1
A blind test of computational technique for predicting peptide sequences
which can be cyclized by the cyanobactin macrocyclase PatGmac
.
Materials, Experimental Methods, and Experimental Data.
Peptide Synthesis
Fmoc amino acid derivatives, 2-(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (HBTU) and 2-chlorotrityl chloride resin were purchased from Novabiochem, Merck Biosciences, UK. Trifluoroacetic acid (TFA), N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF) and piperidine were obtained from Alfa Aesar, UK, and used without further purification.
The linear peptide substrates were synthesized manually in-house using the standard Fmoc-based solid phase peptide synthesis (SPPS) strategy Ref: (Chan, W.; White, P.D (1999).Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Clarendon Press, Oxford: Oxford University Press). In brief, the attachment of the first amino acid was conducted using a molar ratio of amino acid: DIEA: resin of 1.2: 4.8: 1 in DCM. Subsequent amino acids were sequentially coupled following removal of the Fmoc protecting group at each cycle. Fmoc deprotection steps were carried out with 20% piperidine in DMF (v/v) for 6 min (3x: 2min each); coupling reactions were performed in DMF using a molar ratio of amino acid: HBTU: DIEA: resin of 5: 5: 10: 1. Reactions were monitored using the Kaiser test.
The peptides were cleaved from the support and side chain protecting groups removed by treatment with a mixture consisting of 95% TFA, 2.5% triisopropylsilane (TIPS), 2.5% H2O (20 mL of mixture per g of peptide
resin, 5 hours at room temperature). The resin was then filtered and washed twice with TFA. The combined filtrates were concentrated under reduced pressure. The peptide was precipitated with cold diethyl ether and recovered by centrifugation. The peptide was submitted to LC-MS analysis and the sequence was verified by MS-MS analysis.
Protein Purification
PatGmac and LynGmac macrocyclases were cloned from genomic DNA into pHISTEV vectors and expressed inEscherichia coliBL21 (DE3) grown on auto-induction medium (Formedium Terrific broth base containing trace elements) for 48 h at 20 °C, with shaking at 200 rpm. Cells were harvested by centrifugation at 4,000 x g, 4 °C, for 15 min, and re-suspended in lysis buffer (500 mM NaCl, 20 mM Tris, pH 8.0, 20 mM Imidazole,
3mM BME, DTA-free protease inhibitor tablets (Roche) and 0.4 mg DNase (Sigma) g−1 wet cells). Cells were
lysed by passage through a cell disruptor at 30 kPSI and the lysate was cleared by centrifugation at 20,000 x g, 4 °C for 45 min. It was then filtered through a 0.45 µm membrane filter and then to a Ni-sepharose FF column (GE Healthcare) prewashed with lysis buffer. Protein was eluted with 250 mM Imidazole and passed through a HiPrep 26/10 Desalting column (GE Healthcare) into buffer containing 350mM NaCl and 10mM Bicine. Desalted protein was concentrated using Vivaspin 20 MWCO 30 000 concentrators (GE Healthcare) down to required concentrations for future enzymatic assays.
Enzyme assays
List of peptides tested
Table 1: List of peptides tested; J = Fmoc-L-propargylglycine; Pip = Piperidine; ΨP = pseudo Proline; Z =
amino Alanine; Fmoc = Fmoc protected; TFA = TFA protected; Ac = Acetylated; Amino acids inredare in D conformation.
The table also contains the reference to the origin of the peptides.
A: From the reference: (Leikoski, David P. Fewer, Jouni Jokela, Matti Wahlsten, Leo Rouhiainen, and Kaarina Sivonen, Highly Diverse Cyanobactins in Strains of the GenusAnabaena_Niina APPLIED AND
ENVIRONMENTAL MICROBIOLOGY, 2010 76 (Feb.1) pp. 701–709) B: Randomly generated sequences from RandSeq (Expasy)
C: sequences designed to generate analogues of homophymine A. See ref: (Angela Zampella, Valentina
Peptide
original
order
number
Peptide Sequence and reference to
experimental data
P(PCC)
prediction
BXD
Experimental
result
correct?
BXD
2 VPAPIPFP C (Fig. S1, S2) 8.70E-01 Yes Yes Yes
20 VTR(ThH)VTM(ThH) C (Fig. S3, S4) 8.50E-01 Yes Yes Yes
18 RTV(ThH)MTV(ThH) C (Fig. S5, S6) 8.40E-01 Yes Yes Yes
21 VTM(ThH)VTR(ThH) C (Fig. S7, S8) 5.80E-01 Yes Yes Yes
8 ZSKLQIDP C (Fig. S9, S10) 5.50E-01 Yes Yes Yes
19 MTV(ThH)RTV(ThH) C (Fig. S11, S12) 3.40E-01 Yes Yes Yes
3 QENHVFIQFP B (Fig. S13, S14) 1.80E-01 Yes Yes Yes
5 VGAGIGF(Pip) C (Fig. S15) 1.30E-01 Yes No No
4 EDWYFDHP B (Fig. S16, S17) 9.20E-02 Yes Yes Yes
22 NEFMQTGSYSGP A (Fig. S18) 9.00E-02 Yes No No
14 Z(Ac)SKLQIDP C (Fig. S19, S20) 8.30E-02 Yes Yes Yes
6 VGAGIGF(ΨP) C (Fig. S21) 7.40E-02 No No Yes
23 DCSPAKCSLLCSNP C (Fig. S22, S23) 2.40E-02 No Yes No
24 LTPGQWHMKWVP B (Fig. S24) 1.90E-02 No No Yes
10 Z(Fmoc)SKLQIDP C (Fig. S25, S26) 1.3E-02 No Yes No
1 CITJC C (Fig. S27) 7.80E-03 No No Yes
7 GSKLQIDP C (Fig. S28) 5.50E-03 No No Yes
25 VALKLALKLALPRGPRP C (Fig. S29) 1.50E-03 No No Yes
12 Z(TFA)SKLQIDP C (Fig. S30) 3.90E-04 No No Yes
13 Z(TFA)SKLQIDP C (Fig. S31) 8.00E-05 No No Yes
16 S(Ac)SKLQIDP C (Fig. S32) 4.70E-05 No No Yes
17 T(Ac)SKLQIDP C (Fig. S33) 1.10E-05 No No Yes
15 Z(Ac)SKLQIDP C (Fig. S34) 9.40E-07 No No Yes
9 ZSKLQIDP C (Fig. S35) 5.70E-10 No No Yes
3 Petek, Marie-Thérèse Adeline, Olivier Laprévôte, Anne-Marie Aubertin, Cécile Debitus, Christiane Poupat and Alain Ahond, Homophymine A, an Anti-HIV Cyclodepsipeptide from the SpongeHomophymiasp. J. Org Chem 2008 73 (14) pp 5319-5327)
The reference to the experimental data figures is also given
LC-MS data
Fi
g.
S1
:
ES
IL
C-M
S
fo
rP
ep
tid
e
2:
Li
ne
ar
VP
AP
IP
FP
-A
YD
G
+
p
E
S
IF
ul
ll
oc
k
m
s
[2
00
.0
0-40
00
.0
0]
50
0
10
00
15
00
20
00
25
00
30
00
35
00
40
00
m
/z
12
43
.6
36
4
z=
1
C61 H87 O16 N12
=
12
43
.6
35
8
24
.5
R
D
B
E
0.
48
38
pp
m
62
2.
32
31
z=
2
C47 H42
O
=
62
2.
32
30
27
.0
R
D
B
E
0.
06
69
pp
m
12
97
.5
57
1
z=
1
24
87
.2
64
9
z=
1
18
66
.4
57
2
z=
2
27
94
.9
90
7
z=
?
34
86
.4
46
0
z=
?
32
60
.6
76
5
z=
?
[M
+2
H
]
2+
[M
+H
]
5
74
0
75
0
76
0
77
0
78
0
79
0
80
0
81
0
82
0
83
0
84
0
85
0
86
0
87
0
88
0
89
0
90
0
91
0
92
0
m
/z
81
9.
47
77
z=
1
C43 H63 O8 N8
=
81
9.
47
63
16
.5
R
D
B
E
1.
41
19
m
m
u
84
1.
46
01
z=
1
C43 H62 O8 N8
N
a
=
84
1.
45
83
16
.5
R
D
B
E
1.
83
82
m
m
u
75
8.
45
46
z=
?
C37 H63 O9 N6
N
a
=
75
8.
45
49
9.
0
R
D
B
E
-0
.2
69
2
m
m
u
80
2.
48
12
z=
1
C39 H67 O10 N6
N
a
=
80
2.
48
11
9.
0
R
D
B
E
0.
11
50
m
m
u
89
0.
53
34
z=
1
C43 H67 O9 N10
N
a
=
89
0.
49
85
15
.0
R
D
B
E
34
.8
96
6
m
m
u
92
3.
12
89
z=
?
86
6.
96
02
z=
?
Fi
g.
S2
:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
2:
Cy
cl
o
[V
PA
PI
PF
P]
[M
+H
]
12
24
12
26
12
28
12
30
12
32
12
34
12
36
12
38
12
40
m
/z
12
25
.5
2
z=
1
C51 H81 O15 N14 S3
=
12
25
.5
2
18
.5
R
D
B
E
1.
09
pp
m
12
27
.5
2
z=
1
C51 H83 O15 N14 S3
=
12
27
.5
3
17
.5
R
D
B
E
-1
1.
47
pp
m
12
29
.5
2
z=
1
C51 H85 O15 N14 S3
=
12
29
.5
5
16
.5
R
D
B
E
-2
2.
19
pp
m
12
23
.6
5
z=
?
51
H95 O14 N14 S3
=
12
23
.6
3
11
.5
R
D
B
E
14
.4
5
pp
m
12
39
.3
3
z=
?
12
38
.3
2
z=
?
12
34
.5
1
z=
2
12
36
.0
3
z=
2
12
37
.4
3
z=
?
Fi
g.
S3
:
ES
IL
C-M
S
fo
rP
ep
tid
e
20
:L
in
ea
rV
TR
(T
hH
)V
TM
(T
hH
)-AY
D
G
[M
+H
]
+
H2N O HN O
OH NH S
NH HN
NH2 N O
NH O HN
O OH
NH S S
N
O NH
O HN O
OH NH
7
85
5
85
6
85
7
85
8
85
9
86
0
86
1
86
2
86
3
86
4
86
5
86
6
86
7
86
8
86
9
m
/z
85
8.
38
z=
1
C35 H60 O8 N11 S3
=
85
8.
38
11
.5
R
D
B
E
-0
.7
8
pp
m
85
9.
38
z=
1
C35 H61 O8 N11 S3
=
85
9.
39
11
.0
R
D
B
E
-6
.9
8
pp
m
85
8.
17
z=
?
86
1.
38
z=
1
C35 H63 O8 N11 S3
=
86
1.
40
10
.0
R
D
B
E
-2
9.
73
pp
m
85
7.
28
z=
?
C35 H59 O8 N11 S3
=
85
7.
37
12
.0
R
D
B
E
-1
09
.6
3
pp
m
86
5.
70
z=
?
86
4.
74
z=
?
Fi
g.
S4
:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
20
:C
yc
lo
[V
TR
(T
hH
)V
TM
(T
hH
)]
[M
+H
]
+
NH
O
N
H
O
OH
NH
S
NH
HN
NH
2
N
O
HN
O
H
N
O
HO
HN
S
S
N
12
22
12
24
12
26
12
28
12
30
12
32
12
34
12
36
12
38
12
40
m
/z
12
25
.5
16
4
z=
1
12
27
.5
18
3
z=
1
C51 H83 O15 N14 S3
=
12
27
.5
31
9
17
.5
R
D
B
E
-1
1.
06
76
pp
m
12
29
.5
20
5
z=
1
C51 H85 O15 N14 S3
=
12
29
.5
47
5
16
.5
R
D
B
E
-2
1.
99
10
pp
m
12
37
.5
04
0
z=
2
12
38
.5
07
2
z=
2
Fi
g.
S5
:
ES
IL
C-M
S
fo
rP
ep
tid
e
18
:L
in
ea
rR
TV
(T
hH
)M
TV
(T
hH
)-AY
D
G
[M
+H
]
9
85
4
85
6
85
8
86
0
86
2
86
4
86
6
86
8
87
0
87
2
87
4
87
6
87
8
88
0
88
2
88
4
88
6
88
8
89
0
89
2
m
/z
85
8.
38
54
z=
1
88
0.
36
77
z=
?
88
5.
34
89
z=
1
86
2.
09
69
z=
3
86
9.
83
51
z=
2
87
4.
69
56
z=
4
88
3.
54
44
z=
?
88
7.
34
99
z=
1
85
7.
94
81
z=
?
86
5.
69
04
z=
?
87
3.
31
63
z=
?
87
8.
42
96
z=
?
88
9.
40
58
z=
?
Fi
g.
S6
:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
18
:C
yc
lo
[R
TV
(T
hH
)M
TV
(T
hH
)]
[M
+H
]
12
22
12
23
12
24
12
25
12
26
12
27
12
28
12
29
12
30
12
31
12
32
12
33
12
34
12
35
m
/z
12
25
.5
2
z=
1
C51 H81 O15 N14 S3
=
12
25
.5
2
18
.5
R
D
B
E
-0
.4
1
pp
m
12
26
.5
2
z=
1
C51 H82 O15 N14 S3
=
12
26
.5
2
18
.0
R
D
B
E
-5
.3
9
pp
m
12
28
.5
2
z=
1
C51 H84 O15 N14 S3
=
12
28
.5
4
17
.0
R
D
B
E
-1
8.
92
pp
m
12
23
.6
5
z=
?
C51 H95 O14 N14 S3
=
12
23
.6
3
11
.5
R
D
B
E
15
.1
4
pp
m
12
31
.5
2
z=
1
C51 H87 O15 N14 S3
=
12
31
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6
15
.5
R
D
B
E
-3
3.
97
pp
m
12
35
.2
0
z=
?
Fi
g.
S7
:
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IL
C-M
S
fo
rP
ep
tid
e
21
:L
in
ea
rV
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((T
hH
)V
TR
(T
hH
)-AY
D
G
[M
+H
]
11
85
5
85
6
85
7
85
8
85
9
86
0
86
1
86
2
86
3
86
4
86
5
86
6
86
7
86
8
86
9
87
0
87
1
87
2
87
3
87
4
m
/z
85
8.
38
z=
1
C35 H60 O8 N11 S3
=
85
8.
38
11
.5
R
D
B
E
-1
.1
4
pp
m
86
0.
38
z=
1
C35 H62 O8 N11 S3
=
86
0.
39
10
.5
R
D
B
E
-2
0.
03
pp
m
86
2.
38
z=
1
C35 H64 O8 N11 S3
=
86
2.
41
9.
5
R
D
B
E
-3
8.
07
pp
m
85
5.
96
z=
?
86
9.
43
z=
1
C35 H71 O8 N11 S3
=
86
9.
46
6.
0
R
D
B
E
-4
2.
91
pp
m
86
7.
82
z=
?
87
1.
30
z=
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[M
+H
]
+
Fi
g.
S8
:
ES
IL
C-M
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fo
rc
yc
lic
Pe
pt
id
e
21
:C
yc
lo
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TM
((T
hH
)V
TR
(T
hH
86
12
88
12
90
12
92
12
94
12
96
12
98
13
00
13
02
13
04
13
06
13
08
13
10
13
12
13
14
13
16
13
18
m
/z
12
92
.6
50
0
z=
1
C56 H90 O20 N15
=
12
92
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48
1
19
.5
R
D
B
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1.
48
39
pp
m
[M
+H
]
+
Fi
g.
S9
:
ES
IL
C-M
S
fo
rP
ep
tid
e
8:
Li
ne
ar
ZS
KL
Q
ID
P
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13
86
5
86
6
86
7
86
8
86
9
87
0
87
1
87
2
87
3
87
4
87
5
87
6
87
7
m
/z
86
5.
36
z=
1
C38 H63 O12 N11
=
86
5.
47
13
.0
R
D
B
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5
pp
m
86
8.
49
z=
1
C38 H66 O12 N11
=
86
8.
49
11
.5
R
D
B
E
1.
61
pp
m
86
9.
49
z=
1
C38 H67 O12 N11
=
86
9.
50
11
.0
R
D
B
E
-3
.9
3
pp
m
86
6.
36
z=
1
C38 H64 O12 N11
=
86
6.
47
12
.5
R
D
B
E
-1
28
.7
4
pp
m
86
7.
36
z=
1
C38 H65 O12 N11
=
86
7.
48
12
.0
R
D
B
E
-1
36
.7
5
pp
m
87
0.
28
z=
1
86
4.
29
z=
1
86
5.
28
z=
1
87
2.
26
z=
?
87
1.
28
z=
1
87
4.
40
z=
?
86
8.
85
z=
?
87
4.
86
z=
?
87
7.
41
z=
?
87
5.
40
z=
?
Fi
g.
S1
0:
ES
IL
C-M
S
fo
rP
ep
tid
e
8:
Cy
cl
o
[Z
SK
LQ
ID
P]
[M
+H
]
12
20
12
22
12
24
12
26
12
28
12
30
12
32
12
34
12
36
12
38
12
40
12
42
12
44
12
46
12
48
12
50
12
52
m
/z
12
25
.5
2
z=
1
C51 H81 O15 N14 S3
=
12
25
.5
2
18
.5
R
D
B
E
0.
19
pp
m
12
28
.5
2
z=
1
C51
H84
O15
N14
S3
=
12
28
.5
4
17
.0
R
D
B
E
-1
8.
72
pp
m
12
22
.4
1
z=
?
C51 H78 O15 N14 S3
=
12
22
.4
9
20
.0
R
D
B
E
-6
7.
38
pp
m
12
44
.3
9
z=
1
12
47
.4
9
z=
1
12
31
.5
2
z=
1
C51 H87 O15 N14 S3
=
12
31
.5
6
15
.5
R
D
B
E
-3
9.
12
pp
m
12
49
.5
0
z=
1
12
51
.8
3
z=
?
12
40
.4
9
z=
1
C51 H96 O15 N14 S3
=
12
40
.6
3
11
.0
R
D
B
E
-1
12
.2
4
pp
m
12
35
.1
6
z=
?
Fi
g.
S1
1:
ES
IL
C-M
S
fo
rP
ep
tid
e
19
:L
in
ea
rM
TV
(T
hH
)R
TV
(T
hH
)-AY
D
G
[M
+H
]
15
85
2
85
3
85
4
85
5
85
6
85
7
85
8
85
9
86
0
86
1
86
2
86
3
86
4
86
5
86
6
86
7
86
8
86
9
87
0
87
1
87
2
m
/z
85
8.
38
z=
1
C35 H60 O8 N11 S3
=
85
8.
38
11
.5
R
D
BE
-0
.2
8
pp
m
86
0.
38
z=
1
C35
H62
O8
N11
S3
=
86
0.
39
10
.5
R
D
B
E
-1
9.
11
pp
m
86
2.
38
z=
1
C35 H64 O8 N11 S3
=
86
2.
41
9.
5
R
D
BE
-3
9.
27
pp
m
85
6.
56
z=
?
C35 H74 O7 N11 S3
=
85
6.
49
4.
5
R
D
B
E
73
.9
5
pp
m
86
9.
42
z=
?
C35
H71
O8
N11
S3
=
86
9.
46
6.
0
R
D
BE
-4
9.
37
pp
m
86
6.
81
z=
?
[M
+H
]
+
Fi
g.
S1
2:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
19
:C
yc
lo
[M
TV
(T
hH
)R
TV
(T
hH
Fi
g.
S1
3:
ES
IL
C-M
S
fo
rP
ep
tid
e
3:
Li
ne
ar
Q
EN
H
VF
IQ
FP
-A
YD
G
[M
+2
H
]
2+
2.
5
83
3.
0
83
3.
5
83
4.
0
83
4.
5
83
5.
0
83
5.
5
83
6.
0
83
6.
5
83
7.
0
m
/z
83
3.
39
z=
2
C77 H10
8
O23 N19
=
83
3.
39
33
.5
R
D
B
E
-2
.8
7
pp
m
83
3.
89
z=
2
C77 H10
9
O23 N19
=
83
3.
90
33
.0
R
D
B
E
-6
.9
2
pp
m
83
4.
39
z=
2
C77 H11
0
O23 N19
=
83
4.
40
32
.5
R
D
B
E
-1
1.
12
pp
m
83
5.
39
z=
2
C77 H11
2
O23 N19
=
83
5.
41
31
.5
R
D
B
E
-1
8.
02
pp
m
83
6.
38
z=
2
C77 H11
4
O23 N19
=
83
6.
42
30
.5
R
D
B
E
-3
7.
35
pp
17 39 .0 12 39 .5 12 40 .0 12 40 .5 12 41 .0 12 41 .5 12 42 .0 12 42 .5 12 43 .0 12 43 .5 12 44 .0 12 44 .5 12 45 .0 m /z 12 40 .6 12 4 z= 1 C59 H82 O15 N15 = 12 40 .6 10 9 26 .5 R D B E 1. 20 40 pp m 12 41 .6 14 9 z= 1 C59 H83 O15 N15 = 12 41 .6 18 8 26 .0 R D B E -3 .1 33 0 pp m 12 42 .6 18 3 z= 1 C59 H84 O15 N15 = 12 42 .6 26 6 25 .5 R D B E -6 .6 77 0 pp m 12 43 .6 19 6 z= 1 C59 H85 O15 N15 = 12 43 .6 34 4 25 .0 R D B E -1 1. 88 41 pp m 12 41 .1 07 1 z= ? 12 44 .6 16 0 z= 1 C59 H86 O15 N15 = 12 44 .6 42 2 24 .5 R D B E -2 1. 10 40 pp m 12 39 .2 27 1 z= ? 12 40 .2 51 7 z= ? 12 43 .0 24 5 z= ? 12 42 .1 18 3 z= ?
Fi
g.
S1
4:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
3:
Cy
cl
o
[Q
EN
H
VF
IQ
FP
]
[M
+H
]
+H
HN
O
O
NH
O
OH
O
NH
O
O
NH
2NH
O
N
HN
NH
O
HN
O
NH
O
H
HN
O
H
2N
O
HN
19
[M
+2
H
]
2 +Fi
g.
S1
6:
ES
IL
C-M
S
fo
rP
ep
tid
e
4:
Li
ne
ar
ED
W
YF
D
H
P
–
AY
D
G
T M S + p N S IF ul ll oc k m s [5 00 .0 0-35 00 .0 0] 75 7. 5 75 8. 0 75 8. 5 75 9. 0 75 9. 5 76 0. 0 76 0. 5 76 1. 0 76 1. 5 76 2. 0 m /z 0 10 20 30 40 50 60 70 80 90 10 0 75 7. 80 z= 2 C71 H85 O23 N15 = 75 7. 80 37 .0 R D B E 1. 86 pp m 75 8. 80 z= 2 C71 H87 O23 N15 = 75 8. 80 36 .0 R D B E -7 .7 8 pp m 75 9. 80 z= 2 76 0. 30 z= 2 76 0. 80 z= 2 76 1. 30 z= 2 76 0. 04 z= ? 76 1. 76 z= ? C71 H93 O23 N15 = 76 1. 83 33 .0 R D B E -9 0. 96 pp m 76 0. 68 z= ? 76 1. 09 z= ?H
2N
S
IF
ul
ll
oc
k
m
s
[5
00
.0
0-35
00
.0
0]
10
90
10
91
10
92
10
93
10
94
10
95
10
96
10
97
10
98
m
/z
10
90
.4
3
z=
1
C53 H60 O15 N11
=
10
90
.4
3
29
.5
R
D
B
E
0.
62
pp
m
10
91
.4
3
z=
1
C53 H61 O15 N11
=
10
91
.4
3
29
.0
R
D
B
E
-4
.1
1
pp
m
10
92
.4
3
z=
1
C53 H62 O15 N11
=
10
92
.4
4
28
.5
R
D
B
E
-9
.9
4
pp
m
10
93
.4
3
z=
1
10
97
.2
2
z=
3
10
94
.4
7
z=
?
10
96
.8
8
z=
3
10
97
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5
z=
3
10
95
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6
z=
6
10
93
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7
z=
?
10
91
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6
z=
?
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9
?
10
90
.7
1
z=
?
10
95
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0
z=
?
10
90
.2
4
z=
?
10
91
.1
0
z=
?
10
92
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4
z=
?
10
98
.2
9
z=
?
10
96
.0
5
z=
?
[M
+H
]
+
Fi
g.
S1
7:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
4:
Cy
cl
o
[E
D
W
YF
D
H
21
17
23
17
24
17
25
17
26
17
27
17
28
17
29
17
30
m
/z
17
23
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85
7
z=
1
C74 H10
3
O28 N18
S
=
17
23
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90
4
32
.5
R
D
B
E
-2
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68
6
pp
m
17
25
.6
90
1
z=
1
C74 H10
5
O28 N18
S
=
17
25
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06
1
31
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R
D
B
E
-9
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87
7
pp
m
17
27
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88
4
z=
1
C74 H10
7
O28 N18
S
=
17
27
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21
7
30
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R
D
B
E
-1
9.
32
46
pp
m
Fi
g.
S1
8:
ES
IL
C-M
S
fo
rP
ep
tid
e
22
:L
in
ea
rN
EF
M
Q
TG
SY
SG
P
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G
[M
+H
]
66
7.
5
66
8.
0
66
8.
5
66
9.
0
66
9.
5
67
0.
0
67
0.
5
m
/z
66
7.
83
39
z=
2
C58 H93 O21 N15
=
66
7.
83
30
20
.0
R
D
B
E
1.
33
04
pp
m
66
8.
83
58
z=
2
C58 H95 O21 N15
=
66
8.
84
08
19
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R
D
B
E
-7
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42
1
pp
m
66
9.
33
70
z=
2
C58 H96 O21 N15
=
66
9.
34
47
18
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R
D
B
E
-1
1.
55
81
pp
m
67
0.
34
03
z=
2
C58 H98 O21 N15
=
67
0.
35
25
17
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R
D
B
E
-1
8.
20
62
pp
m
[M
+2
H
]
2+
Fi
g.
S1
9:
ES
IL
C-M
S
fo
rP
ep
tid
e
14
:L
in
ea
rZ
(A
c)
SK
LQ
ID
P
-A
YD
G
H
N
NH
2NH
O
O
HN
O
HN
O
NH
NH
O
HN
O
O
O
OH
N
NH
O
O
HN
NH
O
OH
O
O
NH
O
HO
O
H
2N
OH
H
2N
HO
23
90
9
91
0
91
1
91
2
91
3
91
4
91
5
91
6
91
7
91
8
91
9
92
0
m
/z
91
0.
50
z=
1
C40 H68 O13 N11
=
91
0.
50
12
.5
R
D
B
E
1.
55
pp
m
91
1.
50
z=
1
C40 H69 O13 N11
=
91
1.
51
12
.0
R
D
B
E
-5
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6
pp
m
91
2.
51
z=
1
C40 H70 O13 N11
=
91
2.
51
11
.5
R
D
B
E
-1
0.
79
pp
m
91
0.
81
z=
?
91
0.
30
z=
?
91
1.
14
z=
?
91
6.
73
z=
?
C40 H90 O12 N11
=
91
6.
68
1.
5
R
D
B
E
61
.8
1
pp
m
[M
+H
]
+
Fi
g.
S2
0:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
14
:C
yc
lo
[Z
(A
c)
SK
LQ
ID
11
52
11
53
11
54
11
55
11
56
11
57
11
58
11
59
m
/z
11
53
.5
49
2
z=
1
C53 H77 O17 N12
=
11
53
.5
52
4
21
.5
R
D
B
E
-2
.7
92
1
pp
m
11
54
.5
52
0
z=
1
C53 H78 O17 N12
=
11
54
.5
60
2
21
.0
R
D
B
E
-7
.1
35
4
pp
m
11
55
.5
54
8
z=
1
C53 H79 O17 N12
=
11
55
.5
68
1
20
.5
R
D
B
E
-1
1.
47
12
pp
m
11
55
.2
57
3
z=
?
11
57
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60
4
z=
1
C53 H81 O17 N12
=
11
57
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83
7
19
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R
D
B
E
-2
0.
12
03
pp
m
11
51
.8
42
8
z=
?
O17 N12
=
11
51
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24
6
8.
5
R
D
B
E
10
2.
62
48
pp
m
11
52
.8
37
3
z=
?
C51 H10
0
O17 N12
=
11
52
.7
32
4
8.
0
R
D
B
E
90
.9
83
8
pp
m
11
58
.3
99
3
z=
?
Fi
g.
S2
1:
ES
IL
C-M
S
fo
rP
ep
tid
e
6
: L
in
ea
r V
G
AG
IG
F(
Ψ
P)
-A
YD
G
[M
+H
]
25
99
6
99
8
10
00
10
02
10
04
10
06
10
08
10
10
10
12
10
14
10
16
10
18
m
/z
10
00
.9
32
1
z=
2
C82 H13
3
O30 N22 S3
=
10
00
.9
35
5
27
.5
R
D
B
E
-3
.3
84
1
pp
m
10
11
.4
21
3
z=
2
10
12
.4
22
2
z=
2
10
02
.4
32
5
z=
2
10
13
.4
23
7
z=
2
10
18
.4
63
0
z=
?
10
08
.9
31
9
z=
?
10
03
.4
30
2
z=
2
Fi
g.
S2
2:
ES
IL
C-M
S
fo
rP
ep
tid
e
23
:L
in
ea
rD
CS
PA
KC
SL
LC
SN
P
-A
YD
G
[M
+2
H
]
81
5
81
6
81
7
81
8
81
9
82
0
82
1
82
2
82
3
82
4
m
/z
81
6.
86
z=
2
C66 H11
1
O23 N19 S3
=
81
6.
86
21
.0
R
D
B
E
-0
.9
7
m
m
u
81
8.
36
z=
2
C66 H11
4
O23 N19 S3
=
81
8.
37
19
.5
R
D
B
E
-1
1.
82
m
m
u
82
4.
01
z=
?
C64 H14
9
O23 N19 S3
=
82
4.
01
0.
0
R
D
B
E
2.
29
m
m
u
81
5.
86
z=
?
C66 H10
9
O23 N19 S3
=
81
5.
85
22
.0
R
D
B
E
0.
70
m
m
u
81
9.
49
z=
?
C66 H14
4
O23 N17 S3
=
81
9.
49
3.
5
R
D
B
E
2.
66
m
m
u
Fi
g.
S2
3:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
23
:C
yc
lo
[D
CS
PA
KC
SL
LC
SN
P]
[M
+2
H
]
27
18
84
.5
18
85
.0
18
85
.5
18
86
.0
18
86
.5
18
87
.0
18
87
.5
18
88
.0
18
88
.5
18
89
.0
18
89
.5
18
90
.0
18
90
.5
18
91
.0
18
91
.5
m
/z
18
86
.9
05
2
z=
1
C89 H12
6
O22 N22
S
=
18
86
.9
13
2
38
.0
R
D
B
E
-4
.2
78
9
pp
m
18
87
.9
07
1
z=
1
C89 H12
7
O22 N22
S
=
18
87
.9
21
1
37
.5
R
D
B
E
-7
.3
86
9
pp
m
18
88
.9
11
1
z=
1
C89 H12
8
O22 N22
S
=
18
88
.9
28
9
37
.0
R
D
B
E
-9
.3
93
0
pp
m
Fi
g.
S2
4:
ES
IL
C-M
S
fo
rP
ep
tid
e
24
:L
in
ea
rL
TP
G
Q
W
H
M
KW
VP
-A
YD
G
[M
+H
]
S
IF
ul
lm
s
[5
00
.0
0-35
00
.0
0]
75
5
75
6
75
7
75
8
75
9
76
0
76
1
76
2
76
3
76
4
76
5
76
6
76
7
m
/z
75
7.
86
z=
2
C71 H10
1
O22 N15
=
75
7.
86
29
.0
R
D
B
E
1.
08
pp
m
75
9.
37
z=
2
C71 H10
4
O22 N15
=
75
9.
37
27
.5
R
D
B
E
-1
0.
84
pp
m
76
0.
37
z=
2
C71 H10
6
O22 N15
=
76
0.
38
26
.5
R
D
B
E
-1
7.
35
pp
m
76
4.
38
z=
2
C71 H11
4
O22 N15
=
76
4.
41
22
.5
R
D
B
E
-4
7.
83
pp
m
76
6.
36
z=
2
C71 H11
8
O22 N15
=
76
6.
43
20
.5
R
D
B
E
-8
6.
84
pp
m
75
6.
85
z=
?
C71 H99 O22 N15
=
75
6.
85
30
.0
R
D
B
E
1.
18
pp
m
Fi
g.
S2
5:
ES
IL
C-M
S
fo
rP
ep
tid
e
10
:L
in
ea
rZ
(F
m
oc
)S
KL
Q
ID
P
-A
YD
G
[M
+2
H
]
29 M S + p N S IF ul lm s [5 00 .0 0-35 00 .0 0] 10 88 10 89 10 90 10 91 10 92 10 93 10 94 10 95 10 96 10 97 10 98 10 99 11 00 11 01 11 02 11 03 m /z 0 10 20 30 40 50 60 70 80 90 10 0 10 90 .5 6 z= 1 C53 H76 O14 N11 = 10 90 .5 6 21 .5 R D B E 0. 28 pp m 10 91 .5 6 z= 1 C53 H77 O14 N11 = 10 91 .5 6 21 .0 R D B E -4 .0 4 pp m 10 92 .5 6 z= 1 C53 H78 O14 N11 = 10 92 .5 7 20 .5 R D B E -7 .8 4 pp m 10 98 .8 3 z= 3 C53 H10 0 O13 N11 = 10 98 .7 5 9. 5 R D B E 73 .1 5 pp m 11 02 .4 7 z= ? 10 89 .5 8 z= ? C53 H75 O14 N11 = 10 89 .5 5 22 .0 R D B E 24 .0 8 pp m 10 95 .4 8 z= 2 C53 H81 O14 N11 = 10 95 .6 0 19 .0 R D B E -1 01 .4 6 pp m 10 91 .0 6 z= ? 10 91 .9 7 z= ?
Fi
g.
S2
6:
ES
IL
C-M
S
fo
rc
yc
lic
Pe
pt
id
e
10
:C
yc
lo
[Z
(F
m
oc
)S
KL
Q
ID
P]
[M
+H
]
+NH
N
H
HN
O
O
HN
O
N
H
O
HN
NH
O
NH
O
O
O
HO
N
O
O
H
2N
NH
2OH
93
8
93
9
94
0
94
1
94
2
94
3
94
4
94
5
94
6
94
7
94
8
94
9
95
0
95
1
m
/z
94
0.
35
z=
1
C39 H58 O14 N9 S2
=
94
0.
35
15
.5
R
D
B
E
0.
35
pp
m
94
1.
36
z=
1
C39 H59 O14 N9 S2
=
94
1.
36
15
.0
R
D
B
E
-4
.7
8
pp
m
94
3.
36
z=
1
C39 H61 O14 N9 S2
=
94
3.
38
14
.0
R
D
B
E
-2
2.
66
pp
m
93
8.
34
z=
?
39
H56 O14 N9 S2
=
93
8.
34
16
.5
R
D
B
E
0.
77
pp
m
95
0.
43
z=
1
C39 H68 O14 N9 S2
=
95
0.
43
10
.5
R
D
B
E
-0
.4
9
pp
m
94
8.
32
z=
?
C39 H66 O14 N9 S2
=
94
8.
42
11
.5
R
D
B
E
-1
01
.0
6
pp
m
94
2.
24
z=
?
[M
+H
]
+
Fi
g.
S2
7:
ES
IL
C-M
S
fo
rP
ep
tid
e
1:
Li
ne
ar
CI
TJ
C
-A
YD
31
Fi
g.
S2
8:
ES
IL
C-M
S
fo
rP
ep
tid
e
7:
Li
ne
ar
G
SK
LQ
ID
P
-A
YD
G
[M
+H
]
+
12
63
12
64
12
65
12
66
12
67
12
68
12
69
12
70
12
71
12
72
m
/z
0
0
12
63
.6
2
z=
1
C
55H
87O
20N
14=
12
63
.6
2
19
.5
R
D
B
E
1.
13
pp
m
12
65
.6
3
z=
1
12
66
.6
3
z=
1
12
64
.1
2
z=
?
12
65
.1
2
z=
?
12
70
.9
3
z=
?
12
62
.9
4
z=
?
12
72
.1
3
z=
?
12
68
.2
7
z=
?
12
67
.6
3
z=
?
12
66
.0
6
z=
[M
+H
]
+
S
IF
ul
lm
s
[1
00
.0
0-20
00
.0
0]
10
81
.0
10
81
.5
10
82
.0
10
82
.5
10
83
.0
10
83
.5
10
84
.0
10
84
.5
m
/z
10
82
.1
61
9
z=
2
C10
1
H17
5
O24 N28
=
10
82
.1
66
2
28
.5
R
D
B
E
-3
.9
62
2
pp
m
10
82
.6
63
1
z=
2
C10
1
H17
6
O24 N28
=
10
82
.6
70
1
28
.0
R
D
B
E
-6
.4
46
6
pp
m
10
83
.1
64
1
z=
2
10
83
.6
65
9
z=
2
10
84
.1
67
6
z=
2
10
81
.1
07
1
z=
?
C10
1
H17
3
O24 N28
=
10
81
.1
58
3
29
.5
R
D
B
E
-4
7.
42
57
pp
m
Fi
g.
S2
9:
ES
IL
C-M
S
fo
rP
ep
tid
e
25
:L
in
ea
rV
AL
KL
AL
KL
AL
PR
G
PR
P
–
AY
D
G
NH2 O
HN O NH O
HN O
NH2 NH
O HN
O NH O HN
O
NH2 NH
O HN
O NH O
N O HN
O N
H HN
NH2
N H
O N O
N H
O NH HN
NH2
N O HN O
NH O HO HN
O
33
[M
+H
]
+
Fi
g.
S3
0:
ES
IL
C-M
S
fo
rP
ep
tid
e
12
:L
in
ea
rZ
(T
FA
)S
KL
Q
ID
P
-A
YD
69
4.
5
69
5.
0
69
5.
5
69
6.
0
69
6.
5
69
7.
0
69
7.
5
69
8.
0
69
8.
5
69
9.
0
69
9.
5
70
0.
0
70
0.
5
70
1.
0
70
1.
5
m
/z
69
4.
82
z=
2
C58 H90 O21 N15 F3
=
69
4.
82
20
.0
R
D
B
E
1.
41
pp
m
69
5.
82
z=
2
C58 H92 O21 N15 F3
=
69
5.
83
19
.0
R
D
B
E
-1
1.
41
pp
m
70
0.
33
z=
?
C58 H10
1
O21 N15 F3
=
70
0.
36
14
.5
R
D
B
E
-4
4.
54
pp
m
69
7.
86
z=
2
C58
H96
O21
N15
F3
=
69
7.
84
17
.0
R
D
B
E
22
.8
7
pp
m
Fi
g.
S3
1:
ES
IL
C-M
S
fo
rP
ep
tid
e
13
:L
in
ea
rZ
(T
FA
)
SK
LQ
I
D
P
-A
YD
G
[M
+2
H
]
35
13
30
13
32
13
34
13
36
13
38
13
40
13
42
13
44
13
46
13
48
13
50
13
52
13
54
13
56
m
/z
13
35
.6
4
z=
1
C58 H91 O22 N14
=
13
35
.6
4
20
.5
R
D
B
E
-1
.4
5
pp
m
13
37
.6
5
z=
1
C58 H93 O22 N14
=
13
37
.6
6
19
.5
R
D
B
E
-9
.2
3
pp
m
13
39
.6
5
z=
1
C58 H95 O22 N14
=
13
39
.6
7
18
.5
R
D
B
E
-1
5.
52
pp
m
13
33
.4
7
z=
?
C58 H89 O22 N14
=
13
33
.6
3
21
.5
R
D
B
E
-1
19
.9
8
pp
m
Fi
g.
S3
2:
ES
IL
C-M
S
fo
rP
ep
tid
e
16
:L
in
ea
rS
(A
c)
SK
LQ
ID
P
-A
YD
G
[M
+H
]
13
48
13
49
13
50
13
51
13
52
13
53
13
54
13
55
13
56
13
57
13
58
13
59
13
60
m
/z
13
49
.6
6
z=
1
C59 H93 O22 N14
=
13
49
.6
6
20
.5
R
D
B
E
-1
.8
2
pp
m
13
51
.6
6
z=
1
C59 H95 O22 N14
=
13
51
.6
7
19
.5
R
D
B
E
-8
.1
5
pp
m
13
53
.6
6
z=
1
C59 H97 O22 N14
=
13
53
.6
9
18
.5
R
D
B
E
-1
8.
71
pp
m
13
50
.1
6
z=
?
13
48
.5
1
z=
?
H92 O22 N14
=
13
48
.6
5
21
.0
R
D
B
E
-1
01
.9
3
pp
m
13
51
.1
6
z=
?
13
55
.6
6
z=
?
C59
H99
O22
N14
=
13
55
.7
1
17
.5
R
D
B
E
-3
5.
27
pp
m
13
57
.9
4
z=
?
C58 H12
9
O21 N14
=
13
57
.9
5
1.
5
R
D
B
E
-0
.1
3
pp
m
[M
+H
]
+
