Proteomics and antivenom immunoprofiling of Russell’s viper (<i>Daboia siamensis</i>) venoms from Thailand and Indonesia

Lingam, Thava Malar Changra; Tan, Kae Yi; Tan, Choo Hock

doi:10.1590/1678-9199-JVATITD-2019-0048

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Journal of Venomous Animals and Toxins including Tropical Diseases

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Research • J. Venom. Anim. Toxins incl. Trop. Dis 26 • 2020 • https://doi.org/10.1590/1678-9199-JVATITD-2019-0048 copy

Proteomics and antivenom immunoprofiling of Russell’s viper (Daboia siamensis) venoms from Thailand and Indonesia

Authorship SCIMAGO INSTITUTIONS RANKINGS

Background

The Eastern Russell’s viper, Daboia siamensis, is a WHO Category 1 medically important venomous snake. It has a wide but disjunct distribution in Southeast Asia. The specific antivenom, D. siamensis Monovalent Antivenom (DsMAV-Thailand) is produced in Thailand but not available in Indonesia, where a heterologous trivalent antivenom, Serum Anti Bisa Ular (SABU), is used instead. This study aimed to investigate the geographical venom variation of D. siamensis from Thailand (Ds-Thailand) and Indonesia (Ds-Indonesia), and the immunorecognition of the venom proteins by antivenoms.

Methods:

The venom proteins were decomplexed with reverse-phase high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by in-solution tryptic digestion, nano-liquid chromatography-tandem mass spectrometry and protein identification. The efficacies of DsMAV-Thailand and SABU in binding the various venom fractions were assessed using an enzyme-linked immunosorbent assay optimized for immunorecognition profiling.

Results:

The two most abundant protein families in Ds-Thailand venom are phospholipase A₂ (PLA₂) and Kunitz-type serine protease inhibitor (KSPI). Those abundant in Ds-Indonesia venom are PLA₂ and serine protease. KSPI and vascular endothelial growth factor were detected in Ds-Thailand venom, whereas L-amino acid oxidase and disintegrin were present in Ds-Indonesia venom. Common proteins shared between the two included snaclecs, serine proteases, metalloproteinases, phosphodiesterases, 5’nucleotidases and nerve growth factors at varying abundances. DsMAV-Thailand exhibited strong immunorecognition of the major protein fractions in both venoms, but low immunoreactivity toward the low molecular weight proteins e.g. KSPI and disintegrins. On the other hand, SABU was virtually ineffective in binding all fractionated venom proteins.

Conclusion:

D. siamensis venoms from Thailand and Indonesia varied geographically in the protein subtypes and abundances. The venoms, nevertheless, shared conserved antigenicity that allowed effective immunorecognition by DsMAV-Thailand but not by SABU, consistent with the neutralization efficacy of the antivenoms. A specific, appropriate antivenom is needed in Indonesia to treat Russell’s viper envenomation.

Keywords:
Venomics; Antivenomics; Eastern Russell's viper; Neutralization

Frac.	Protein score	Protein name*	Accession number^#	Species	Relative abundance (%)
4					9.83%
	192.8	Kunitz-type serine protease inhibitor C1	A8Y7N4	D. siamensis	7.37
	52.94	Kunitz-type serine protease inhibitor C6	A8Y7N9	D. siamensis	0.54
	38.91	Kunitz-type serine protease inhibitor B6	A8Y7P6	D. siamensis	1.92
5					9.14%
	67.75	Kunitz-type serine protease inhibitor C1	A8Y7N4	D. siamensis	0.65
	85.22	Kunitz-type serine protease inhibitor C2	A8Y7N5	D. siamensis	0.54
	48.9	Kunitz-type serine protease inhibitor C4	A8Y7N7	D. siamensis	2.59
	30.32	Kunitz-type serine protease inhibitor C6	A8Y7N9	D. siamensis	4.29
	85.09	Kunitz-type serine protease inhibitor DrKIn-II	H6VC06	D. russelii	0.25
	72.37	Kunitz-type serine protease inhibitor 2	P00990	D. siamensis	0.82
6					3.14%
	63.41	Kunitz-type serine protease inhibitor C1	A8Y7N4	D. siamensis	0.20
	70.78	Kunitz-type serine protease inhibitor C2	A8Y7N5	D. siamensis	0.08
	52.04	Kunitz-type serine protease inhibitor C4	A8Y7N7	D. siamensis	1.10
	29.22	Kunitz-type serine protease inhibitor C6	A8Y7N9	D. siamensis	0.20
	31.54	Kunitz-type serine protease inhibitor B5	A8Y7P5	D. siamensis	0.66
	39.97	Kunitz-type serine protease inhibitor DrKIn-II	H6VC06	D. russelii	0.07
	63.82	Kunitz-type serine protease inhibitor 2	P00990	D. siamensis	0.59
	34.41	Coagulation factor X-activating enzyme heavy chain	Q7LZ61	D. siamensis	0.23
7					0.92%
	44.81	Kunitz-type serine protease inhibitor C2	A8Y7N5	D. siamensis	0.07
	31.75	Kunitz-type serine protease inhibitor DrKIn-II	H6VC06	D. russelii	0.04
	47.6	Snake venom vascular endothelial growth factor toxin VR-1	P67861	D. russelii	0.39
	79.52	Venom nerve growth factor 1	V9I1K1	D. russelii	0.43
8					3.19%
	46.88	Kunitz-type serine protease inhibitor C1	A8Y7N4	D. siamensis	0.10
	45.33	Kunitz-type serine protease inhibitor C2	A8Y7N5	D. siamensis	0.05
	36.24	Kunitz-type serine protease inhibitor C4	A8Y7N7	D. siamensis	0.05
	56.41	Phospholipase A₂	B2YHV5	D. siamensis	0.24
	53.05	Basic phospholipase A₂ beta-bungarotoxin A1 chain	P00617	B. multicinctus	0.05
	50.79	Basic phospholipase A₂ beta-bungarotoxin A1 chain	P00617	B. multicinctus	0.05
	35.5	Snake venom vascular endothelial growth factor toxin VR-1'	P0DL42	D. siamensis	2.64
9					26.23%
	174.64	Basic phospholipase A₂	B3RFI7	D. r. limitis	12.63
	43.61	Kunitz-type serine protease inhibitor C1	A8Y7N4	D. siamensis	0.10
	21.86	Kunitz-type serine protease inhibitor C2	A8Y7N5	D. siamensis	0.06
	65.41	Phospholipase A₂	B2YHV5	D. siamensis	0.88
	52.49	Beta-fibrinogenase	E0Y419	M. lebetina	1.38
	52.09	Serine protease VLSP-3	E0Y420	M. lebetina	4.22
	32.37	Beta-fibrinogenase-like	E5L0E4	D. siamensis	3.10
	24.5	Snake venom vascular endothelial growth factor toxin VR-1	P67861	D. russelii	2.00
	56.87	Venom serine proteinase-like protein 2	Q9PT40	M. lebetina	1.75
	35.87	Venom nerve growth factor 1	V9I1K1	D. russelii	0.09
10					23.34%
	36.23	Beta-fibrinogenase-like	E5L0E4	D. siamensis	0.12
	133.44	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	11.00
	195.54	Acidic phospholipase A2 RV-7	P31100	D. siamensis	9.52
	30.91	Phospholipase A₂ 1	P86529	D. russelii	2.69
11					4.17%
	94.05	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.25
	291.13	Factor V activator RVV-V gamma	P18965	D. siamensis	1.95
	274.2	Factor V activator RVV-V gamma	P18965	D. siamensis	1.86
	58.76	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.12
12					0.54%
	37.01	Beta-fibrinogenase	E0Y419	M. lebetina	0.02
	29.97	Serine protease VLSP-3	E0Y420	M. lebetina	0.02
	72.73	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.03
	244.98	RVV-V gamma-like protein precursor	P18965	D. siamensis	0.10
	181.46	Factor V activator RVV-V gamma	P18965	D. siamensis	0.14
	84.44	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.09
	37.42	Venom serine proteinase-like protein 2	Q9PT40	M. lebetina	0.01
	78.54	Serine beta-fibrinogenase	CL2958.Contig2_DrSL	D. russelii	0.13
13					1.17%
	42	Kunitz-type serine protease inhibitor C1	A8Y7N4	D. siamensis	0.002
	25.6	Kunitz-type serine protease inhibitor B6	A8Y7P6	D. siamensis	0.01
	34.12	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.31
	153.15	Factor V activator RVV-V gamma	P18965	D. siamensis	0.28
	133.87	Factor V activator RVV-V gamma	P18965	D. siamensis	0.23
	75.58	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.03
	46.83	Factor V activator RVV-V gamma	Unigene26743_DrSL	D. russelii	0.32
14					1.48%
	37.58	Serine protease VLSP-3	E0Y420	M. lebetina	0.03
	192.24	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.29
	32.79	P31 alpha subunit	K9JBU9	D. siamensis	0.02
	146.91	Factor V activator RVV-V gamma	P18965	D. siamensis	0.18
	134.03	Factor V activator RVV-V gamma	P18965	D. siamensis	0.13
	86.12	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.12
	21.67	Snake venom vascular endothelial growth factor toxin VR-1	P67861	D. russelii	0.07
	51.59	Basic phospholipase A₂ RV-4	Q02471	D. siamensis	0.10
	48.78	Snaclec 4	Q4PRC9	D. siamensis	0.02
	68.15	Serine beta-fibrinogenase	CL2958.Contig2_DrSL	D. russelii	0.16
	115.48	RVV-V gamma-like protein precursor	CL310.Contig22_NnSL	N. naja	0.23
	41.42	Serine protease VLSP-1	CL310.Contig24_NnSL	N. naja	0.13
15					5.17%
	25.78	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.02
	28.57	P31 beta subunit	K9JBV3	D. r. limitis	0.35
	32.42	P31 beta subunit	K9JDF6	D. siamensis	0.23
	85.94	Factor V activator RVV-V gamma	P18965	D. siamensis	0.13
	96.25	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.33
	58.85	Snaclec coagulation factor X-activating enzyme light chain 1	Q4PRD1	D. siamensis	0.21
	120.28	Coagulation factor X-activating enzyme heavy chain	Q7LZ61	D. siamensis	0.12
	31.13	P31 alpha subunit	CL1101.Contig2_DrSL	D. russelii	3.54
	41.97	P68 alpha subunit	CL2900.Contig2_NnSL	N. naja	0.03
	56.67	Serine protease VLSP-1	CL2958.Contig2_DrSL	D. russelii	0.07
	33.15	Serine protease VLSP-1	CL310.Contig24_NnSL	N. naja	0.13
16					11.68%
	82.86	RVV-X light chain 1	A0A2H4Z2X7	D. siamensis	1.35
	35.54	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.04
	36.29	P31 alpha subunit	K9JBU9	D. siamensis	0.17
	41.85	P68 alpha subunit	K9JBV0	D. siamensis	1.01
	95.46	Factor X activator light chain 2	K9JDJ1	D. siamensis	0.25
	21.91	Snake venom vascular endothelial growth factor toxin VR-1'	P0DL42	D. siamensis	0.32
	54.47	Factor V activator RVV-V gamma	P18965	D. siamensis	0.11
	43.48	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.08
	72.99	Snaclec 3	Q4PRD0	D. siamensis	0.32
	129.18	Snaclec coagulation factor X-activating enzyme light chain 1	Q4PRD1	D. siamensis	1.17
	48.29	Snaclec coagulation factor X-activating enzyme light chain 2	Q4PRD2	D. siamensis	0.38
	193.33	Coagulation factor X-activating enzyme heavy chain	Q7LZ61	D. siamensis	1.32
	172.13	Coagulation factor X-activating enzyme heavy chain	Q7LZ61	D. siamensis	1.37
	115.48	5'-nucleotidase	U3T7C6	O. okinavensis	0.54
	118.19	Phosphodiesterase	U3TBJ5	O. okinavensis	0.08
	229.87	5'-nucleotidase	W8EFS0	M. ebetina	0.39
	58.32	Factor X activator light chain 1	CL290.Contig28_NnSL	N. naja	1.58
	199.35	Snake venom 5'-nucleotidase OS	CL2941.Contig2_EcSL	E. carinatus	0.53
	49.82	Serine beta-fibrinogenase	CL2958.Contig2_DrSL	D. russelii	0.05
	71.65	RVV-V gamma-like protein precursor	CL310.Contig22_NnSL	N. naja	0.08
	29.49	Serine protease VLSP-1	CL310.Contig24_NnSL	N. naja	0.05
	288.98	Snake venom 5'-nucleotidase	CL3322.Contig2_DrSL	D. russelii	0.39
	297.24	Phosphodiesterase 1	CL3655.Contig2_DrSL	D. russelii	0.08

Frac.	Protein score	Protein name*	Accession number^#	Species	Relative abundance (%)
4					6.21%
	20.01	Disintegrin jerdostatin	Q7ZZM2	P. jerdonii	6.21
5					0.85%
	66.21	Venom nerve growth factor 1	V9I1K1	D. russelii	0.85
6		(Unidentified)			7.47%
7					32.61%
	119.66	Basic phospholipase A₂	B3RFI7	D. r. limitis	21.25
	56.61	Venom serine proteinase-like protein 2	Q9PT40	M. lebetina	11.37
8					31.47%
	76.93	Acidic phospholipase A₂ Drk-a1	A8CG86	D. russelii	4.95
	50.37	Basic phospholipase A₂	B3RFI7	D. r. limitis	0.17
	35.19	Serine protease VLSP-3	E0Y420	M. lebetina	0.13
	56.33	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.12
	38.5	Beta-fibrinogenase-like	E5L0E4	D. siamensis	0.18
	224.61	Factor V activator RVV-V gamma	P18965	D. siamensis	2.03
	182.85	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	16.83
	32.28	Phospholipase A₂ 1	P86529	D. russelii	4.89
	217.36	RVV-V gamma-like protein precursor Daboia-russelii	CL29580.Contig2_DrSL	D. russelii	2.18
9					9.06%
	21.24	Serine protease VLSP-3	E0Y420	M. lebetina	4.53
	92.33	Alpha-fibrinogenase-like	E5L0E3	D. siamensis	0.35
	168.8	Factor V activator RVV-V gamma	P18965	D. siamensis	1.48
	171.75	Factor V activator RVV-V gamma	P18965	D. siamensis	0.92
	36	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.24
	74.09	Factor V activator RVV-V gamma	Unigene26743_EsM	E. schistosa	1.54
10					2.78%
	45.57	Factor V activator RVV-V gamma	P18965	D. siamensis	0.08
	45.95	Acidic phospholipase A₂ RV-7	P31100	D. siamensis	0.05
	74.97	Snaclec coagulation factor X-activating enzyme light chain 1	Q4PRD1	D. siamensis	0.21
	43.43	Snaclec coagulation factor X-activating enzyme light chain 2	Q4PRD2	D. siamensis	0.29
	40.23	Factor X activator light chain 2	T1P647	D. russelii	0.32
	50.26	Serine protease VLSP-1	CL29580.Contig2_DrSL	D. russelii	1.79
	64.24	RVV-V gamma-like protein precursor	CL3102.Contig2_NnSL	N. naja	0.04
11		(Unidentified)			8.97%
12					4.90%
	47.46	Coagulation factor X-activating enzyme heavy chain	Q7LZ61	D. siamensis	1.83
	142.43	L-amino-acid oxidase	G8XQX1	D. russelii	0.53
	96.77	P68 alpha subunit	K9JDF2	D. r. limitis	0.44
	71.25	L-amino-acid oxidase	P0C2D7	V. berus berus	0.94
	53.81	Snaclec 3	Q4PRD0	D. siamensis	0.09
	96.88	Phosphodiesterase	U3TBJ5	O. okinavensis	0.60
	113.5	Snake venom 5'-nucleotidase	CL3322.Contig21_DrSL	D. russelii	0.18
	305.76	Phosphodiesterase 1	CL3655.Contig2_DrSL	D. russelii	0.29

Protein family and subtype	Accession number	Relative abundance (% total venom protein)
Protein family and subtype	Accession number	D. siamensis (Thailand)	D. siamensis (Indonesia)
Kunitz-type serine protease inhibitor (KSPI)		22.38	-
Kunitz-type serine protease inhibitor 2	P00990	1.4	-
Kunitz-type serine protease inhibitor B5	A8Y7P5	0.66	-
Kunitz-type serine protease inhibitor B6	A8Y7P6	1.93	-
Kunitz-type serine protease inhibitor C1	A8Y7N4	8.43	-
Kunitz-type serine protease inhibitor C2	A8Y7N5	0.81	-
Kunitz-type serine protease inhibitor C4	A8Y7N7	3.75	-
Kunitz-type serine protease inhibitor C6	A8Y7N9	5.04	-
Kunitz-type serine protease inhibitor DrKIn-II	H6VC06	0.36	-
Phospholipase A₂ (PLA₂)		37.92	48.37
Acidic phospholipase A₂ Drk-a1	A8CG86	-	4.95
Acidic phospholipase A₂ RV-7	P31100	21.27	17.12
Basic phospholipase A₂	B3RFI7	12.63	21.41
Basic phospholipase A2 beta-bungarotoxin A1 chain	P00617	0.1	-
Basic phospholipase A₂ RV-4	Q02471	0.1	-
Phospholipase A2	B2YHV5	1.13	-
Phospholipase A2 1	P86529	2.69	4.89
Snake venom C-type lectin/lectin-like protein (snaclec)	10.63	1.03
Factor X activator light chain 1	CL290.Contig28_NnSL	1.58	-
Factor X activator light chain 2	K9JDJ1	0.25	-
P31 alpha subunit	K9JBU9	0.19	-
P31 alpha subunit	CL1101.Contig2_DrSL	3.54	-
P31 beta subunit	K9JBV3	0.35	-
P31 beta subunit	K9JDF6	0.23	-
P68 alpha subunit	K9JBV0	1.01	-
P68 alpha subunit	K9JDF2	-	0.44
P68 alpha subunit	CL2900.Contig2_NnSL	0.03	-
RVV-X light chain 1	A0A2H4Z2X7	1.35	-
Snaclec 3	Q4PRD0	0.32	0.09
Snaclec 4	Q4PRC9	0.02
Snaclec coagulation factor X-activating enzyme light chain 1	Q4PRD1	1.38	0.21
Snaclec coagulation factor X-activating enzyme light chain 2	Q4PRD2	0.38	0.29
Snake venom serine protease (SVSP)		18.07	22.41
Alpha-fibrinogenase-like	E5L0E3	0.95	0.47
Beta-fibrinogenase	E0Y419	1.41	-
Beta-fibrinogenase-like	E5L0E4	3.22	0.18
Factor V activator RVV-V gamma	P18965	5.11	4.5
Factor V activator RVV-V gamma	Unigene26743_DrSL	0.32	-
Factor V activator RVV-V gamma	Unigene26743_EsM	-	1.54
RVV-V gamma-like protein precursor	CL3102.Contig2_NnSL	0.31	0.04
Serine beta-fibrinogenase	CL2958.Contig2_DrSL	0.4	3.97
Serine protease VLSP-1	CL310.Contig24_NnSL	0.32	-
Serine protease VLSP-3	E0Y420	4.27	0.34
Venom serine proteinase-like protein 2	Q9PT40	1.76	11.37
Snake venom metalloproteinase (SVMP)		3.04	2.15
Coagulation factor X-activating enzyme heavy chain	Q7LZ61	3.04	1.83
Factor X light activator chain 2	T1P647	-	0.32
L-amino acid oxidase (LAAO)		-	1.47
L-amino-acid oxidase	G8XQX1	-	0.53
L-amino-acid oxidase	P0C2D7	-	0.94
Snake venom vascular endothelial growth factor (svVEGF)	5.42
Snake venom vascular endothelial growth factor toxin VR-1	P67861	2.46	-
Snake venom vascular endothelial growth factor toxin VR-1'	P0DL42	2.96	-
Snake venom nerve growth factor (svNGF)		0.52	0.85
Venom nerve growth factor 1	V9I1K1	0.52	0.85
Snake venom 5′-nucleotidase (5′NUC)		1.85	0.18
5’-nucleotidase	U3T7C6	0.54	-
5'-nucleotidase	W8EFS0	0.39	-
Snake venom 5’-nucleotidase	CL3322.Contig1_DrSL	-	0.18
Snake venom 5'-nucleotidase	CL3322.Contig2_DrSL	0.39	-
Snake venom 5'-nucleotidase OS	CL2941.Contig2_EcSL	0.53	-
Phosphodiesterase (PDE)		0.16	0.89
Phosphodiesterase	U3TBJ5	0.08	0.6
Phosphodiesterase 1	CL3655.contig2_DrSL	0.08	0.29
Disintegrin		-	6.21
Disintegrin jerdostatin	Q7ZZM2	-	6.21
Unidentified proteins	-	-	16.44

Species and locale	D. siamensis (Thailand - pooled sample)	D. siamensis (Java, Indonesia - pooled sample)	D. siamensis (Insular Taiwan - pooled sample)	D. siamensis (Taiwan - captive - polled sample)	D. siamensis (Guangxi, China - n ?#61619; 10 adults)	D. siamensis (Myanmar - pooled sample)	D. russelii (Western, India - n = 4 adults)	D. russelii (Tamil Nadu, India - pooled sample)	D. russelii (Pakistan - wild - n ?#61619;10 adults)	D. russelii (Pakistan - captive - n = 3 adults)	D. russelii (Sri Lanka - n ?#61619;5 adults)
LD₅₀ (µg/g mouse)	0.34 (0.30-0.38) (i.v.)	0.22 (0.20-0.24) (i.v.)	0.09 (0.06-0.14) (i.v.)	0.47 (i.p.)	0.18 (0.12-0.27) (i.v.)	NA	NA	NA	0.19 (0.17-0.25) (i.v.)	NA	NA
Reference	Current study	Current study	[1818. Tan KY, Tan NH, Tan CH. Venom proteomics and antivenom neutralization for the Chinese eastern Russell's viper, Daboia siamensis from Guangxi and Taiwan. Sci Rep. 2018;8(1):8545.]	[1717. Sanz L, Quesada-Bernat S, Chen PY, Lee CD, Chiang JR, Calvete JJ. Translational venomics: third-generation antivenomics of anti-siamese Russell's viper, Daboia siamensis, antivenom manufactured in Taiwan CDC's Vaccine Center. Trop Med Infect Dis. 2018 Jun 15;3(2):pii: E66. ]	[1818. Tan KY, Tan NH, Tan CH. Venom proteomics and antivenom neutralization for the Chinese eastern Russell's viper, Daboia siamensis from Guangxi and Taiwan. Sci Rep. 2018;8(1):8545.]	[1919. Risch M, Georgieva D, von Bergen M, JehmLich N, Genov N, Arni RK, et al. Snake venomics of the Siamese Russell's viper (Daboia russelli siamensis) -- relation to pharmacological activities. J Proteomics. 2009 Mar 6;72(2):256-69. ]	[66. Kalita B, Patra A, Mukherjee AK. Unraveling the proteome composition and immuno-profiling of Western India Russell’s viper venom for in-depth understanding of its pharmacological properties, clinical manifestations, and effective antivenom treatment. J Proteome Res. 2017 Feb 3;16(2):583-98.]	[3434. Sharma M, Das D, Iyer JK, Kini RM, Doley R. Unveiling the complexities of Daboia russelii venom, a medically important snake of India, by tandem mass spectrometry. Toxicon. 2015 Dec 1;107(Pt B):266-81.]	[55. Faisal T, Tan KY, Sim SM, Quraishi N, Tan NH, Tan CH. Proteomics, functional characterization and antivenom neutralization of the venom of Pakistani Russell's viper (Daboia russelii) from the wild. J Proteomics. 2018 Jul 15;183:1-13.]	[77. Mukherjee AK, Kalita B, Mackessy SP. A proteomic analysis of Pakistan Daboia russelii russelii venom and assessment of potency of Indian polyvalent and monovalent antivenom. J Proteomics. 2016 Jul 20;144:73-86.]	[88. Tan NH, Fung SY, Tan KY, Yap MKK, Gnanathasan CA, Tan CH. Functional venomics of the Sri Lankan Russell's viper (Daboia russelii) and its toxinological correlations. J Proteomics. 2015 Oct 14;128:403-23.]
Method	C18 rpHPLC, in-solution tryptic digestion, LC-MS/MS	C18 rpHPLC, in-solution tryptic digestion, LC-MS/MS	In-gel tryptic digestion, LC-MS/MS	C18 rpHPLC, in-gel tryptic digestion, LC-MS/MS	In-gel tryptic digestion, LC-MS/MS	2D gel, tryptic digestion	Gel filtration chromatography, in-solution tryptic digestion, LC-MS/MS	C18 rpHPLC, in-gel tryptic digestion, LC-MS/MS	Gel filtration chromatography, in-solution tryptic digestion, LC-MS/MS	C18 rpHPLC, in-solution tryptic digestion, LC-MS/MS	In-gel tryptic digestion, LC-MS/MS
KSPI	22.38	-	28.20	9.4	23.17	-	12.50	P	16.00	28.4	4.70
PLA₂	37.92	48.37	24.47	47.5	22.18	P	32.50	P	63.80	32.8	35.00
Acidic	25.09	26.96	10.87	21.2	14.76	P	P	P	21.51	P	34.7
Basic	12.83	21.41	13.61	26.3	7.42	P	P	P	42.30	P	0.3
Neutral	-	-	-	-	-	-	-	-	-	-	-
Snaclec	10.64	1.03	16.52	5.2	16.89	P	1.80	P	1.30	6.4	22.40
SVSP	18.07	22.41	17.5	19.1	13.61	P	8.00	P	5.50	3.2	16.00
SVMP	3.04	2.15	5.85	9.2	8.92	P	24.80	P	2.50	21.8	6.90
LAAO	-	1.47	-	-	5. 95	P	0.30	P	0.80	0.6	5.20
svNGF	0.52	0.85	2.13	0.3	2.11	-	4.80	P	1.10	0.4	3.42
svVEGF	5.42	-	4.84	7.2	4.79	P	1.80	P	4.30	1.5	-
5’NUC	1.85	0.18	0.17	-	0.82	-	0.40	P	0.10	0.5	3.00
CRiSP	-	-	-	0.1	0.95	-	6.80	P	1.30	2.6	2.00
PDE	0.16	0.89	0.31	0.9	0.25	-	1.40	P	2.50	0.6	1.30
Disintegrin	-	6.21	-	-	-	-	4.90	P	P	0.4	-
Aminopeptidase		-	0.15	-	0.35	-	-	-	-	-	-
PLB	-	-	-	-	-	-	0.10	-	-	-	-
HLP	-	-	-	-	-	-	-	P	-	-	-
Unknown	-	16.44	-	-	-	-	-	-	-	-	-

Centro de Estudos de Venenos e Animais Peçonhentos (CEVAP/UNESP) Av. Universitária, 3780, Fazenda Lageado, Botucatu, SP, CEP 18610-034, Brasil, Tel.: +55 14 3880-7693 - Botucatu - SP - Brazil
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[1] *Correspondence to: tanch@um.edu.my