|P82896.1|PA2A5TRIST were present in high quantities partially contributing to more acidic properties of the venom. These were previously found to trigger oedema [35]. PLA2 elicits inflammatory responses through the overproduction of pro-inflammatory cytokines (such as TNFα, IL-1β and IL-6) largely by immunocompetent cells (monocytes, neutrophils and mast cells) [36].

LAAOs can act in concert with PLA2 in local inflammatory reactions. LAAOs from Calloselasma rhodosthoma venom were shown to induce superoxide anion and hydrogen peroxide production by human neutrophils [37]. The most abundant LAAO in O. monticola venom is sp|P0C2D5.2|OXLA_PROFL, also known as Okinawa habu apoxin protein-1. This protein was first characterized from the venom of Protobothrops flavoridis to induce apoptosis in glioma cells [38]. The roles of both PLA2 and LAAOs in snake venoms are multi-faceted. Their catalytic as well as cytotoxic properties have been extensively investigated for pharmaceutical potential against cancers and other diseases [39].

Due to the unavailability of homospecific antivenom to Ovophis spp. venoms, all pit viper envenoming victims usually receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) [16]. The latter gave considerably higher immunoreactive levels (30–50%) to O. monticola venom proteins than the former. Relatively greater levels of reactivity of the polyvalent antivenom was previously reported with the venoms of C. rhodostoma, Hypnale hypnale and Trimeresurus hageni, and even Trimeresurus albolabris when compared with those of monovalent antivenom [40]. With the combination of 2DE immunoblotting and LC-MS/MS analyses, we found that hematotoxic polyvalent antivenom reacted with a wider range of proteins and peptides accounting for 58% of the entire range of proteins and covering all major enzymatic groups. Nonetheless, we were able to observe that an array of LAAOs and SVSPs did not react with a monovalent antivenom specific only to T. albolabris venom. This finding suggests the shared antigenic epitopes particularly from Ovophis, Calloselasma and Trimeresurus venoms used to generate antivenom. In this context, proteomic analysis of Malayan pit viper C. rhodostoma venom revealed a similar SVMP dominance (41.17%), with other major constituents of snaclec (26.3%) and SVSP (14.9%) [41]. A study of phylogenetic relationships based on geographic distribution and mitochondrial and nuclear gene sequences also demonstrated that O. monticola is less distantly separated from C. rhodostoma than from T. albolabris [42]. Thus, the antivenom against immunogenic epitopes from C. rhodostoma venom should be further investigated for the possible adjunctive treatment of O. monticola bite victims.

Our current study revealed that 72 proteins (42% of venom proteins) were left unrecognized by both antivenoms. The majority of immunologically non-reactive proteins have low molecular mass. They include a number of SVSPs, PLA2 and certain SVMP. The poor immunogenicity of these low molecular venomic proteins has been obviously reported, although some possess high toxicity [43, 44]. In addition, an array of non-enzymatic CTLs such as galactose-binding lectins, snaclec, alboaggregin A, and CRISPs such as triflin, okinavin, catrin, and piscivotin were found unrecognizable by antivenom. This reflects the difference in antigenic abundance between O. monticola venom and those venoms employed to generate horse immunoglobulins, as the proportions of CTLs and CRISPs in our O. monticola venom were only 1.6% and 1.2%, respectively. Nonetheless, their biological impacts on host cells and tissues should not be neglected. In the context of CTLs, alboaggregin A was shown to bind strongly with platelet glycoproteins IB and VI, and hence, activated platelet aggregation [45]. In addition, evidence of enhanced platelet activation and thrombotic microangiopathy-like symptoms has been documented with other related snaclecs [46]. In terms of CRISPs, the unique pdb|1WVR|A chain A triflin, as well as sp|P0DL18.1|CRVP_OVOOK okinavin from the related hime habu O. okinavensis were not reactive with the antivenoms. They have been previously described to have a calcium channel-impairing effect, leading to aberrations in muscle contraction [47, 48]. Considering the pathophysiological effects, our findings address the suite of protein targets which could be additional antigens for future antivenomic design. Furthermore, in order to alleviate the symptoms of mountain pit viper envenomation, these would facilitate the development of specific drug schemes allowing patients to recover more quickly. The proteomic profile of O. monticola venom not only provides insight into the venomic phenotypes reflecting the evolutionary path among Viperid snakes, but also accelerates the discovery of novel candidates for medical and pharmaceutical use.

(PDF)

Click here for additional data file.

List of all proteins found in Ovophis monticola venom.

(XLSX)

Click here for additional data file.

List of proteins in 89 spots in Ovophis monticola venom.

Crude venom was subjected to 2DE gel. Proteins were separated in the first dimension in the pH range 3–10.

(DOCX)

Click here for additional data file.

13 Jul 2021

PONE-D-21-18686

Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venoms

PLOS ONE

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Reviewer #1: Referee comments:

In this work, the authors demonstrate by venomics approaches, a comprehensive characterization of the venom proteome and immunocharacterization of Asian Mountain pit viper (Ovophis monticola) venoms, a medically important snake from Thailand, found at high altitude areas of several mountain ranges of East, South and Southeast Asia. Moreover the authors analyzed the immunoreactivity of monospecific (monovalent) and polyspecific (polyvalent) antivenoms against green pit viper Trimeresurus albolabris and hemotoxic venoms, respectively. The authors present a study with satisfactory quality in methodology and data generation. However the manuscript has an inventory of toxins and immunoreactive proteins without coherence with the scientific literature and poor consistency with some cited references. Furthermore, in many topics of the manuscript the authors present a lot of information without any reference. For example, the authors don't compare the results regarding 2D immunoblotting with any previously study. I suggest that this manuscript should be reword to improve the quality about the data presentation. I'm sending some notes

- Abstract

Line 33  ..... venoms was investigated by indirect ELISA..... Line 37.....were subjected to 2D immunoblotting

These related methodologies would be more appropriate if they were introduced together.

- Introduction

. Line 46-50 - The geographical distribution data deserve references. The same for the sentences introducing biological data about pit viper from lines 51-58.... length of 110 cm.

Lines 59: Incidence of pit vipers  bites, including those from mountain pit vipers, have been documented in their known ranges???.

The lack of references should be revised in all manuscript...

-Materials and methods

Line 92 : Snake venom,

- Are there more information about the venom's sample used in this study? Number of specimens? Localities? size/age...

Line 121: ...a non-linear immobilized pH gradient (IPG) strip (pH 3-10 Amersham Bioscience, USA)....

- Why the authors choose the non-linear gradient instead of linear gradient, since the propose were excised immunoreactive spots for mass spectrometric analysis? Just for curious

- Lack references in almost all methods. The references for 2D immunoblotting is required

Results

Line 208: Change Ovophis / Ovophis sp or genera Ovophis

- Are there any published information about neutralize activity of monovalent and polyvalent antivenoms against O. monticola venom? This information should be provided or I suggest that an experiment to determine the lethal dose 50% should be done.

Table 2. List of identified proteins ... immunologically reacted ...

- If the MW of the spots content reacted/ non-reacted proteins could be provided, this information can improve the quality table.

Discussion

The Authors should revise the references from the first paragraph.... For example: the statement about hematotoxic potential of SVMP, SVSP and PLA2 is already a scientific consensus. Therefore, there are many sources specially from the seminal studies that corroborate with this question. However, the Authors present references from venom proteomic analysis of other snakes to support this question. On the other hand, the authors present coherency in the literature proposed as reference to address the mechanism of action of P-III metalloproteinase. I suggest that his paragraph must be reworded.

Line 208: The presence of class P-II metalloproteinase, which possesses metalloproteinase and disintegrin-like domians was also observed.????

Line 303: The combination of SVMP and SVSP observed in O. monticola venom is responsible for prey attack, incapacitation, and digestion.???

Reviewer #2: It is an interesting work to be published in PLOS. This article describes describe proteomics and immuno characterization of snake Ovophis monticola venom. I have the following comments that the authors may like to consider:

Line 95

- Provide the total number of individuals, age, sex, locality (adults or young people, males, females)

- It is important to provide some functional analysis

Discussion:

Despite little published literature on Ovophis monticola:

Please, include in the discussion this reference:

Pandey, D. P., Chaudhary, B., & Ram Shrestha, B. (2021). Documentation of a proven Mountain Pitviper (Ovophis monticola) envenomation in Kathmandu, Nepal, with its distribution ranges: implications for prevention and control of pitviper bites in Asia. Journal of venom research, 11, 1–6.

Mainly about clinical characteristic of poisoning by this specie and lack of specific treatment.

Provide conclusion or future perspectives in the last paragraphy about immunocharacterization results/ envenomation treatment.

Line 527

Exclude the number 75.

**********

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20 Aug 2021

Responses to reviewers

Reviewer #1:

In this work, the authors demonstrate by venomics approaches, a comprehensive characterization of the venom proteome and immunocharacterization of Asian Mountain pit viper (Ovophis monticola) venoms, a medically important snake from Thailand, found at high altitude areas of several mountain ranges of East, South and Southeast Asia. Moreover the authors analyzed the immunoreactivity of monospecific (monovalent) and polyspecific (polyvalent) antivenoms against green pit viper Trimeresurus albolabris and hemotoxic venoms, respectively. The authors present a study with satisfactory quality in methodology and data generation. However the manuscript has an inventory of toxins and immunoreactive proteins without coherence with the scientific literature and poor consistency with some cited references. Furthermore, in many topics of the manuscript the authors present a lot of information without any reference. For example, the authors don't compare the results regarding 2D immunoblotting with any previously study. I suggest that this manuscript should be reword to improve the quality about the data presentation.

Response: We thank the reviewer for suggestion. We rewrote introduction and discussion to improve data presentation. Relevant references were cited in every part to enhance literature coherence. Biological and geographical details regarding all snakes were also provided in materials and methods (Table 1; page 5, line 105).

- Abstract

Line 33 ..... venoms was investigated by indirect ELISA..... Line 37.....were subjected to 2D immunoblotting

These related methodologies would be more appropriate if they were introduced together.

Response: We edited an abstract according to reviewer’s suggestion.

- Introduction

. Line 46-50 - The geographical distribution data deserve references. The same for the sentences introducing biological data about pit viper from lines 51-58.... length of 110 cm.

Lines 59: Incidence of pit vipers bites, including those from mountain pit vipers, have been documented in their known ranges???.

The lack of references should be revised in all manuscript...

Response : We rewrote introduction with relevant references cited in a revised version of manuscript; ref no.6 and 8).

-Materials and methods

Line 92 : Snake venom,

- Are there more information about the venom's sample used in this study? Number of specimens? Localities? size/age...

Response: Biological and geographical information regarding all snakes is provided as a table in materials and methods (Table 1; page 5 line 105).

Line 121: ...a non-linear immobilized pH gradient (IPG) strip (pH 3-10 Amersham Bioscience, USA)....

- Why the authors choose the non-linear gradient instead of linear gradient, since the propose were excised immunoreactive spots for mass spectrometric analysis? Just for curious

Response: According to our previous experiments, we found that the non-linear immobilized pH gradient (IPG) could provide the well separation of protein in venoms. Therefore, we chose the NL strip for this research.

- Lack references in almost all methods. The references for 2D immunoblotting is required.

Response: In a revised materials and methods of manuscripts, we provided relevant references for all methods used. For 2DE immunoblotting, a reference is doi: 10.3390/toxins6051526 as reference page 7, line 137 (ref no.14)

Results

Line 208: Change Ovophis / Ovophis sp or genera Ovophis

- Are there any published information about neutralize activity of monovalent and polyvalent antivenoms against O. monticola venom? This information should be provided or I suggest that an experiment to determine the lethal dose 50% should be done.

Table 2. List of identified proteins ... immunologically reacted ...

- If the MW of the spots content reacted/ non-reacted proteins could be provided, this information can improve the quality table.

Response: We added a column of MW of proteins in a revised Table 2. In this particular study, we focused mainly on providing the inventory of all protein constituents of Asian mountain pit viper O. monticola venom. Those with antigenic properties reacted with monovalent and hematotoxic polyvalent antivenom were given. However, we did not perform neutralizing experiments.

Discussion

The Authors should revise the references from the first paragraph.... For example: the statement about hematotoxic potential of SVMP, SVSP and PLA2 is already a scientific consensus. Therefore, there are many sources specially from the seminal studies that corroborate with this question. However, the Authors present references from venom proteomic analysis of other snakes to support this question. On the other hand, the authors present coherency in the literature proposed as reference to address the mechanism of action of P-III metalloproteinase. I suggest that his paragraph must be reworded.

Line 208: The presence of class P-II metalloproteinase, which possesses metalloproteinase and disintegrin-like domians was also observed.????

Line 303: The combination of SVMP and SVSP observed in O. monticola venom is responsible for prey attack, incapacitation, and digestion.???

Response: We thank the reviewer for comments on discussion. We rewrote discussion by updating and improving literature coherence.

Reviewer #2:

It is an interesting work to be published in PLOS. This article describes describe proteomics and immuno characterization of snake Ovophis monticola venom. I have the following comments that the authors may like to consider:

Line 95

- Provide the total number of individuals, age, sex, locality (adults or young people, males, females)

- It is important to provide some functional analysis

Response: Biological and geographical information regarding all snakes is provided as a table in materials and methods (Table 1; page 5 line 105). In this particular study, we focused mainly on providing the inventory of all protein constituents of Asian mountain pit viper O. monticola venom. Those with antigenic properties reacted with monovalent and hematotoxic polyvalent antivenom were given. However, we did not perform the experiments to analyse the functions.

Discussion:

Despite little published literature on Ovophis monticola:

Please, include in the discussion this reference:

Pandey, D. P., Chaudhary, B., & Ram Shrestha, B. (2021). Documentation of a proven Mountain Pitviper (Ovophis monticola) envenomation in Kathmandu, Nepal, with its distribution ranges: implications for prevention and control of pitviper bites in Asia. Journal of venom research, 11, 1–6.

Mainly about clinical characteristic of poisoning by this specie and lack of specific treatment.

Provide conclusion or future perspectives in the last paragraphy about immunocharacterization results/ envenomation treatment.

Line 527

Exclude the number 75.

Response: We cited the above literature as a reference in a revised version of manuscript (ref no.5).

Submitted filename: Response to reviewers.docx

Click here for additional data file.

6 Oct 2021

PONE-D-21-18686R1Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venomsPLOS ONE

Dear Dr. Kumkate,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Nov 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Rafael Ximenes

Academic Editor

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Based on the new round of peer-round some minor concerns have arisen. I recommend a minor revision before acceptance of the manuscript.

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: No

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors rewrote the manuscript and provide relevant references and endeavored to comply with all referee requests.I just emphasize that only Ovophis don't belong to a taxonomic system. This task dont were revide for authors. However. The authors present a study with high quality in methodology, data generation and data analysis, producing significant contribution for toxinology field. Thus, this work is suitable for publication on PLOS ONE.

Reviewer #3: The article highlights the spectrometry-based proteomics and the immunoreactivity of the venom of the Ovophis monticola viper. It is a work based on an excellent premise and the methodologies are very well defined. I recommend the work for publication. However, some minor revisions are needed.

1. In the session "Two-dimensional polyacrylamide gel electrophoresis (2DE)" (Material and Methods), the authors state that a 100 µg sample of venom was used. However, since the protein concentrations were measured using the Lowry method, please review whether these 100 µg are venom or protein.

2. In the session “Proteomic analysis of O. monticola venom” (Results), the first paragraph deals with a methodological aspect.

3. The same observation applies to the following topics. Instead of repeating the concepts of the methodology, the objective could be presented if evaluating that result, since this information is not present in the text.

4. In “Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenoms by indirect ELISA”, the information “Since there is no homospecific antivenom to Ovophis venoms currently available, all pit viper envenoming victims usually receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) to alleviate symptoms” is applicable to the discussion, requiring references.

5. Figures and tables should be self-explanatory. In the case of table 2, a legend stating the meaning of MW, PI, emPI must be provided.

6. Discussion: What is the toxinological consequence related to the fact that the venom has more acidic than basic protein spots? How does this interfere with symptoms related to accident? Is this positive or negative regarding the immunoreactivity of the venom, especially with the polyvalent serum? Present references.

7. Why are some references in numerical format (ex (22) (23)) and others are in nominal format (ex Damm et al, 2021)?

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Reviewer #1: Yes: Carlos Correa-Netto

Reviewer #3: No

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9 Nov 2021

Additional Editor Comments (if provided):

Based on the new round of peer-round some minor concerns have arisen. I recommend a minor revision before acceptance of the manuscript.

Reviewers' comments:

Reviewer #1: The authors rewrote the manuscript and provide relevant references and endeavored to comply with all referee requests. I just emphasize that only Ovophis don't belong to a taxonomic system. This task dont were revide for authors. However. The authors present a study with high quality in methodology, data generation and data analysis, producing significant contribution for toxinology field. Thus, this work is suitable for publication on PLOS ONE.

Response: We corrected Ovophis to Ovophis spp. as suggested by the reviewer (e.g. lines 68, 83, 91, 288, 305, 313, 396)

Reviewer #3: The article highlights the spectrometry-based proteomics and the immunoreactivity of the venom of the Ovophis monticola viper. It is a work based on an excellent premise and the methodologies are very well defined. I recommend the work for publication. However, some minor revisions are needed.

1. In the session "Two-dimensional polyacrylamide gel electrophoresis (2DE)" (Material and Methods), the authors state that a 100 µg sample of venom was used. However, since the protein concentrations were measured using the Lowry method, please review whether these 100 µg are venom or protein.

Response: : It was 100 �g of protein. We corrected the sentence in line 135.

2. In the session “Proteomic analysis of O. monticola venom” (Results), the first paragraph deals with a methodological aspect.

Response: We rewrote this paragraph according to the reviewer’s suggestion.

3. The same observation applies to the following topics. Instead of repeating the concepts of the methodology, the objective could be presented if evaluating that result, since this information is not present in the text.

Response: We revised result session and eliminated repetition particularly on methodology.

4. In “Immunoreactivity of protein antigens in O. monticola venom to monovalent and polyvalent antivenoms by indirect ELISA”, the information “Since there is no homospecific antivenom to Ovophis venoms currently available, all pit viper envenoming victims usually receive either monovalent antivenom (raised against T. albolabris venom) or hematotoxic polyvalent antivenom (produced against venoms of C. rhodostoma, D. siamensis and T. albolabris) to alleviate symptoms” is applicable to the discussion, requiring references.

Response: We clarified this point by providing a reference (line 232, ref no. 16 )

5. Figures and tables should be self-explanatory. In the case of table 2, a legend stating the meaning of MW, PI, emPI must be provided.

Response: We added the explanation in the table 2 legend (lines 238-241, 244-246) and the definition of abbreviation is provided as in a legend as suggested.

6. Discussion: What is the toxinological consequence related to the fact that the venom has more acidic than basic protein spots? How does this interfere with symptoms related to accident? Is this positive or negative regarding the immunoreactivity of the venom, especially with the polyvalent serum? Present references.

Response: In this revised version of manuscript, we discussed the above points raised by the reviewer with relevant references, lines 323-327, 391-395. In addition, to make table 4 more readable, we added a column of MW of non-immunoreactive peptides.

7. Why are some references in numerical format (ex (22) (23)) and others are in nominal format (ex Damm et al, 2021)?

Response: We corrected all references appeared in text in numerical format according to Plos guideline.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

11 Nov 2021

Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venom

PONE-D-21-18686R2

Dear Dr. Kumkate,

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Rafael Ximenes

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

19 Nov 2021

PONE-D-21-18686R2

Proteomics and immunocharacterization of Asian Mountain pit viper (Ovophis  monticola) venom

Dear Dr. Kumkate:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Rafael Ximenes

Academic Editor

PLOS ONE