Quantitative Proteomic Analysis of Venoms from Russian Vipers of Pelias Group: Phospholipases A₂ are the Main Venom Components

Abstract

Venoms of most Russian viper species are poorly characterized. Here, by quantitative chromato-mass-spectrometry, we analyzed protein and peptide compositions of venoms from four Vipera species (V. kaznakovi, V. renardi, V. orlovi and V. nikolskii) inhabiting different regions of Russia. In all these species, the main components were phospholipases A₂, their content ranging from 24% in V. orlovi to 65% in V. nikolskii. Altogether, enzyme content in venom of V. nikolskii reached ~85%. Among the non-enzymatic proteins, the most abundant were disintegrins (14%) in the V. renardi venom, C-type lectin like (12.5%) in V. kaznakovi, cysteine-rich venom proteins (12%) in V. orlovi and venom endothelial growth factors (8%) in V. nikolskii. In total, 210 proteins and 512 endogenous peptides were identified in the four viper venoms. They represented 14 snake venom protein families, most of which were found in the venoms of Vipera snakes previously. However, phospholipase B and nucleotide degrading enzymes were reported here for the first time. Compositions of V. kaznakovi and V. orlovi venoms were described for the first time and showed the greatest similarity among the four venoms studied, which probably reflected close relationship between these species within the “kaznakovi” complex.

1. Introduction

Venomous snakes inhabit all continents of the globe except Antarctica. They are particularly abundant in tropical areas of Asia, Africa, South America and Australia. Russia, despite its large territory, is inhabited by only a small number of poisonous snake species, which belong to three genera: Gloydius, Macrovipera and Vipera. The Vipera genus is the most speciose in Russia and includes more than ten species, the systematics within this genus being constantly updated [1,2]. The most abundant species is common (or European) adder Vipera berus, which has a very large habitat in Russia, ranging from its western borders to Sakhalin and the Ussuri region. V. berus is also spread throughout Europe—between 68 and 45 degrees north latitude. The venom of this species is fairly well studied. Biological activities of this venom were characterized and proteolytic, fibrinolytic, anticoagulant, and phospholipolytic ones were demonstrated by in vitro experiments [3]. Several toxic proteins were isolated from V. berus venom, including phospholipase A₂ (PLA2) [4], metalloproteinase (SVMP) [5], l-amino acid oxidase (LAAO) [6] and several others. Recently, we have partially characterized the steppe viper V. renardi venom, the PLA2s and Kunitz type protease inhibitors were isolated from this venom and sequenced [7]. The isolated PLA2s were studied in more details and found to exert their action both on lipid membranes [8] and on nicotinic acetylcholine receptor [9]. The venom of Nikolsky’s viper was also partially characterized and several proteins including heterodimeric neurotoxic PLA2s were identified [10,11]. The venoms of other Russian viper species are characterized very poorly. Thus, for Caucasian viper V. kaznakovi and Orlov’s viper V. orlovi, only the toxicity of venoms to insects was determined [12]. Here, we used proteomic chromato-mass-spectrometry analysis to obtain more detailed information on the composition of Russian viper venoms.

Modern proteomic analysis allows both qualitative and quantitative characterization of the venom proteins, leading to suggestions about venom biological effects. So far, among Vipera genus, the venoms of only three species, i.e., V. ammodytes, V. anatolica and V. raddei, were thus studied [13,14,15]. Semi-quantitative venom analysis of V. anatolica showed that the most abundant toxin family was SVMPs (41.5%), followed by two cysteine-rich secretory protein (CRISP) isoforms (15.9%); other proteins represented less than 10% per family [13]. SVMPs (31.6%) were also the most abundant in V. raddei venom, followed by PLA2s (23.8%), and, again, the contents of other toxin families did not exceed 10% each [14]. There is no quantitative analysis of the V. ammodytes venom, however monomeric and heterodimeric Group II PLA2s; serine proteinases (SVSPs); Group I, II, and III SVMPs; l-amino acid oxidases (LAAOs); CRISPs; disintegrins (Dis); and growth factors were found [15]. On the whole, the above data indicate that the composition of different viper venoms might be different. It should also be noted that V. raddei in some publications is classified as Montivipera raddei and attributed to Montivipera genus [16], thus some differences might be attributed to the discrepancy in classification. Using quantitative proteomic, we have studied the venoms from four Vipera species (V. kaznakovi, V. nikolskii, V. orlovi and V. renardi) that inhabit different regions of Russia. In contrast to the venoms of earlier studied Vipera species where the SVMP were found to be predominant [13,14,15], we have observed that the main components of the venoms studied are PLA2s, the content of which ranged between 24 and 65%.

2. Results

2.1. Venom Proteins Identification

In this work, venom proteomes and peptidomes for four species of Vipera snakes were analyzed. Venom proteomes were analyzed by LC-MS/MS after in-solution trypsin proteolysis. In total, for the four Vipera species venoms, the search against the Serpentes database resulted in the identification of 210 proteins (

Table 1. List of proteins identified in Russian viper venoms.

Protein No. in SuppData	Protein Name	Taxon	Protein Family 1	MW, KDa	Seq Cov, %	V. nikolskii		V. kaznakovi		V. orlovi		V. renardi
						Prot Abun INT, %	Seq Cov, %	Prot Abun INT, %	Seq Cov, %	Prot Abun INT, %	Seq Cov, %	Prot Abun INT, %	Seq Cov, %
9	Natriuretic peptide	Pseudonaja textilis	B-NAP	14	9.8	0.007	9.8	0.029	9.8	0.012	9.8	-	-
2	Hemoglobin subunit alpha	Vipera aspis	BP	15	6.4	0	6.4	-	-	-	-	0	6.4
3	Hemoglobin subunit beta-2	Naja naja	BP	16	7.5	0.016	7.5	-	-	-	-	-	-
4	Hemoglobin subunit beta	Erythrolamprus miliaris	BP	15	23.3	0.018	19.2	-	-	-	-	0	11.6
33	Alpha globin	Hydrophis melanocephalus	BP	16	19.7	0.011	9.2	0	9.2	-	-	0.010	19.7
34	Alpha globin	Elaphe climacophora	BP	11	25.2	-	-	-	-	-	-	0.032	25.2
116	Murinoglobulin-2	Ophiophagus hannah	BP	143	2.7	-	-	-	-	0.008	1.8	0.002	2.2
124	Serum albumin	Protobothrops flavoviridis	BP	69	5.4	0.013	5.4	-	-	0.002	5.2	-	-
158	Hemoglobin subunit alpha	Hydrophis gracilis	BP	15	15.6	0.013	15.6	0.002	15.6	-	-	0.017	15.6
165	Hemoglobin subunit alpha	Crotalus horridus	BP	15	11.3	0.012	11.3	0.003	11.3	-	-	0.013	11.3
171	Hemoglobin subunit beta-1	Boiga irregularis	BP	16	21.8	0	21.8	-	-	-	-	0	21.8
177	Transferrin	Crotalus adamanteus	BP	77	3.1	0.006	3.1	-		0.004	3.1	0.009	3.1
199	Hemoglobin subunit beta-2	Thamnophis sirtalis	BP	16	24.5	0.021	18.4	-	-	-	-	0.020	12.9
200	Hemoglobin subunit beta-1	Thamnophis sirtalis	BP	16	19.7	0.002	19.7	-	-	-	-	-	-
206	Serum albumin-like	Thamnophis sirtalis	BP	57	8.0	0.012	8.0	-	-	-	-	-	-
180	Cathepsin B-like protein	Crotalus adamanteus	CP	37	5.0	0	5.0	-	-	-	-	-	-
7	Cysteine-rich venom protein	Philodryas patagoniensis	CRISP	1	64.3	-	-	0.037	64.3	0.002	64.3	-	-
18	Cysteine-rich seceretory protein Dr-CRPK	Daboia russelii	CRISP	26	24.7	-	-	0.170	24.7	0.107	21.8	-	-
19	Cysteine-rich seceretory protein Dr-CRPB	Daboia russelii	CRISP	25	17.6	-	-	0.033	17.6	0.006	15.8	0	17.6
89	Cysteine-rich venom protein	Protobothrops jerdonii	CRISP	26	22.5	-	-	0.148	22.5	0.149	22.5	-	-
90	Cysteine-rich venom protein	Vipera nikolskii	CRISP	24	83.3	-	-	0.112	77.4	0.050	80.1	0.116	68.3
91	Cysteine-rich venom protein	Vipera berus	CRISP	26	77	0.345	74.1	7.916	71.5	9.924	74.1	5.125	63.2
145	Cysteine-rich venom protein triflin	Protobothrops flavoviridis	CRISP	24	23.1	0.070	23.1	2.474	23.1	2.065	23.1	3.016	23.1
22	Snaclec 1	Sistrurus catenatus edwardsii	CTL	17	6.2	-	-	1.404	6.2	0.168	6.2	-	-
23	C-type lectin lectoxin-Thr1	Thrasops jacksonii	CTL	18	7.0	0.010	6.3	0.060	7.0	0.110	7	0.069	7
24	Snaclec A13	Macrovipera lebetina	CTL	15	33.6	-	-	2.042	29.0	2.555	33.6	-	-
25	Snaclec A15	Macrovipera lebetina	CTL	17	46.8	0.710	46.8	0.867	46.8	0.783	46.8	0.524	46.8
26	Snaclec B7	Macrovipera lebetina	CTL	15	27.2	-	-	3.475	27.2	1.274	27.2	-	-
101	Snaclec VP12 subunit A	Daboia palaestinae	CTL	12	26.2	-	-	0.008	26.2	-	-	-	-
102	Snaclec VP12 subunit B	Daboia palaestinae	CTL	15	25.6	-	-	0.084	25.6	0.029	17.6	-	-
153	C-type lectin J	Echis coloratus	CTL	18	14.6	0.589	14.6	0.740	14.6	0.579	14.6	0.477	14.6
154	C-type lectin H	Echis coloratus	CTL	18	10.8	0.050	10.1	0.935	10.8	0.909	10.8	0.172	10.8
155	C-type lectin E	Echis coloratus	CTL	11	12.1	0.011	7.1	-	-	0.008	7.1	0.045	12.1
156	C-type lectin B	Echis coloratus	CTL	13	7.1	-	-	0.048	7.1	0.005	7.1	-	-
157	C-type lectin A	Echis coloratus	CTL	18	10.1	0.014	10.1	-	-	-	-	-	-
159	Snaclec coagulation factor X-activating enzyme light chain 2	Macrovipera lebetina	CTL	18	6.3	0.039	6.3	0.199	6.3	0.340	6.3	0.051	6.3
173	C-type lectin-like protein 3B	Macrovipera lebetina	CTL	17	42.6	2.457	42.6	2.758	42.6	2.500	42.6	1.545	42.6
174	C-type lectin-like protein 4B	Macrovipera lebetina	CTL	17	14.7	0.018	12.0	-	-	0.017	12.0	0.353	14.7
175	Snaclec dabocetin subunit alpha	Daboia siamensis	CTL	17	6.5	-	-	0.443	6.5	0.064	6.5	-	-
181	Snaclec tokaracetin subunit beta	Protobothrops tokarensis	CTL	4	32.5	-	-	0.026	32.5	-	-	-	-
210	Snaclec anticoagulant protein subunit B	Deinagkistrodon acutus	CTL	14	12.2	-	-	0.352	12.2	0.124	12.2	-	-
14	Disintegrin VB7A	Vipera berus berus	Dis	7	76.6	-	-	0.111	23.4	0.008	23.4	12.932	76.6
15	Disintegrin VB7B	Vipera berus berus	Dis	6	70.3	-	-	0.008	29.7	-	-	0.935	70.3
16	Disintegrin VLO4	Macrovipera lebetina obtusa	Dis	7	38.5	-	-	0.011	24.6	0.006	24.6	0.034	38.5
17	Disintegrin VA6	Vipera ammodytes ammodytes	Dis	7	23.4	-	-	-	-	-	-	0.133	23.4
191	Disintegrin lebein-1-alpha	Macrovipera lebetina	Dis	12	30.6	-	-	0.389	9.0	0.578	9.0	0.006	21.6
86	Hyaluronidase	Echis ocellatus	Hya	52	13.6	0.007	13.6	0.011	7.3	0.004	2.7	-	-
176	Hyaluronidase	Crotalus adamanteus	Hya	52	6.7	0.004	6.7	0.007	6.7	0.003	2.0	-	-
27	Inhibitor, chymotrypsin	Vipera ammodytes	Kunitz	7	40.0	0.002	24.6	-	-	0.047	29.2	0.478	29.2
80	Protease inhibitor 3	Walterinnesia aegyptia	Kunitz	8	21.0	-	-	-	-	0	21.0	0.005	21.0
81	Kunitz-type serine protease inhibitor Vur-KIn	Vipera renardi	Kunitz	7	39.4	0.015	39.4	-	-	0.074	39.4	0.241	39.4
87	KP-Sut-1	Suta fasciata	Kunitz	13	9.4	0.072	9.4	-	-	-	-	0.077	9.4
142	Kunitz-type serine protease inhibitor ki-VN	Vipera nikolskii	Kunitz	10	34.7	0.610	34.7	-	-	-	-	-	-
5	l-amino-acid oxidase	Macrovipera lebetina	LAAO	12	42.1	0.041	41.1	1.388	42.1	1.631	41.1	1.786	41.1
65	l-amino-acid oxidase	Echis ocellatus	LAAO	56	6.0	-	-	-	-	0	6.0	-	-
66	l-amino-acid oxidase	Vipera ammodytes ammodytes	LAAO	54	13.2	-	-	0.009	11.2	0.012	11.2	0.007	13.0
75	l-amino-acid oxidase	Daboia russelii	LAAO	56	11.7	0	5.8	0.316	7.7	0.045	11.7	0.049	9.9
92	Kunitz-type serine protease inhibitor PIVL	Macrovipera lebetina transmediterranea	LAAO	10	8.4	-	-	-	-	0	8.4	0.021	8.4
94	l-amino-acid oxidase	Gloydius halys	LAAO	55	9.9	-	-	-	-	0	7.4	-	-
107	l-amino acid oxidase	Ovophis okinavensis	LAAO	58	11.0	0.001	3.9	1.893	6.8	2.106	6.8	1.378	10.9
144	l-amino acid oxidase	Protobothrops elegans	LAAO	57	5.7	-	-	0	5.7	-	-	-	-
152	l-amino acid oxidase B variant 1	Echis coloratus	LAAO	56	17.1	-	-	0.425	13.1	0.213	12.9	0.216	13.9
164	l-amino-acid oxidase	Crotalus horridus	LAAO	58	11.2	0.001	4.5	0.042	6.6	0.005	5.8	0	5.8
183	l-amino-acid oxidase	Bothrops moojeni	LAAO	54	12.1	0.022	6.5	0.256	7.1	0.178	9.4	0.098	9.2
62	Venom nerve growth factor 2	Daboia russelii	NGF	27	14.4	-	-	0	14.4	-	-	-	-
63	Venom nerve growth factor	Vipera ursinii	NGF	27	25.5	0.285	18.1	0.122	18.1	0.254	25.5	0.093	18.1
76	Snake venom 5′-nucleotidase	Gloydius blomhoffii blomhoffii	Nuc	6	27.8	0.013	27.8	-	-	-	-	-	-
110	5′-nucleotidase	Ovophis okinavensis	Nuc	55	26.6	0.091	26.6	0.012	9.1	0.018	16.5	0.057	22.4
125	Phosphodiesterase	Macrovipera lebetina	Nuc	96	35.4	0.243	35.4	0.103	21.4	0.088	20.7	0.038	19.5
126	5′-nucleotidase	Macrovipera lebetina	Nuc	45	58.1	0.563	54.4	0.046	14.5	0.097	30.4	0.226	39.2
127	Venom phosphodiesterase 2	Crotalus adamanteus	Nuc	91	14.0	0.007	14.0	-	-	-	-	-	-
132	Ectonucleotide pyrophosphatase/phosphodiesterase family member 3-like	Python bivittatus	Nuc	93	7.5	0.004	7.5	0.002	3.5	0.003	3.5	-	-
170	Ectonucleotide pyrophosphatase/phosphodiesterase family member 3	Boiga irregularis	Nuc	100	4.5	-	-	0.441	3.3	1.521	2.6	-	-
73	Proactivator polypeptide-like	Crotalus adamanteus	OP	58	19.7	0.036	19.7	0.011	5.2	0.024	8.7	0.006	2.5
104	ArfGAP with SH3 domain ankyrin repeat and PH domain 3	Micrurus fulvius	OP	107	2.3	-	-	0.027	2.3	0.033	2.3	-	-
113	Uncharacterized protein	Ophiophagus hannah	OP	46	3.4	-	-	0.040	3.4	0.015	3.4	-	-
115	78 kDa glucose-regulated protein	Ophiophagus hannah	OP	67	6.4	0	2.8	0.004	4.4	0	2.0	-	-
117	WD repeat-containing protein 67	Ophiophagus hannah	OP	113	0.8	-	-	-	-	-	-	0.652	0.8
118	Pituitary adenylate cyclase-activating polypeptide type I receptor	Ophiophagus hannah	OP	7	18.2	0.008	18.2	-	-	-	-	-	-
119	Iron-responsive element-binding protein 2	Ophiophagus hannah	OP	90	1.8	-	-	0.536	1.8	0.482	1.8	0.224	1.8
122	Glutathione peroxidase	Ophiophagus hannah	OP	29	21.6	0.078	18.9	0.047	16.7	0.095	21.6	0.081	16.7
128	PiggyBac transposable element-derived protein 5	Python bivittatus	OP	69	2.7	-	-	-	-	-	-	0.067	2.7
129	Calmodulin-lysine N-methyltransferase	Python bivittatus	OP	14	12.3	-	-	-	-	0	12.3	-	-
130	Dipeptidase 2	Python bivittatus	OP	46	7.0	-	-	-	-	-	-	0	7.0
131	Serine/threonine-protein phosphatase 6 regulatory subunit 1 isoform X3	Python bivittatus	OP	92	1.9	-	-	0.199	1.9	0.117	1.8	0.279	1.8
133	E3 ubiquitin-protein ligase MARCH8-like isoform X8	Python bivittatus	OP	30	7.2	-	-	-	-	-	-	0.702	7.2
134	Nucleolar and coiled-body phosphoprotein 1 isoform X5	Python bivittatus	OP	104	1.4	-	-	-	-	0	1.4	-	-
135	E3 ubiquitin-protein ligase UBR4	Python bivittatus	OP	555	0.2	0	0.2	-	-	-	-	-	-
136	Extracellular matrix protein 1	Python bivittatus	OP	24	8.6	0.019	8.6	-	-	0.004	8.6	-	-
137	Receptor-type tyrosine-protein phosphatase gamma-like	Python bivittatus	OP	102	1.9	-	-	0.026	1.9	0.018	1.9	0.018	1.9
138	Nurim-like	Python bivittatus	OP	32	7.7	0.010	7.7	-	-	-	-	-	-
162	Dickkopf-related protein 3-like	Crotalus horridus	OP	31	5.0	-	-	0.005	5	0.007	5.0	-	-
168	RNA binding motif protein 6	Boiga irregularis	OP	59	3.5	0.007	3.5	-	-	-	-	-	-
172	Filamin-B isoform 15	Boiga irregularis	OP	282	0.9	-	-	0.010	0.9	-	-	-	-
197	Leucine-rich repeats and immunoglobulin-like domains protein 1	Thamnophis sirtalis	OP	48	1.4	0.010	1.4	0.028	1.4	0.026	1.4	-	-
201	Peroxiredoxin-4-like	Thamnophis sirtalis	OP	31	8.7	0.002	8.7	-	-	-	-	-	-
202	Obscurin	Thamnophis sirtalis	OP	1024	0.5	0.109	0.4	0.107	0.2	-	-	-	-
203	CCR4-NOT transcription complex subunit 3	Callithrix jacchus	OP	12	15.7	-	-	0.008	15.7	0.046	15.7	-	-
205	Tyrosine-protein phosphatase non-receptor type 20	Thamnophis sirtalis	OP	50	4.0	-	-	0.019	4.0	0.020	4.0	-	-
208	Protein BANP	Thamnophis sirtalis	OP	40	4.4	-	-	0.064	4.4	0.076	4.4	0.119	4.4
209	Microtubule-associated serine/threonine-protein kinase 1-like	Thamnophis sirtalis	OP	94	0.7	-	-	-	-	-	-	0	0.7
10	Basic phospholipase A2 chain HDP-1P	Vipera nikolskii	PLA2	13	86.9	20.289	86.9	5.460	9.0	0.230	9.0	0.288	27.0
13	Basic phospholipase A2 B chain	Vipera aspis zinnikeri	PLA2	13	80.3	0.130	80.3	-	-	-	-	-	-
28	Phospholipase A2 II	Vipera aspis	PLA2	5	40.4	0.018	19.2	-	-	0.009	21.2	-	-
29	Phospholipase A2 III	Vipera aspis	PLA2	5	66.0	0.002	66.0	6.579	66.0	5.099	66.0	0	66.0
30	Acidic phospholipase A2 PLA-1	Eristicophis macmahoni	PLA2	13	22.3	-	-	-	-	-	-	0.419	22.3
31	Acidic phospholipase A2 PLA-2	Eristicophis macmahoni	PLA2	13	29.8	0	28.9	-	-	0	21.5	0.388	29.8
32	Acidic phospholipase A2 homolog vipoxin A chain	Vipera ammodytes meridionalis	PLA2	13	94.3	34.017	94.3	-	-	-	-	0.048	18.9
56	Basic phospholipase A2 3	Daboia russelii	PLA2	13	27.3	-	-	0.003	12.4	0.002	12.4	0.048	27.3
57	Phospholipase A2	Agkistrodon piscivorus	PLA2	13	27.6	0	16.3	-	-	-	-	-	-
58	Phospholipase A2 homolog P-elapitoxin-Aa1a beta chain	Acanthophis antarcticus	PLA2	3	22.6	-	-	0.165	22.6	0.127	22.6	0.030	22.6
67	Acidic phospholipase A2 RV-7	Daboia siamensis	PLA2	13	45.1	1.078	45.1	-	-	-	-	-	-
78	Basic phospholipase A2 Pla2Vb	Vipera berus berus	PLA2	15	46.4	-	-	0	13.8	0.056	46.4	-	-
79	Acidic phospholipase A2 Vur-PL3	Vipera renardi	PLA2	15	69.3	-	-	12.956	67.2	12.705	67.2	7.113	58.4
82	Acidic phospholipase A2 PL1	Vipera renardi	PLA2	15	75.4	4.057	70.3	5.097	59.4	4.080	52.9	10.603	75.4
83	Acidic phospholipase A2 Vur-PL2B	Vipera renardi	PLA2	15	72.3	-	-	0	19.7	0.041	19.7	14.999	72.3
84	Basic phospholipase A2 homolog Vur-S49	Vipera renardi	PLA2	15	63.0	-	-	0.009	18.8	0.055	18.8	7.676	63.0
85	Basic phospholipase A2 vurtoxin	Vipera renardi	PLA2	15	50.0	-	-	-	-	-	-	4.220	50.0
95	Ammodytin I1	Vipera aspis aspis	PLA2	15	56.5	0.032	56.5	0.048	56.5	0.040	44.9	0.026	39.9
96	Ammodytin I1	Vipera ammodytes montandoni	PLA2	15	75.4	1.389	75.4	1.428	75.4	0.524	63.8	1.710	59.4
97	Ammodytin I2	Vipera aspis aspis	PLA2	15	19.7	-	-	-	-	-	-	0	19.7
98	Ammodytin I2	Vipera berus berus	PLA2	15	36.5	-	-	0.021	31.4	-	-	-	-
99	Ammodytin I2	Vipera ursinii	PLA2	15	45.3	-	-	-	-	-	-	0.017	45.3
100	Ammodytin L	Vipera ammodytes ammodytes	PLA2	15	14.5	-	-	-	-	-	-	0.009	14.5
139	Basic phospholipase A2 vipoxin B chain	Vipera ammodytes meridionalis	PLA2	13	80.3	0.086	80.3	-	-	-	-	-	-
151	Phospholipase A2 Group IIE	Echis coloratus	PLA2	13	5.8	-	-	-	-	-	-	0.020	5.8
161	Basic phospholipase A2	Azemiops feae	PLA2	15	7.2	-	-	-	-	0.012	7.2	0.016	7.2
193	Phospholipase A2-III	Daboia russelii	PLA2	13	13.1	0.019	13.1	-	-	-	-	-	-
195	Phospholipase A2 ammodytin I1	Vipera nikolskii	PLA2	15	75.4	4.779	75.4	4.663	75.4	4.289	63.8	-	-
196	Basic phospholipase A2 chain HDP-2P	Vipera nikolskii	PLA2	15	69.6	0.068	69.6	-	-	-	-	0.006	31.2
109	Phospholipase b	Ovophis okinavensis	PLB	64	20.8	-	-	0.015	13.6	0.037	20.1	0.057	15.0
114	Phospholipase B-like 1	Ophiophagus hannah	PLB	58	16.8	0.022	7.0	0.085	12.0	0.104	16.2	0.088	10.2
169	Phospholipase B	Boiga irregularis	PLB	64	15.6	-	-	0.020	11.6	0.034	11.2	0.034	9.9
179	Phospholipase B	Crotalus adamanteus	PLB	64	26.9	0.080	16.5	0.218	15.9	0.321	21.0	0.334	12.7
6	Unassigned	Calloselasma rhodostoma	SP	24	9.4	-	-	0.007	5.5	0.041	8.1	0.006	8.1
11	Snake venom serine protease pallase	Gloydius halys	SP	26	13.1	0.002	9.3	0.005	10.6	0.002	11.9	0	10.6
12	Snake venom serine protease ussurase	Gloydius ussuriensis	SP	26	5.6	0.077	5.6	-	-	-	-	-	-
21	Thrombin-like enzyme KN-BJ 2	Bothrops jararaca	SP	27	11.3	1.017	11.3	0.329	11.3	1.944	11.3	0.050	11.3
35	Serine protease	Echis coloratus	SP	28	8.5	-	-	0.043	8.5	0.172	8.5	0.087	8.5
36	Serine protease	Echis ocellatus	SP	24	6.3	0.515	6.3	1.980	3.6	2.401	6.3	0.694	6.3
37	Serine protease	Echis coloratus	SP	25	7.3	-	-	0.079	4.7	0.047	7.3	-	-
38	Serine protease	Echis coloratus	SP	25	12.0	0.004	12.0	0	9.4	0.007	12.0	-	-
39	Serine protease	Echis coloratus	SP	26	5.9	0.110	5.9	0.048	3.4	0.015	5.9	0.005	5.9
40	Serine protease	Echis carinatus sochureki	SP	25	8.1	0.021	5.5	-	-	-	-	0.036	5.5
64	Factor V activator RVV-V gamma	Daboia siamensis	SP	25	13.7	0.006	11.1	0.473	8.1	0.075	5.6	-	-
69	Serine protease VLSP-3	Macrovipera lebetina	SP	28	15.5	3.526	15.5	2.548	12.0	3.479	14.3	1.303	14.3
70	Beta-fibrinogenase	Macrovipera lebetina	SP	28	18.3	0.017	18.3	0.008	9.3	0.078	11.7	0.224	11.7
71	Chymotrypsin-like protease VLCTLP	Macrovipera lebetina	SP	28	29.6	0.069	29.6	-	-	-	-	-	-
74	Snake venom serine protease nikobin	Vipera nikolskii	SP	28	43.2	12.595	43.2	2.334	34.2	8.515	32.3	1.732	28.0
77	Snake venom serine protease pallabin	Gloydius halys	SP	28	12.3	0.002	8.8	-	-	-	-	0.010	10.0
103	Kallikrein-CohID-1	Crotalus oreganus helleri	SP	28	10.8	-	-	-	-	-	-	0	10.8
105	Serine protease	Protobothrops flavoviridis	SP	18	17.4	0	17.4	-	-	-	-	-	-
106	Serine protease	Ovophis okinavensis	SP	10	23.3	0.061	20.0	0.028	23.3	0.352	20.0	0.067	23.3
140	Factor V activator	Macrovipera lebetina	SP	28	18.9	0.049	13.9	0.776	11.6	0.088	11.2	0.005	5.0
141	Venom serine proteinase-like protein 2	Macrovipera lebetina	SP	28	30.8	1.028	30.8	0.896	26.5	1.680	25.4	1.595	26.5
163	Serine proteinase 1	Crotalus horridus	SP	28	13.2	1.023	10.9	0.274	4.3	2.042	6.6	-	-
187	Snake venom serine protease HS112	Bothrops jararaca	SP	27	14.9	-	-	0.157	12.5	0.323	14.9		-
188	Snake venom serine protease KN6	Trimeresurus stejnegeri	SP	28	3.5	0.036	3.5	-	-	-	-	-	-
189	Snake venom serine protease 5	Trimeresurus stejnegeri	SP	28	11.6	0	11.6	-	-	-	-	-	-
190	Snake venom serine protease catroxase-2	Crotalus atrox	SP	27	17.4	0.352	17.4	0.162	8.5	0.531	10.9	0.012	10.9
198	Rho GTPase-activating protein 28-like	Thamnophis sirtalis	SP	24	3.2	-	-	0.811	3.2	0.822	3.2	0.207	3.2
41	Metalloproteinase	Echis carinatus sochureki	SVMP	69	14.6	-	-	1.488	8.7	1.023	11.5	0.401	14.6
42	Metalloproteinase	Echis carinatus sochureki	SVMP	68	6.9	-	-	-	-	-	-	0.107	6.9
43	Metalloproteinase	Echis carinatus sochureki	SVMP	27	6.5	-	-	2.987	6.5	2.973	6.5	1.419	6.5
44	Metalloproteinase	Echis coloratus	SVMP	56	6.3	-	-	-	-	-	-	0.004	6.3
45	Metalloproteinase	Echis coloratus	SVMP	56	11.2	-	-	0.109	9.4	0.080	9.4	0.116	9.0
46	Metalloproteinase	Echis coloratus	SVMP	66	10.7	-	-	0	9.2	0	9.2	0	10.7
47	Metalloproteinase	Echis coloratus	SVMP	69	3.1	-	-	-	-	-	-	0.008	3.1
48	Metalloproteinase	Echis coloratus	SVMP	69	7.4	-	-	0.208	7.4	0.435	4.7	0.058	4.9
49	Metalloproteinase	Echis coloratus	SVMP	68	8.8	-	-	-	-	-	-	0	8.8
50	Metalloproteinase	Echis coloratus	SVMP	68	13.8	-	-	0	8.9	0	5.2	0.488	4.9
51	Metalloproteinase	Echis coloratus	SVMP	61	5.4	-	-	2.479	5.4	2.296	5.4	1.071	5.4
52	Metalloproteinase	Echis coloratus	SVMP	68	6.4	-	-	0.019	6.4	0.012	5.6	0.021	6.4
53	Metalloproteinase	Echis pyramidum leakeyi	SVMP	62	8.1	0.009	2.5	-	-	-	-	-	-
54	Metalloproteinase	Echis pyramidum leakeyi	SVMP	46	5.4	-	-	0.019	5.4	0.014	5.4	-	-
55	Metalloproteinase	Echis carinatus sochureki	SVMP	54	5.6	-	-	-	-	-	-	0.010	5.6
59	Group III snake venom metalloproteinase	Echis ocellatus	SVMP	62	10.7	0	3.1	1.254	6.0	1.744	9.0	0.651	10.7
61	Snake venom metalloproteinase VMP1	Agkistrodon piscivorus leucostoma	SVMP	46	6.8	-	-	-	-	-	-	0.833	6.8
72	Snake venom metalloproteinase	Crotalus adamanteus	SVMP	68	9.0	-	-	0.005	3.4	0.016	4.7	0.085	7.7
93	H3 metalloproteinase 1	Vipera ammodytes ammodytes	SVMP	68	43.5	0.611	32.0	3.415	33.3	2.970	35.9	2.737	37.3
108	P-III metalloprotease	Ovophis okinavensis	SVMP	16	12.1	-	-	2.110	12.1	1.999	12.1	0.945	12.1
112	Metalloproteinase H4-A	Vipera ammodytes ammodytes	SVMP	68	14.2	0.022	11.4	0.342	4.2	0.006	4.2	-	-
143	Snake venom metalloproteinase lebetase-4	Macrovipera lebetina	SVMP	24	19.4	-	-	-	-	0.008	12.4	0.091	19.4
146	Zinc metalloproteinase-disintegrin-like daborhagin-K	Daboia russelii	SVMP	69	6.2	-	-	-	-	-	-	0.112	6.2
147	Coagulation factor X-activating enzyme heavy chain	Daboia siamensis	SVMP	69	10.0	0.003	2.9	0.473	7.3	0.088	7.1	0.010	7.3
160	Coagulation factor X-activating enzyme heavy chain	Macrovipera lebetina	SVMP	68	11.4	0.013	5.4	1.243	11.4	0.105	6.5	0.020	5.4
166	Metalloproteinase F1	Vipera ammodytes ammodytes	SVMP	68	25.7	-	-	-	-	0.001	4.1	0.498	25.7
182	Antihemorrhagic factor cHLP-A	Gloydius brevicaudus	SVMP	36	4.0	-	-	-	-	-	-	0	4.0
184	Zinc metalloproteinase/disintegrin	Macrovipera lebetina	SVMP	53	11.9	-	-	0.008	4.8	0.027	8.8	0.657	7.9
185	Zinc metalloproteinase-disintegrin-like VLAIP-B	Macrovipera lebetina	SVMP	68	9.3	0	4.6	-	-	-	-	0.052	6.5
186	Zinc metalloproteinase-disintegrin-like VLAIP-A	Macrovipera lebetina	SVMP	68	25.2	0.005	17.7	0.138	14.9	0.110	17.5	0.048	19.0
192	Group III snake venom metalloproteinase	Echis ocellatus	SVMP	69	10.7	-	-	0	7.9	0	10.7	-	-
194	Zinc metalloproteinase-disintegrin-like bothrojarin-2	Bothrops jararaca	SVMP	24	11.9	-	-	0.015	11.9	0.017	11.9	0.089	11.9
1	Renin-like aspartic protease	Echis ocellatus	TBP	43	9.4	0.009	4.6	0.040	4.8	0.039	4.8	0.055	4.8
8	Aminopeptidase A	Gloydius brevicaudus	TBP	110	7.6	0	2.1	0.004	2.5	0.007	2.5	0.029	7.6
20	Aminopeptidase N	Gloydius brevicaudus	TBP	106	1.3	-	-	-	-	-	-	0	1.3
68	Glutaminyl-peptide cyclotransferases	Daboia russelii	TBP	42	37.8	0.109	37.8	0.092	29.1	0.055	32.1	0.085	37.8
111	Glutaminyl-cyclase	Ovophis okinavensis	TBP	40	33.2	0.012	33.2	-	-	-	-	-	-
120	Cathepsin D	Ophiophagus hannah	TBP	30	16.3	0.013	16.3	-	-	-	-	-	-
121	Endoplasmic reticulum aminopeptidase 1	Ophiophagus hannah	TBP	91	1.6	0.003	1.6	-	-	-	-	-	-
123	Renin	Ophiophagus hannah	TBP	40	5.5	0.016	2.2	0.059	5.5	0.048	5.5	0.023	5.5
149	Renin	Echis coloratus	TBP	12	23.9	-	-	-	-	0.013	23.9	-	-
167	Xaa-Pro aminopeptidase 2	Boiga irregularis	TBP	76	25.7	0.420	25.7	0.041	17.3	0.114	23.6	0.042	17.3
178	Peptidyl-prolyl cis-trans isomerase	Crotalus adamanteus	TBP	22	12.9	0.006	12.9	-	-	-	-	-	-
204	Dipeptidase 2-like	Thamnophis sirtalis	TBP	33	7.4	0.002	7.4	-	-	0.003	7.4	0.019	7.4
207	Xaa-Pro aminopeptidase 2-like	Thamnophis sirtalis	TBP	27	19.4	0.057	19.4	0.011	11.3	0.016	19.4	-	-
60	Snake venom vascular endothelial growth factor toxin vammin	Vipera ammodytes ammodytes	VEGF	16	49.7	5.315	31.0	4.239	44.1	5.109	36.6	2.446	32.4
88	Snake venom vascular endothelial growth factor toxin HF	Vipera aspis aspis	VEGF	12	65.5	0.083	65.5	0.002	58.2	0.002	48.2	-	-
148	Vascular endothelial growth factor A	Echis coloratus	VEGF	22	34.4	0.017	34.4	-	-	0.004	17.2	-	-
150	Vascular endothelial growth factor F	Echis coloratus	VEGF	16	28.5	-	-	0.390	28.5	0.640	28.5	0.030	18.8

¹ B-NAP: Bradykinin potentiating and C-type natriuretic peptides; BP: Blood protein; CP: Cysteine Proteases; CRISP: Cysteine-rich secretory protein; CTL: C-type lectin like; Dis: Disintegrin; Hya: Hyaluronidase; Kunitz: Kunitz type proteinase inhibitor; LAAO: l-amino acid oxidase; NGF: Nerve growth factor; Nuc: Nucleic acid degrading enzymes; OP: Other protein; PLA2: Phospholipase A₂; PLB: Phospholipase B; SP: Serine proteinase; SVMP: Metalloproteinase; TBP: Toxin biosynthesis proteins (including aminopeptidases); VEGF: Vascular endothelial growth factor.

and

Table 2. Protein families found in the venoms of Russian vipers.

Protein Family 3	# of Identified Proteins	Protein Abundance 1 LFQ/INT, % (# of Identified Proteins 2)
		V. nikolskii	V. kaznakovi	V. orlovi	V. renardi
PLA2	(29)	64.68/65.96 (14)	41.03/36.43 (11)	24.21/27.27 (14)	44.05/47.64 (18)
SVMP	(32)	0.66/0.66 (8)	16.15/16.31 (19)	14.77/13.92 (21)	11.98/10.53 (28)
CTL	(18)	4.01/3.9 (9)	12.48/13.44 (15)	11.2/9.46 (15)	3.46/3.24 (8)
SP	(27)	19.34/20.51 (20)	10.79/10.96 (18)	23.97/22.61 (19)	7.87/6.03 (15)
CRISP	(7)	0.66/0.41 (2)	9.72/10.89 (7)	12.2/12.3 (7)	7.98/8.26 (4)
LAAO	(11)	0.08/0.07 (5)	3.99/4.33 (8)	4.59/4.19 (10)	4.21/3.56 (8)
VEGF	(4)	7.57/5.42 (3)	3.96/4.63 (2)	4.2/5.76 (4)	2.92/2.48 (2)
Dis	(5)	0/0 (0)	0.53/0.52 (4)	0.56/0.59 (3)	13.43/14.04 (5)
OP	(28)	0.17/0.28 (11)	0.49/1.13 (13)	0.95/0.96 (16)	1.8/2.15 (8)
PLB	(4)	0.12/0.1 (2)	0.32/0.34 (3)	0.52/0.5 (3)	0.54/0.51 (4)
Nuc	(7)	0.88/0.92 (6)	0.21/0.6 (4)	2.12/1.73 (5)	0.47/0.32 (3)
TBP	(13)	0.68/0.65 (11)	0.17/0.25 (5)	0.3/0.3 (8)	0.33/0.25 (7)
NGF	(2)	0.33/0.28 (1)	0.14/0.12 (2)	0.25/0.25 (1)	0.12/0.09 (1)
Hya	(2)	0/0.01 (2)	0.01/0.02 (2)	0/0.01 (2)	0/0 (0)
BP	(14)	0.15/0.12 (12)	0/0.01 (2)	0.01/0.01 (3)	0.06/0.1 (9)
B-NAP	(1)	0.01/0.01 (0)	0/0.03 (1)	0/0.01 (1)	0/0 (0)
Kunitz	(5)	0.66/0.7 (4)	0/0 (0)	0.15/0.12 (3)	0.79/0.8 (4)
CP	(1)	0/0 (1)	0/0 (0)	0/0 (0)	0/0 (0)
total	(210)	(111)	(116)	(135)	(124)

¹ Protein abundance was calculated on the basis of peptide abundances for the peptides identified by MS/MS, as well as the peptides identified by MS1 matching between chromatograms. Protein abundances were calculated either on the basis of the MaxLFQ (Label-Free Quantification) algorithm (LFQ) or on the basis of the comparison of total protein intensities (sums of peptide intensities were calculated for each protein) within a single venom (INT). ² Numbers of proteins were calculated on the basis of the peptides identified by MS/MS only (no MS1 matching hits were used). Therefore the protein might not be listed as identified, but it would still be quantified ”By Matching” with non-zero abundance value (e.g., B-NAP in V. nikolskii venom). ³ B-NAP: Bradykinin potentiating and C-type natriuretic peptides; BP: Blood protein; CP: Cysteine Proteases; CRISP: Cysteine-rich secretory protein; CTL: C-type lectin like; Dis: Disintegrin; Hya: Hyaluronidase; Kunitz: Kunitz type proteinase inhibitor; LAAO: l-amino acid oxidase; NGF: Nerve growth factor; Nuc: Nucleic acid degrading enzymes; OP: Other protein; PLA2: Phospholipase A₂; PLB: Phospholipase B; SP: Serine proteinase; SVMP: Metalloproteinase; TBP: Toxin biosynthesis proteins (including aminopeptidases); VEGF: Vascular endothelial growth factor.

, and Tables S1 and S2): 116 proteins were identified in V. kaznakovi, 124 in V. renardi, 135 in V. orlovi and 111 in V. nikolskii venoms. Most proteins could be matched to previously reported snake toxins. To minimize individual variations, venoms from several individual animals were pooled for analysis [12].

The proteins were categorized into 14 known venom protein families (Table 2). The most numerous classes were PLA2, SVMP, C-type lectin like (CTL) and serine protease (SP). Eleven families were represented in all viper venoms, while disintegrins (Dis) were absent in V. nikolskii. There were no Kunitz type proteinase inhibitors in V. kaznakovi, no hyaluronidase (Hya) in V. renardi and no bradykinin potentiating and C-type natriuretic peptides (B-NAP) in V. renardi and V. nikolskii. Besides, in the V. nikolskii venom, a single low abundance protein was identified belonging to Cysteine Proteases (CP), which are not common for snake venoms. Along with venom proteins, several Blood Proteins (BP) (up to 0.15% of the total protein abundance) and proteins with unclear family annotation (Other Proteins (OP)) (up to ~2% of the total protein content) were also found.

While the most numerous venom protein families were fairly similar in all snakes studied, individual protein composition was quite different (Figure 1). From 210 proteins only 46 were common for all four species and each species featured unique proteins: six in V. kaznakovi, 26 in V. renardi, eight in V. orlovi and 29 in V. nikolskii. These differences did not correlate with the total number of individual proteins identified in each venom.

2.2. Composition of Russian Viper Venoms

As a result of venom protein quantification, it was found that the main venom components were PLA2s; their content ranged from about 24% in V. orlovi venom to more than 60% in V. nikolskii (Table 2, Figure 2). The overwhelming majority of PLA2s belonged to D49 subgroup of group IIA as it might be expected for the snakes from Viperidae family. The venom of V. nikolskii contained PLA2s only from this group. One PLA2 of S49 subgroup was highly represented in V. renardi. One PLA2 of group IA was observed in small amounts in three venoms and a low quantity of group IIE PLA2 was detected in V. renardi venom.

Altogether, the enzyme content in venom of V. nikolskii reached about 85%, however this venom was characterized by a very low content of SVMPs (less than 1%) and LAAO (less than 0.1%). PLA2s accounted for more than 40% in V. kaznakovi and V. renardi venoms. While the content of SVMPs was less than 1% in the V. nikolskii venom, they comprised 12%–16% in V. kaznakovi, V. orlovi and V. renardi. The highest content of SPs was in V. orlovi venom (24%) and the lowest in V. renardi (8%). LAAO was at the level of 4%–5% in all the analyzed venoms with the exception of V. nikolskii. Nucleic acid degrading enzymes (Nuc) represented about 2% in V. orlovi venom and less that 1% in all the others. Phospholipase B (PLB) was found in all venoms (less than 1%) and very low amount of Hya (0.01%) was detected in three venoms. Among the non-enzymatic proteins, Dis (13%) in the V. renardi venom, CTL (12%) in V. kaznakovi, CRISPs (12%) in V. orlovi and vascular endothelial growth factors (VEGF, 8%) in V. nikolskii were the most abundant ones in the venoms studied. The total amount of non-enzymatic proteins was about 13% in the V. nikolskii venom and about 27%–28% in all the others. In addition to the proteins mentioned above, nerve growth factor (NGF) and Kunitz were present in all the venoms (less than 1%) with exception of V. kaznakovi, where Kunitz was absent.

Interestingly, comparison of both the nature of the identified proteins and their abundance showed very close venom compositions for the species V. kaznakovii and V. orlovii. The Pearson correlation coefficient for individual protein abundance LFQ was 0.83, while for the rest of pairs the correlation coefficient varied from 0.16 to 0.34 (Figure 3).

2.3. Identification of Endogenous Peptides in the Venoms

It is well known that snake venoms may contain various peptides: several peptide families including bradykinin-potentiating peptides, natriuretic peptides, sarafotoxin, etc. were identified [17]. Moreover, the venoms studied in this work contain proteases, therefore their proteins may undergo proteolysis leading to generation of peptides. To study endogenously generated peptides in the venoms of interest, high molecular weight (MW) proteins were separated by ultrafiltration (10 KDa cut-off). The peptide fractions obtained were analyzed by LC-MS/MS in the same fashion as proteins, but without preliminary proteolysis. Peptides were searched at first against a full NCBI Serpentes database by Mascot search engine with 10% protein FDR (False Discovery Rate). A fusion database containing the peptidogenic proteins from Mascot search and the SwissProt Serpentes database was used for the final search in MaxQuant. Full NCBI database search with unspecific digestion failed in MaxQuant due to internal software limitations. In summary, 512 endogenous peptides from 80 proteins belonging to 13 protein families were found (Table S3). As expected, most of the peptides (462 peptides) belonged to proteins (48 proteins) which were earlier found in proteome, thus most likely representing venom protein degradation in vivo as a result of proteases and peptidases activity. At the same time, 50 peptides (Table S4) belonged to 32 unique proteins from nine protein families (

Table 3. Snake venom protein families for which peptides were found in peptidome only.

Family	Number of Proteins	Number of Peptides
CTL	4	5
Dis	3	7
Kunitz	2	2
LAAO	2	3
NAP	6	14
PLA2	1	1
SP	2	2
SVMP	11	22
VEGF	1	1

). Among these, proteins in six families mostly had one peptide per protein, which can explain their identification only in the peptidome analysis as a result of a very low concentration of original proteins before degradation, so they were missed in the shotgun MS/MS selection in proteome analysis (or were beyond the taken FDR cut off). The largest number of unique peptides was found in proteins belonging to Dis and SVMP/Dis families: 25 peptides from 14 proteins were found. The peptides identified were mainly fragments of larger venom proteins. However, we found 12 peptides from proteins belonging to B-NAP family (Figure 4). These peptides may represent real endogenous peptides and possess their own biological activity. This is the first indication for the presence of bradykinin-potentiating and natriuretic peptides in venoms of vipers from the Pelias group.

3. Discussion

We have analyzed venom proteomes and peptidomes for four species of Vipera, for which there is no genomic or transcriptomic data published. For each species, the venoms of at least 15 individual animals were pooled for the analysis. Protein identification for such “non-sequenced” species is problematic for inherently database oriented bottom-up LC-MS/MS-based proteomics [18]. A possible solution is to use the protein sequences of closely related species, based on the assumption of their high homology level [18]. Thus, when the exact protein sequence is missing in the database, the protein might still be identified by partial/full homology with a known protein of another species. Here, we searched LC-MS/MS data against the database containing all the proteins from the taxon Serpentes in the NCBI database on the date of the experiment (the results are given in Table 1).

In the bottom-up proteomics, there are two major approaches for the quantitative analysis: (a) relative quantification of a single protein across samples; and (b) comparison of different proteins within a single sample. Principle (a) is based on the measurement of all the peptides belonging to a protein in several samples (and pair-wise peptide Fold Changes estimation) followed by protein Fold Change calculation as, e.g., mean or median value of the peptide fold changes. Principle (b) is based on the assumption that the sum of peptide peak areas (either all or just some of them, like in the top 3 theory [19,20]) for a given protein is proportional to its absolute abundance. Thus, comparison of these sums for two proteins is supposed to give the difference in their content within one sample. What is the most important, when making a comparison between several samples, the two approaches (a) and (b) are supposed to give consistent results.

In case of protein analysis of “non-sequenced” species, both these approaches encounter significant albeit different problems arising from the incomplete peptide identification due to the lack of adequate protein amino acid sequences in the search database. Principle (a) works best when as many as possible shared peptides per protein are identified and quantified for a pair of samples, since individual peptide measurements are prone to err due to possible post-translational modifications or isoforms. When it comes to different species, the number of shared peptides between samples goes down just because of different protein sequences. Besides, this approach works only when there are shared peptide sequences identified and quantified in both samples (recommended number of shared peptides for a reliable quantitation is 2). Thus, if a protein is unique for a sample, it cannot be quantified this way at all. Besides, it provides no data for concentration comparison between different proteins within a single sample.

Principle (b) was developed and verified for systems (artificial protein mixtures) where all the best flyer peptides for a protein (the peptides which have the best proportion between peptide concentration and intensity and thus have the maximum impact on the summed protein intensity) can be easily identified and quantified [19]. For “non-sequenced” species it would mess the final results through protein abundance underestimation if the missed peptides were among the best flyers for the given protein of some particular species but were overlooked because their amino acid sequence was missing in the database. For that, peptide MS/MS sequencing de novo might help a bit, but many peptides would still be missed for the reasons that are not clarified.

Here, we used two approaches to quantify proteins. First, we used MaxLFQ approach [21] which is basically principle (a), but it also uses absolute peptide intensities in addition to peptide FC comparison between samples (such results are labeled LFQ in Table 2). Second, we used direct comparison of sums of peptide intensities to make quantitation within each sample (such results are labeled INT in Table 2). The results of protein quantitation made by different methods gave quite similar results (Table 2, Table S1), especially when potential errors in individual protein contents were compensated by consolidation of proteins into families.

There is also a question of which types of peptides should be used for protein quantitation. Protein identification process deals not with separate proteins, but with protein groups, which are sets of individual proteins (at least partially homologous) sharing a set of identified peptide sequences. In the absence of unique specific peptides, no distinction between these proteins within a group can be made. A standard approach is to take as a hit the protein from a protein group which has a maximum number of assigned identified peptides. Thus, there are three types of peptides for a single protein group in the identification list: unique, razor and other (shared) peptides (MaxQuant terminology) [22,23]. Usually, protein groups have some unique peptides to pinpoint them as “correct” hits, but it is also possible that the number of unique peptides for a protein is zero. Absence of unique peptides means that all the peptides from the current protein group are shared and can be just as successfully assigned to some other protein groups. In such situation, the final set of protein groups shown in the identification list is the minimal one sufficient to explain all the identified peptides (Occam’s razor principle). Shared peptides are named “razor” when they belong to the protein group with the maximum total number of peptides among other possible protein groups. These razor peptides are used for quantitation (along with unique peptides), both LFQ and intensity based [24]. A shared peptide, which is “razor” for some particular group, is counted in “all peptides” in all the protein groups to which it can be potentially assigned, and “all peptides” list is used to calculate Sequence Coverage.

Importantly, any analytical method may prove only that the amount of the compound under investigation is below the method sensitivity, rather than show the absolute absence of the compound in the sample. This is specifically applicable for the LC-MS/MS-based shotgun identification principle which selects peptide ions pseudo-randomly, sometimes missing the peptides with very low intensities just because of a wrong choice. Thus, quantitation is much more reliable for showing the absence of the compound (or, more accurate, the concentration being lower than its Low Limit of Detection). MaxQuant features chromatogram alignment and the possibility to quantify peptides on the basis of similarity of their retention time and m/z in the sample, where they were identified by MS/MS and in another sample where this particular m/z signal got lost during the shot-gun selection for the MS/MS analysis (proteins with such peptides are marked “By matching” in “Identity Type” column in Tables S1 and S2). In this work the protein is considered to be identified (and considered as present) in the sample only if it has an MS/MS spectrum identified in this particular sample. However, for quantitation both MS/MS identified peptides and the peptides identified on the basis of the above described similarity were used. This might lead to apparent contradictions when there are no proteins identified, but the protein abundance is non-zero (like natriuretic peptides (B-NAP) in the V. nikolskii venom—0.01/0.01 (0) in Table 2).

At the present time, the genus Vipera includes 22 species, however it is not homogenous. Molecular phylogeny studies showed that this genus comprises the V. aspis group, the V. ammodytes complex, and the Pelias group as separate clades [25]. Of these clades, only snakes from the Pelias group inhabits Russia. The Pelias was further classified into two subgroups, one comprising V. dinniki, V. kasnakovi, and V. ursinii, and another including V. berus, V. barani, V. nikolskii, and V. seoanei [25]. The first subgroup was further subdivided into the “kaznakovi” complex, including V. kasnakovi, V. orlovi and some other closely related species, and the “ursinii” complex, in which V. renardi was included [26,27]. Earlier, for the vipers of the Pelias group, we have studied the venom toxicity towards crickets Gryllus assimilis [12] and found that it differed depending on feeding preferences. The snakes from the V. renardi, V. lotievi, V. kaznakovi, and V. orlovi species feed on a wide range of animals including insects, whereas the snakes from V. berus and V. nikolskii species do not include insects in their diet. The venom from vipers which hunt insects was found to possess a greater toxicity towards crickets. This suggests that the venom composition may greatly differ among these species. As concerns the toxicity to other animals, it was shown that the venom of V. nikolskii was more toxic than that of V. berus to frogs (9–11 µg/g vs. 30–52 µg/g) and mice (0.93 vs 1.58 µg/g) at intraperitoneal injection [28]. The venom of V. renardi was less toxic to mice (2.96 µg/g) than that of V. berus [28]. We were not able to find any data about toxicity of V. orlovi and V. kaznakovi venoms.

Regarding the danger to humans, the data about bites by these snakes are sparse. Most of the documented cases refer to steppe viper V. renardi and report that it usually has calm and timid behavior, is reluctant to bite, and seeks to escape. This viper bites only when it is in danger, for example, if the snake is suddenly stepped on or picked up. V. renardi is considered less dangerous to humans than common adder. The human fatalities as a consequence of steppe viper bites are not reliably known [29], though there are some cases of the death of horses and small ruminants. A picture of human envenomation is characterized mainly by local signs which include severe pain at the site of the bite, redness, swelling that spreads far beyond the site of the biting. In severe cases, drowsiness, dizziness, nausea, increase of heart rate, and reduction in body temperature may be observed [30].

Records of the bites of humans by the Caucasian viper V. kaznakovi and Nikolsky’s viper V. nikolskii are practically absent. However, V. kaznakovi may be dangerous. Solitary human deaths and livestock losses after Caucasian viper bites were mentioned [30]. We were able to find only one report about human fatalities after the Nikolsky’s viper bites [31]. No information on the V. orlovi bites is available.

It should be noted that the venoms of not all Pelias species were studied equally well. The venom of V. berus is the best characterized. As mentioned earlier, the V. berus venom displayed in vitro proteolytic, fibrinolytic, anticoagulant, and phospholipolytic activities. In mice, significant local tissue-damaging effects, including edema, hemorrhage and myonecrosis were observed for this venom [3]. Several proteins involved in manifestation of those effects were isolated from V. berus venom. These proteins included basic PLA2 [4], SVMP [5], LAAO [6] and some others.

The V. nikolskii species is phylogenetically very close to V. berus and is included in the same subgroup within the Pelias group. It is regarded as a V. berus subspecies in some publications. However, the analysis of the V. nikolskii venom has shown it to differ greatly from that of V. berus. Thus, two heterodimeric PLA2s were isolated from the V. nikolskii venom [10], but similar proteins are absent in V. berus. The data obtained in the present work are in good agreement with the published results; the basic and acidic PLA2 subunits forming heterodimeric enzymes account for more than 50% of the V. nikolskii venom (Table 1). Earlier, cDNA encoding SP nikobin and Kunitz type inhibitor in the V. nikolskii venom gland was cloned and sequenced [11]. In this study we have found that nikobin is the main SP in the V. nikolskii venom (more than 12% of the total protein content, Table 1) and Kunitz-type serine protease inhibitor ki-VN was also the main protein of the Kunitz family in this venom (about 0.6%, Table 1). CRISP, the sequence of which was also deduced from cDNA analysis [32], was found in the venom in fairly low amount (0.66%, Figure 2). Interestingly, the content of CRISPs was much higher in other venoms studied and accounted for 8%, 10% and 12% in V. renardi, V. kaznakovi and V. orlovi venoms, respectively (Figure 2).

The steppe viper V. renardi is included in the “ursinii” complex [33] while the other two vipers, V. kaznakovi and V. orlovi, belong to the “kaznakovi” complex. Among these vipers only the composition of the V. renardi venom was in some way studied [7]. The amino acid sequences for several PLA2s and Kunitz-type inhibitor were deduced from the cloned cDNA of venom gland. Some PLA2s and Kunitz protein were isolated from the venom. The most abundant was ammodytin I2d analogue. In this work we have found all the PLA2s described by Tsai et al. [7] in the V. renardi venom, Vur-PL2 having the highest content (Table 1). Interestingly, this viper venom has very high content of disintegrins which accounts for about 13% of total protein, while the V. kaznakovi and V. orlovi venoms contain less than 1% and in the V. nikolskii venom no disintegrins were detected.

There are no published data on the composition of the V. kaznakovi and V. orlovi venoms and they are characterized for the first time in this work. These two venoms have the highest similarity among the four ones studied (Figure 3) that confirms the inclusion of V. orlovi in the “kaznakovi” complex. They have a fairly high content of SVMPs (15%–16%), CTL (11%–12%) and CRISPs (11%–12%) (Figure 3). The V. orlovi venom has the highest amount of SP (24%) among the four venoms studied (Figure 3) and only V. kaznakovi contains a small quantity of hyaluronidase (Hya) at the level of 0.01%. However no Kunitz type proteins were detected in the latter venom.

Although a limited number of B-NAP proteins (one in V. kaznakovi and one in V. orlovi, Table 2) were detected in the proteome analysis, several peptides derived from proteins of this family were found in the peptidomes of all the venoms studied (Figure 4). The mature bradykinin-potentiating peptide QGGLPRPGPEIPP was observed in the V. nikolskii venom and several fragments of similar peptides were detected in the other analyzed venoms. Several fragments of C-type natriuretic peptides were found in all four venoms as well (Figure 4). It should be noted that no bradykinin-potentiating and C-type natriuretic peptides from the vipers of Pelias group were reported so far.

In total, 210 proteins (Table 1) and 512 endogenous peptides (Table S3) were identified in four viper venoms. The overwhelming majority of the proteins (98%–99% of the total protein content) and the peptides represented 14 snake venom protein families (Table 2). The comparison of our results with those for other snakes of the Vipera genus shows higher representation of venom protein families in our data (

Table 4. Snake venom protein families represented in viper venoms.

Snake Venom Protein Family	V. kaznakovi Venom	V. renardi Venom	V. orlovi Venom	V. nikolskii Venom	V. anatolica Venom 1	V. raddei Venom 2	V. a. ammodytes Venom 3	V. a. meridionalis Venom 3,4
PLA2	+	+	+	+	+	+	+	+ 3
SP	+	+	+	+	+	+	+	+ 3
Dis	+	+	+	−	+	+	+	+ 3
CRISP	+	+	+	+	+	+	+	−
Kunitz	−	+	+	+	+	+	−	+ 4
LAAO	+	+	+	+	−	+	+	+ 3
SVMP	+	+	+	+	+	+	+	+ 3
NGF	+	+	+	+	−	+	+	+ 3
CTL	+	+	+	+	+	+	−	−
PLB	+	+	+	+	−	−	−	−
VEGF	+	+	+	+	−	+	+	+ 3
Nuc	+	+	+	+	−	−	−	−
B-NAP	+	+	+	+	−	+	−	+ 4
Hya	+	−	+	+	−	−	−	−

¹ Taken from [13]; ² Taken from [14]; ³ Taken from [15]; ⁴ Taken from [34].

). For example, while Nuc and PLB were found in all venoms studied in this work, no proteins of these families were reported for other venoms from the Vipera species (Table 4).

Hya was observed in three of the studied venoms and this is also the first indication for the presence of this enzyme in the venoms of the Vipera species. We have found that the main components of all venom studied are PLA2s, while SVMPs were prevailing in venoms of V. anatolica [13] and V. raddei [14].

4. Conclusions

In this work, quantitative proteomic and peptidomic characterization of venoms from four vipers inhabiting Russia was done; the compositions of the venoms from V. kaznakovi and V. orlovi, which showed the highest similarity among the four studied species, were analyzed for the first time.

More than 200 proteins and over 500 peptides were detected in total in all four venoms. They represented 14 snake venom protein families. In all venoms studied, over 70% of the total proteins were enzymes, the highest enzyme content (85.7%) being in the V. nikolskii venom. The main components of the venoms were PLA2s, which accounted for 65% of total protein content in the V. nikolskii venom. For the first time, bradykinin-potentiating and C-type natriuretic peptides were reported for vipers of the Pelias group. Nucleic acid degrading enzymes and phospholipase B were found in the venoms of Vipera species for the first time.

Due to the low toxicity of the steppe viper, or a limited habitat of the Caucasian and Orlov’s vipers, these snakes do not pose an epidemiological threat to Russian population. However, the envenomation by Nikolsky’s viper, the venom of which was shown in this study to contain a considerable amount of neurotoxic phospholipase A₂, may represent certain danger. An antiserum “Antigadyuka” (“Antiviper”) produced by Russian company “Allergen” is based on the venom of the common viper and may not be effective against the Nikolsky’s viper bites due to strong differences in the composition of the venoms. The need to consider the differences in the composition of the venoms in the antivenom production is discussed in recent publications [28,35] and should be taken into account by antiserum manufacturers.

5. Materials and Methods

The venoms of V. kaznakovi, V. nikolskii, V. orlovi and V. renardi vipers were obtained as described earlier [12]. The venoms from several individual animals were pooled as described in [12]. Snakes were captured in their natural habitat: V. kaznakovi in Krasnodar Territory near Adler, V. nikolskii in Penza region near Zubrilovo village, V. orlovi in Krasnodar Territory at Mikhaylovskiy mountain pass and V. renardi in Krasnodar Territory near Beysugskiy firth.

5.1. In-Solution Trypsin Digestion of Venom Samples

Lyophilized venom sample (100 μg each) was dissolved in 10 μL of a buffer containing 100 mM ammonium bicarbonate (ABC), 5% sodium deoxycholate (SDC) and 5 mM dithiothreitol (DTT) and incubated for 40 min at 60 °C to reduce cysteine residues. Then, 5 μL of 50 mM iodoacetamide (IAA) water solution was added and the mixture was incubated 30 min at RT, in the dark. Residual IAA was neutralized by 5 μL of 50 mM DTT and sample was diluted with 50 μL 50 mM ABC and trypsin was added in a 1:100 (enzyme/protein) ratio to the final volume 100 μL and the protein concentration ~1 mg/mL. Samples were incubated overnight at 37 °C. Trypsin was deactivated by addition of 5 μL of 10% TFA. Tryptic peptides were desalted using reverse-phase solid extraction cartridges Discovery DSC-18 (100 mg) (Supelco, Bellefonte, PA, USA) according to the manufacturer protocol. Final peptide solution was dried in vacuum and stored at −80 °C prior to LC-MS/MS analysis.

5.2. Endogenous Venom Peptides Isolation

Endogenous peptides from venom samples were isolated using C₁₈ StageTips [36]. To make StageTips, two pieces of C18 Empore extraction disk were cut using blunt-ended 16-gauge needle and packed into a P200 pipette tip. Membranes were conditioned by 20 μL of methanol and equilibrated by 20 μL of 0.1% aqua TFA. Venom solutions were applied onto the conditioned tips, followed by membrane washing with 20 μL of 0.1% aqua TFA. Peptides were eluted by 20 μL of 80% ACN, 0.1% TFA. Eluates were dried in vacuum and stored at −80 °C prior to LC-MS/MS analysis.

5.3. LC-MS/MS Analysis

Analysis was performed on the QExactive HF mass-spectrometer (Thermo Scientific, Waltham, MA, USA) coupled to the Dionex 3000 RSLCnano HPLC system (Thermo Scientific, Waltham, MA, USA). The HPLC system was configured in a trap-elute mode. An analytical column (75 μm × 150 mm) and a precolumn (100 μm × 10 mm) were in-house packed with Aeris Peptide C18 2.6 μm sorbent (Phenomenex, Torrance, CA, USA). Samples were loaded on the precolumn for 10 min at 3 mL/min with buffer A (3% AcN, 96.9% H2O, 0.1% FA), followed by separation at 300 nL/min with the 4%–55% gradient of buffer B (80% AcN, 19.9% H₂O, 0.1% FA).

Mass-spectrometer experiment consisted of one full survey MS1 scan followed by 20 dependent MS2 scans for the most intense ions. MS1 spectra were acquired in the profile mode in mass range 350–1400 m/z, maximum IT time 100 ms, AGC target 3e6, resolution 60000. Dependent MS2 scan were performed at resolution 15000 for 200–2000 m/z mass range, AGC target 1e5, maximum IT 25 ms, isolation window 1.4 m/z. Dynamic exclusion was set to 30 s.

5.4. LC-MS/MS Data Analysis

Data analysis was performed in the MaxQuant software (V. 1.5.3.30, Max Planck Institute of Biochemistry, Martinsried, Germany, 2016). Proteomic LC-MS/MS data was searched with the Andromeda search engine incorporated in the MaxQuant software against NCBI Serpentes DataBase exported from the NCBI web-site [37] for the Taxon Serpentes 2015/11/17 and containing 134677 entries with the following parameters: digestion Trypsin/P; max number of miscleavages 2; include contaminants; fixed modification: carbamidomethyl (Cys); variable modifications: Oxidation (Met), Acetylation (N-term), Deamidation (Asn, Gln); min peptide length 6; max peptide MW 5500; PSM FDR 0.01; protein FDR 0.05; decoy mode: revert; min number of peptides for identification 1; razor protein FDR; second peptide; match between runs; LFQ quantitation with minimum 2 peptide pairs; and stabilize large LFQ ratios. Full set of MaxQuant parameters for the analysis can be found in the Supplementary Data file mqpar_proteins.xml.

Peptidomic LC-MS/MS data were searched with the Mascot search engine against the same full NCBI Serpentes data base with the following parameters: MS tolerance 5 ppm; MS/MS tolerance 0.01 Da; charge: +1, +2, +3; fixed modification: carabamidomethyl (Cys); variable modifications: Oxidation (Met), Deamidation (Asn, Gln); enzyme none. Mascot results were reprocessed in the Scaffold software and identified peptidogenic proteins (protein FDR 10%) for all four venoms were added to the SwissProt Serpentes database exported from the NCBI web-site on 2015/11/30 and contains 2567 sequences to generate a fused database. This database was used for Andromeda search in MaxQuant software with the digestion parameter set to unspecific. Peptide length for unspecific digestion search was from 6 to 50 amino acids. The rest of the parameters were the same as for proteome data analysis, however in the peptidogenic protein features in the in the “Number of Unique and Razor Peptides (NoURP)” column, the number of peptides corresponds to that before PEP-based filtration.

Results were processed in the Perseus (V. 1.5.2.6, Max Planck Institute of Biochemistry, Martinsried, Germany, 2016) and Excel software (V. 12.06743.5000, Microsoft Corporation, Redmond, WA, USA, 2007) and with the use of R.