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Comprehensive Snake Venomics of the Okinawa
Habu Pit Viper, Protobothrops Flavoviridis, by
Complementary Mass Spectrometry-Guided
Approaches Maik Damm 1,┼, Benjamin-Florian Hempel 1,┼, Ayse Nalbantsoy 2 and Roderich D. Süssmuth 1,*
1 Department of Chemistry, Technische Universität Berlin, 10623 Berlin, Germany; [email protected]
(M.D.); [email protected] (B.-F.H.) 2 Department of Bioengineering, Ege University, 35100 Izmir, Turkey; [email protected] (A.N.)
* Correspondence: [email protected]; Tel.: +49-30-314-24205
┼ These authors contributed equal to this work
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1-S5:
Annotated MS2 spectra of BPP-RP, Figure S6-S9: Annotated MS2 spectra of tripeptidic svMP-i, Figure S101:
P. flavoviridis reduced venom MS TIC for IMP and TD, Figure S11: P. flavoviridis venom tested for cytotoxicity
against different human cell lines, Figure S12: SH-SY5Y cells after 48 h treatment with different P. flavoviridis
venom fractions.
Figure S1. MS2 spectra of pEQWMPGGRPPHHIPP. Representative MS/MS spectra of the m/z 859.42
(z=2) precursor ion for the top-down annotation of a BPP.
Figure S2. MS2 spectra of pESKPGRSPPISP. Representative MS/MS spectra of the m/z 617.33 (z=2)
precursor ion for the top-down annotation of a BPP.
Figure S3. MS2 spectra of pESKPGRSPPIS. Representative MS/MS spectra of the m/z 568.80 (z=2)
precursor ion for the top-down annotation of a BPP.
Figure S4. MS2 spectra of pESKPGRSPPI. Representative MS/MS spectra of the m/z 528.29 (z=2)
precursor ion for the top-down annotation of a BPP.
Figure S5. MS2 spectra of pEQWSQGRPR. Representative MS/MS spectra of the m/z 563.28 (z=2)
precursor ion for the top-down annotation of a BPP.
Figure S6. MS2 spectra of pEKW. Representative MS/MS spectra of the m/z 444.22 precursor ion for
the de novo annotation of a small tripeptic svMP inhibitor.
Figure S7. S2 spectra of pENW. Representative MS/MS spectra of the m/z 430.17 precursor ion for the
de novo annotation of a small tripeptic svMP inhibitor.
Figure S8. MS2 spectra of pEQW. Representative MS/MS spectra of the m/z 444.18 precursor ion for
the de novo annotation of a small tripeptic svMP inhibitor.
Figure S9. MS2 spectra of pEQ[-17.03]W/pEEW. Representative MS/MS spectra of the m/z 427.16
precursor ion for the de novo annotation of a small tripeptic svMP inhibitor.
Figure S10. P. flavoviridis reduced venom MS TIC for IMP and TD. The total ion counts from
P. flavoviridis crude venom were measured by an HPLC-ESI-MS of reduced crude venom. The relative
abundance was set to 100% for the highest peak.
Figure S11. P. flavoviridis venom tested for cytotoxicity against different human cell lines. The
effect on cell viability (%) of (A) P. flavoviridis crude venom was determined at various concentrations
(0.2-40.0 µg/mL) and (B) Doxorubicin (0.1-10.0 µg/mL) as control after 48 h treatment by an MTT assay
at 570 nm. One non-cancerous (HEK-293) and six cancerous human cell lines were tested. Error mean
in ±SD.
Figure S12. SH-SY5Y cells after 48 h treatment with different P. flavoviridis venom fractions. Single
RP-HPLC venom fractions of P. flavoviridis with the mentioned concentration in µg/mL were tested
against human neuroblastoma SH-SY5Y cells. Imaged were taken after 48 h treatment at 37 °C.
Doxorubicin was used as positive cytotoxic control drug and no stimulation as negative control.
Table S1. Venom proteins and peptides identified from Protobothrops flavoviridis. Assignment of
venomic components by crude venom intact mass profiling (IMP, method A), bottom-up (BU, method
C) and top-down (TD, method D). Peak numbers are based on the RP-HPLC annotation (Figure 2)
and low abundant peaks in the HPLC, but detectable in the IMP, are marked by #. Sequence tags were
obtained de novo from MS/MS spectra and identified against a non-redundant Protobothrops flavoviridis
protein database (taxid: 88087) by BLASTP. SDS-PAGE and intact mass profile analysis provided the
average molecular weight. Most abundant mass in a IMP TIC are asterisked. Method B with no
distinct RT are marked by °. Monoisotopic TD masses are marked by an indexed m.
N
ati
ve
inta
ct m
ass
33
8.0
9, *
40
7.0
4, 59
6.3
2,
93
6.4
8
11
36
.60
m
40
8.1
4, 6
65
.01
, *
11
36
.60
,
11
58
.58
, 1
38
7.7
3
11
24
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m
10
49
.56
m
72
3.4
1, 7
86
.16
, *
10
49
.56
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44
.22
88
7.4
3
12
33
.64
m
44
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6, 1
11
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9, *1
23
3.6
5
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30
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37
6.1
4, 4
13
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59
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42
7.1
6
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31
6.1
3, 8
87
.36
37
4.2
2, 4
45
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, 8
28
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,
11
05
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, 1
23
3.6
0, 7
34
9.0
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50
9.0
6, 7
65
4.1
3
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622
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, 7
693
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622
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, 7
693
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RT
1.6
1
2.8
8
2.8
8
13
.75
15
.28
15
.45
17
.82
17
.82
19
.28
19
.44
20
.74
20
.74
21
.88
21
.88
21
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24
.90
27
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30
.00
NC
BI
acc
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on
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mb
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BA
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52
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P5
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P0
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52
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E-0
7
5.0
E-0
9
6.0
E-0
4
5.0
E-0
8
9.0
E-0
3
4.0
E-0
4
Pro
tein
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tid
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tifi
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A;
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A
A;
C
A;
C
Pea
k
no
. 1
2
#
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3
4
5
6
7
8
9
10
41
3.2
6, 1
73
3.8
2,
*7
484
.06
42
949
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, 1
161
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17
17
.82
, 7
51
3.1
2, *
79
57
.27
1
16
1.6
2, 1
40
6.7
0, 1
71
7.8
3,
*7
96
1.2
7
14
06
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m
29
32
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m
17
17
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m
42
6.1
7, 9
49
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, 1
16
1.6
2,
17
17
.83
, *
79
43
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15
.47
4
29
4.1
1m
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66
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m
83
11
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m
83
16
.49
16
48
.82
, 4
08
3.9
8,
83
17
.48
,
13
786
.42
33
.61
36
.88
38
.10
38
.39
41
.03
41
.36
39
.79
45
.48
44
.09
44
.71
44
.71
45
.48
BA
P3
99
48
.1
P1
86
19
.2
BA
N8
93
92
.1
BA
P3
99
48
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BA
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99
48
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BA
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99
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BA
P3
99
48
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BA
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99
48
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BA
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93
60
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BA
P3
99
48
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BA
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52
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P1
86
19
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BA
P3
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48
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P2
33
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7
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4
4.0
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4
4.0
E-0
4
4.0
E-0
4
4.0
E-0
4
6.1
E-0
7
2.0
E-0
4
1.6
E-0
3
3.0
E-0
8
5.3
E-0
4
2.9
E-0
3
8.0
E-0
6
9.0
E-0
4
met
allo
pro
teas
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rin
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ote
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atin
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met
allo
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dis
inte
gri
n C
TF
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bas
ic p
ho
spho
lip
ase
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tid
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tid
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P/D
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R
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QIF
PQ
RY
SH
NS
DK
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RV
HQ
MV
NH
INE
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17
.03
]QW
MP
GG
RP
PH
HIP
P
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DF
PD
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)[-1
2.1
7]
WN
DL
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CD
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KG
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)[-1
2.1
7]W
N
DL
Y
TIY
PK
PF
NY
GL
YG
CV
DD
TG
GV
22
20
20
10
20
10
20
15
A;
C
C
A;
C
C
A;
C
C
D
A;
D
C;
D
C
A
11
a
11
b
12
a
12
b
13
a
13
b
14
15
a
15
b
11
16
.60
, 1
37
7.5
4,*
15
31
.79
,
25
48
.41
, 3
85
4.9
3, 4
52
3.2
5,
82
97
.47
, 1
37
86
.38
1
40
3.7
3, *
82
98
.47
30
75
.50
m
18
32
.94
m
*1
375
3.4
2
13
753
.43
, *
140
21
.19
14
013
.19
m
46
.95
48
.73
48
.88
56
.19
59
.24
60
.66
60
.25
BA
P3
99
50
.1
BA
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19
97
.1
BA
P3
99
48
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P0
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9.1
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9.1
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9.1
P0
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25
17
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93
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BA
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26
70
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9.1
P0
DJJ
9.1
Q0
25
17
.1
P5
92
65
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1.6
E-0
9
4.0
E-0
7
9.9
E-0
5
8.0
E-0
6
3.0
E-0
7
5.0
E-0
5
1.0
E-1
0
3.0
E-0
7
3.0
E-0
6
1.0
E-1
0
8.0
E-0
6
3.0
E-0
9
1.0
E-0
7
2.0
E-0
7
9.0
E-0
7
2.0
E-0
6
4.0
E-1
1
1.0
E-0
7
1.0
E-0
7
1.0
E-1
0
3.0
E-0
7
8.0
E-0
6
8.0
E-0
3
7.6
E-0
4
pep
tid
e
pep
tid
e
L-a
min
o a
cid
ox
idas
e
met
allo
pro
teas
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met
allo
pro
teas
e P
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ic p
ho
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lip
ase
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ic p
ho
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lip
ase
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bas
ic p
ho
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lip
ase
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bas
ic p
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lip
ase
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bas
ic p
ho
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lip
ase
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ase
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lip
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ic p
ho
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lip
ase
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bas
ic p
ho
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lip
ase
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'
bas
ic p
ho
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lip
ase
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LA
-B
LA
AO
svM
P/D
I
svM
P/D
I
PL
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PL
A2
PL
A2
PL
A2
PL
A2
PL
A2
PL
A2
PL
A2
TA
HA
HG
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ST
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GL
TA
AR
DV
NR
AS
EN
P
YL
CS
LG
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CR
TP
EQ
QIF
PQ
RY
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A
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KP
F
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SY
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CN
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V
NY
GL
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CN
CG
VG
R
MD
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SY
SW
K
13
1.0
3-L
FQ
ET
GK
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AK
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GL
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CN
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R
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KP
F
CC
FV
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CC
YE
K
AA
AV
CF
GQ
NL
R
30
3.0
4-F
VH
DC
CY
EK
AF
YG
CY
CG
K
33
7.1
1-T
FP
DIF
CT
DS
CK
TG
VII
CG
EG
TE
CE
K
14
2.0
4-A
AA
VC
FG
QN
LR
AA
AV
CF
GQ
NL
R
NY
GL
YG
CN
CG
VG
R
MD
SY
SY
SW
K
YT
IYP
KP
F
CG
DH
LL
QF
R
HL
LQ
FR
KM
IKK
MT
GK
EP
IV(S
YA
FY
GC
YC
GK
GG
RG
KP
KD
AT
DR
CC
FV
HD
CC
Y
EK
VT
GC
DP
KW
SY
YT
YS
LE
NG
DIV
CE
G
DP
YC
TK
VK
CE
CD
KK
AA
ICF
RD
NL
KT
Y
KN
RY
)[+
25
7.9
6]M
TF
PD
IFC
TD
PT
35
25
20
15
10
20
15
10
A
D
C;
D
C
C
A;
C
C
C
A;
C
C
D
#
#
16
a
16
b
16
c
16
d
16
e
17
a
17
b
17
c
14
013
.14
m
*1
394
5.2
6
13
937
.22
m
13
937
.24
m
*2
476
8.2
61
.40
63
.51
62
.57
62
.80
70
.26
Q9
0Y
77
.1
BA
C5
68
93
.1
P0
DJJ
9.1
BA
A0
15
65
.1
P5
92
65
.1
P5
92
65
.1
AJC
52
505
.1
BA
C5
68
93
.1
BA
P3
99
46
.1
BA
C5
68
93
.1
AJC
52
505
.1
BA
P3
99
57
.1
Q8
JI3
9.1
O1
30
57
.1
Q8
JI3
9.1
Q8
JI3
9.1
3.4
E-0
4
1.0
E-0
7
3.0
E-0
7
2.0
E-0
3
8.5
E-0
3
1.1
E-0
9
8.0
E-0
9
1.0
E-0
7
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5
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.4
8.0
E-0
9
5.0
E-0
3
6.0
E-0
7
4.0
E-0
6
3.0
E-0
3
7.0
E-0
7
1.0
E-0
3
2.0
E-1
0
9.0
E-0
9
2.0
E-0
7
3.0
E-0
6
5.0
E-0
7
3.0
E-0
6
3.0
E-0
6
1.0
E-0
3
5.0
E-0
7
8.0
E-0
9
5.0
E-0
5
3.0
E-0
4
2.0
E-1
0
5.0
E-0
6
2.0
E-0
4
2.0
E-0
9
2.0
E-0
7
2.0
E-0
7
1.0
E-0
5
3.0
E-1
0
2.0
E-0
7
seri
ne
pro
teas
e
zin
c m
etal
lop
rote
inas
e-D
I-li
ke
HV
1
met
allo
pro
teas
e P
-IIa
1
met
allo
pro
teas
e P
-II
zin
c m
etal
lop
rote
inas
e/D
I-li
ke
HR
1a
C-t
yp
e le
ctin
alp
ha
sub
un
it
C-t
yp
e le
ctin
A
C-t
yp
e le
ctin
F I
X/X
B
C-t
yp
e le
ctin
bet
a su
bu
nit
un
kno
wn
(p
rote
in)
zin
c m
etal
lop
rote
inas
e-D
I-li
ke
HV
1
zin
c m
etal
lop
rote
inas
e-D
I-li
ke
HV
1
seri
ne
pro
teas
e
zin
c m
etal
lop
rote
inas
e-D
I-li
ke
HV
1
C-t
yp
e le
ctin
alp
ha
sub
un
it
C-t
yp
e le
ctin
F I
X/X
A
C-t
yp
e le
ctin
F I
X/X
B
C-t
yp
e le
ctin
bet
a su
bu
nit
ph
osp
ho
die
ster
ase
zin
c m
etal
lop
rote
inas
e/D
I-li
ke
HR
1b
svS
P
svM
P/D
I
svM
P/D
I
CT
L
CT
L
svM
P/D
I
svM
P/D
I
svS
P
svM
P/D
I
CT
L
CT
L
PD
E
svM
P/D
I
DN
EV
LD
KD
IML
IK
LL
VQ
SV
PD
VL
TK
YIE
LA
IVV
DH
YM
GK
HD
QW
PL
NG
SD
INF
TG
NII
KA
YK
61
6.2
-MS
AV
ISD
GL
NR
ST
FQ
YV
WIG
LR
NW
ED
AE
SH
EG
EE
PG
K
AW
AE
ES
YC
VY
FK
AW
SG
GS
DC
IVS
K
38
4.2
3-Q
SS
EE
EA
FV
GK
WE
WS
DD
AR
LY
CID
SS
PA
K
LL
VQ
SV
PD
VT
LK
LL
VQ
SV
PD
VT
LK
FL
VA
LY
EF
K
LY
CID
SS
PA
K
TS
FQ
YV
WIG
LR
NW
ED
AE
SF
MS
SE
GP
K
12
26
.58
-GD
FV
AQ
LV
TV
K
AW
AE
ES
YC
VY
FK
38
4.2
4-G
AS
SE
EE
AF
VG
K
18
6.7
-EW
SD
DA
R
DF
YT
FD
SE
GIV
K
71
1.3
9-L
TG
LN
FY
SG
LK
LA
NV
LC
SC
SE
DS
WE
K
CS
SIT
EL
EK
LW
YQ
LV
NT
INN
IYR
AIV
VD
HG
IVT
K
30
25
15
12
11
0
50
30
25
15
12
11
0
70
C
C
C
C
C
C
C
C
C
C
C
C
28
c
28
d
28
e
28
f
29
a
29
b
29
c
29
d
29
e
29
f
30
a
30
b
BA
N8
20
13
.1
Q8
JIR
2.1
O1
30
57
.1
Q8
AV
97
.1
BA
N8
19
98
.1
BA
P3
99
48
.1
Q8
AV
97
.1
BA
N8
19
88
.1
AA
B3
48
07
.1
Q8
AV
97
.1
BA
N8
20
10
.1
BA
P3
99
64
.1
BA
P3
99
54
.1
BA
P3
99
68
.1
Q8
AV
97
.1
BA
N8
20
13
.1
BA
K6
43
83
.1
BA
K6
43
83
.1
BA
N8
19
88
.1
BA
N8
19
88
.1
BA
P3
99
68
.1
BA
N8
20
22
.1
5.0
E-0
8
3.0
E-0
4
4.0
E-0
6
1.0
E-0
4
3.0
E-0
7
2.0
E-0
6
8.0
E-0
9
4.0
E-1
1
5.0
E-0
7
8.0
E-0
8
7.0
E-0
4
7.0
E-1
0
3.0
E-0
9
1.0
E-0
7
6.5
E-0
2
2.0
E-0
6
1.4
E-0
2
5.0
E-0
5
4.5
E-0
2
8.0
E-0
7
1.0
E-0
5
4.0
E-0
4
2.0
E-0
4
2.8
E-0
2
8.0
E-0
9
2.0
E-0
6
3.0
E-0
5
1.0
E-0
4
L-a
min
o a
cid
ox
idas
e
zin
c m
etal
lop
rote
inas
e/D
I-li
ke
HR
1a
snak
e ven
om
ser
ine
pro
teas
e 2
Fla
vo
ceti
n-A
alp
ha
sub
un
it
met
allo
pro
teas
e P
-II
met
allo
pro
teas
e P
-IIa
1
Fla
vo
ceti
n-A
alp
ha
sub
un
it
C-t
yp
e le
ctin
alp
ha
sub
un
it
Fla
vo
ceti
n-A
alp
ha
sub
un
it
C-t
yp
e le
ctin
bet
a su
bu
nit
C-t
yp
e le
ctin
F I
X/X
B
C-t
yp
e le
ctin
F I
X/X
A
C-t
yp
e le
ctin
A
Fla
vo
ceti
n-A
alp
ha
sub
un
it
L-a
min
o a
cid
ox
idas
e
Fla
vo
rase
Fla
vo
rase
C-t
yp
e le
ctin
alp
ha
sub
un
it
un
kno
wn
(p
rote
in)
C-t
yp
e le
ctin
alp
ha
sub
un
it
C-t
yp
e le
ctin
A
ph
osp
ho
die
ster
ase
LA
AO
svM
P/D
I
svS
P
CT
L
svM
P/D
I
CT
L
CT
L
CT
L
CT
L
CT
L
LA
AO
svM
P/D
I
svM
P/D
I
CT
L
CT
L
PD
E
KD
GD
AN
FF
QA
LD
FK
59
0.3
0-L
VN
TL
NQ
VY
R
FL
VA
LY
TF
R
TS
FQ
YV
WL
KR
SK
NG
HP
LT
AP
PA
QL
FS
DC
SK
12
62
.57
-NF
TG
QV
IGL
AY
K
TS
FQ
YV
WIG
LR
43
4.2
0-N
VY
CG
TE
NP
FV
CK
18
8.0
8-F
QY
VW
IGL
R
32
3.0
9-Y
CY
QA
FS
KP
K
12
26
.57
-FD
GV
AQ
LV
AE
K
23
1.7
9-S
TL
QY
TS
MW
IGL
K
18
4.1
2-S
IQS
SE
EE
AF
VG
K
25
7.1
1-A
EE
SY
CV
YF
K
14
32
.74
-LV
AQ
LV
TV
K
WC
VK
AA
VA
PD
SG
DF
VA
QL
VA
EK
ST
FQ
YV
WL
KR
50
8.1
8-E
EF
LE
IAR
21
2.1
5-L
VA
TE
VM
VR
26
9.1
8-E
DG
LK
DG
LC
CY
QC
R
FS
IAG
AE
CR
LV
IVA
DD
VH
AP
P
SS
KS
LL
NV
LT
LIY
R
33
5.1
5-Q
YV
WL
KR
TS
FQ
YV
WIG
LR
12
26
.57
-GD
FV
AQ
LV
AE
K
DF
YT
FD
SE
KV
K
71
1.3
8-L
TG
LN
FY
SK
K
60
30
25
15
12
9
11
0
50
25
22
15
11
0
C
C
C
C
C
C
C
C
C
C
C
C
30
c
30
d
30
e
30
f
30
g
30
h
31
a
31
b
31
c
31
d
31
e
32
a
BA
N8
19
99
.1
BA
N8
20
13
.1
P0
C2
D5
.2
BA
N8
20
13
.1
BA
N8
20
09
.1
BA
P3
99
48
.1
BA
N8
19
88
.1
BA
P3
99
64
.1
BA
N8
20
10
.1
Q8
AV
97
.1
9.0
E-1
4
1.0
E-0
9
7.0
E-0
6
4.0
E-0
5
2.0
E-0
7
5.0
E-1
0
3.0
E-0
8
9.0
E-0
4
6.0
E-0
9
8.0
E-0
9
1.8
E-0
2
2.0
E-1
0
4.0
E-0
9
1.0
E-0
4
met
allo
pro
teas
e P
-III
L-a
min
o a
cid
ox
idas
e
Ap
ox
in I
-lik
e pro
tein
OH
AP
-1
L-a
min
o a
cid
ox
idas
e
met
allo
pro
teas
e P
-III
met
allo
pro
teas
e P
-IIa
1
C-t
yp
e le
ctin
alp
ha
sub
un
it
C-t
yp
e le
ctin
F I
X/X
B
C-t
yp
e le
ctin
bet
a su
bu
nit
Fla
vo
ceti
n-A
alp
ha
sub
un
it
svM
P/D
I
LA
AO
LA
AO
svM
P/D
I
svM
P/D
I
CT
L
CT
L
TW
VY
EIV
NT
LN
EIY
R
LY
CS
LG
NQ
LP
CR
FN
SN
LN
EV
K
TC
TG
LV
QD
YS
SR
LF
EL
VIV
AD
YR
ET
DY
EE
FL
EIA
R
34
2.1
4-D
AN
FF
QA
LD
FK
82
0.3
8-V
NL
ILQ
R
12
62
.56
-NF
TG
NII
GL
AY
K
TS
FQ
YV
WIG
LR
HY
ED
AE
SF
-71
6.3
1
AW
AE
ES
YC
VY
FK
AL
SIQ
SS
EE
EA
FV
GK
TS
FQ
YV
WL
KR
70
60
25
15
12
C
C
C
C
C
32
b
32
c
32
d
32
e
32
f
