Supplemental figures corresponding to “Degradation of lipoxygenase-derived
oxylipins by glyoxysomes from sunflower and cucumber cotyledons” by Danilo
Meyer, Cornelia Herrfurth, Florin Brodhun and Ivo Feussner
mAU
80
80
60
60
40
40
20
20
A260
100
0
DAD1 B, Sig=260,4 Ref=off (K:\_UBGB1~1\DATENC~1\DANILO\HPLC5\DM090706\COAMIX.D)
C2-CoA
CoA
C10-CoA
C12-CoA
C14-CoA
0
0
PMP1, Solvent B
100
%B
80
60
40
20
0
C4-CoA
C6-CoA
C8-CoA
20
40
60
80
100
120
140
160
180
min
80
100
120
140
160
180
min
%
80
60
40
20
0
0
0
20
40
60
20
40
60
80 100 120 140 160 180
time (min)
0
30
32,5
37,5
52,5
56,2
57,5
67,2
69,2
79,2
89,2
116,2
131,2
146,2
161,2
191,2
%B
0
0
0,6
3,6
7,2
9,0
9,0
18,0
18,0
30,0
40,0
46,0
62,0
62,0
0,0
0,0
time (min)
Supplemental Figure S1: HPLC gradient for the separation of β-oxidation
intermediates. The complex gradient between A (25 mM phosphate, pH 5.3) and B
(acetonitrile) is shown in the lower panel and the table (right panel). β-Oxidation
intermediates are separated between 75 and 130 min as indicated in the upper panel
with authentic acyl-CoA standards. CoA and acetyl-CoA elute with retention times
between 55 and 65 min.
NH2
N
N
N
-H2O → (Y-H2O)
(Y)
N
O
OH
HO
CH3
O
NH
NH
R
S
O
OHO
O
O H3C
P
O
(Z)
O
P
O
OH
OH
O
P
O
OH
426 (A)
-H2O → 408 (A-H2O)
Supplemental Figure S2: Fragmentation pattern of acyl-CoAs during MS/MS as given
in the product spectra in Supplemental Figures S4 – S9.
1
2 3
4
8:0-CoA
5
6
10:0-CoA
12:0-CoA
hydroxy12:0-CoA
hydroxy14:2-CoA
dihydroxy16:2-CoA
hydroxy16:2-CoA
hydroxy18:2-CoA
dihydroxy18:2-CoA
5
1400
1400
234 nm
260 nm
absorbance
absorbance
1200
1200
1000
1000
800
800
2
3
600
600
4
6
400
400
1
200
0
85 88
90
90
92
9594
96100
98
100
105
RT (min)
Supplemental Figure S3. Preparative HPLC-profile for turnover of 13-HOD by
glyoxysomes from etiolated cucumber. A β-oxidation assay (1 h incubation) was
prepared as described in materials and methods with the only exception that a 6 mL
reaction was used instead of 1 mL. Absorption traces for 260 nm (solid line,
indicating CoA) and 234 nm (dashed line, indicating conjugated hydroxy diene
system) are shown. Peaks 1 to 6 were collected, evaporated under nitrogen-flow and
solved in 10 µL acetonitrile:water:acetic acid (90:10:0.1) before ESI-MS analysis (see
section 2.4). The identified intermediates (Supplemental Figures S4 – S9) are
arranged in the upper panel according to their chemical composition.
8:0-CoA
[M-H]892
100
90
100
90
80
relative abundance [%]
relative abundance [%]
80
70
60
50
40
30
20
S
70
60
50
40
(A)
426
30
(Y-H2O )
545
(Z)
465
10
750
800
850
900
m/z
950
1000
1050
1100
CoA
O
20
10
0
700
(A-H2O)
408
0
400
500
(Y)
563
600
[M-H-PO3H]812
700
800
8:0-CoA
[M-H] 892
900
1000
1100
m/z
Supplemental Figure S4: ESI-MS spectra for peak 1 (RT 89.1 – 89.9 min). Precursor
ion analysis focusing on CoA-esters (m/z 408, left) and the respective MS/MS
spectra from product ion analysis of octanoyl-CoA (m/z 892, right) from a β-oxidation
assay with glyoxysomes from etiolated cucumber and 13-HOD after 1 h incubation.
dihydroxy16:2-CoA
[M-H]1032
100
90
100
S
80
relative abundance [%]
relative abundance [%]
OH
90
80
70
60
50
40
30
20
70
60
OH
(A)
426
50
40
30
10
750
800
850
900
m/z
950
1000
1050
1100
CoA
O
dihydroxy16:2-CoA
[M-H] 1032
(Z)
605
(Y-H2O )
685
(Y)
703
600
700
20
10
0
700
(A-H2O)
408
0
400
500
[M-H-PO3H]952
800
900
1000
1100
m/z
Supplemental Figure S5: ESI-MS spectra for peak 2 (RT 90.7 – 91.3 min). Precursor
ion analysis focusing on CoA-esters (m/z 408, left) and the respective MS/MS
spectra from product ion analysis of dihydroxy hexadecadienoyl-CoA (m/z 1032,
right) from a β-oxidation assay with glyoxysomes from etiolated cucumber and 13HOD after 1 h incubation. Dihydroxy hexadecadienoyl-CoA is one of the
intermediates during the second round of β-oxidation.
hydroxy14:2-CoA
[M-H] 988
100
90
100
S
70
60
50
40
dihydroxy16:2-CoA
[M-H]1032
relative abundance [%]
relative abundance [%]
80
20
(A)
426
70
hydroxy14:2-CoA
[M-H]988
60
50
40
(Y-H2O )
641
30
(Z)
561
10
750
800
850
900
m/z
950
1000
1050
1100
CoA
O
20
10
0
700
OH
90
80
30
(A-H2O)
408
0
400
500
600
[M-H-PO3H] 908
(Y)
659
700
800
900
1000
1100
m/z
Supplemental Figure S6: ESI-MS spectra for peak 3 (RT 91.3 – 92.3 min). Precursor
ion analysis focusing on CoA-esters (m/z 408, left) and the respective MS/MS
spectra from product ion analysis of hydroxy tetradecadienoyl-CoA (m/z 988, right)
from a β-oxidation assay with glyoxysomes from etiolated cucumber and 13-HOD
after 1 h incubation. Hydroxy tetradecadienoyl-CoA is the intermediate after two
rounds of β-oxidation.
10:0-CoA
[M-H] 920
100
90
100
90
50
hydroxy12:0-CoA
[M-H] 964
40
30
relative abundance [%]
relative abundance [%]
dihydroxy18:2-CoA
[M-H]1060
60
60
50
40
20
10
750
800
850
900
m/z
950
1000
1050
1100
S
CoA
(A)
426
0
hydroxy12:0-CoA
[M-H] 964
(Z) (Y-H2O )
537
617
400
500
600
O
700
800
900
1000
1100
m/z
(A-H2O)
408
100
(A-H2O)
408
90
90
CoA
O
(A)
426
50
(Y-H2O )
573
40
30
(Z)
493
20
10
10:0-CoA
[M-H] [M-H-PO3H]
920
840
(Y)
591
80
relative abundance [%]
S
80
0
OH
30
10
0
700
relative abundance [%]
70
20
60
CoA
80
70
70
S
O
OH
80
100
(A-H2O)
408
500
600
700
800
m/z
900
1000
OH
70
S
60
OH
50
dihydroxy18:2-CoA
[M-H] 1060
30
20
(Y-H2O )
713
10
1100
CoA
O
40
0
400
(A)
426
400
500
600
700
800
900
1000
1100
m/z
Supplemental Figure S7: ESI-MS spectra for peak 4 (RT 94.0 – 94.6 min). Precursor
ion analysis focusing on CoA-esters (m/z 408, upper left) and the respective MS/MS
spectra from product ion analysis of decanoyl-CoA (m/z 920, lower left), hydroxy
dodecanoyl-CoA (m/z 964, upper right) and dihydroxy octadecadienoyl-CoA (m/z
1060, lower right) from a β-oxidation assay with glyoxysomes from etiolated
cucumber and 13-HOD after 1 h incubation. Either 3-hydroxy dodecanoyl-CoA or 7hydroxy dodecanoyl-CoA can account for the compound with m/z 964. Dihydroxy
octadecadienoyl-CoA is one of the intermediates during the first round of β-oxidation.
hydroxy16:2-CoA
[M-H] 1016
100
90
100
90
80
relative abundance [%]
relative abundance [%]
80
70
10:0-CoA
[M-H]920
60
50
40
keto16:2-CoA
[M-H] 1014
30
20
OH
(A)
426
S
70
60
50
800
850
900
m/z
950
1000
1050
1100
hydroxy16:2-CoA
[M-H]1016
40
(Y-H2O )
669
30
(Z)
589
10
750
CoA
O
20
10
0
700
(A-H2O)
408
0
400
500
600
(Y)
687
[M-H-PO3H]936
700
800
900
1000
1100
m/z
Supplemental Figure S8: ESI-MS spectra for peak 5 (RT 94.6 – 96.4 min). Precursor
ion analysis focusing on CoA-esters (m/z 408, left) and the respective MS/MS
spectra from product ion analysis of hydroxy hexadecadienoyl-CoA (m/z 1016, right)
from a β-oxidation assay with glyoxysomes from etiolated cucumber and 13-HOD
after 1 h incubation. Hydroxy hexadecadienoyl-CoA is the intermediate after one
round of β-oxidation, and its accumulation was supposed by Gerhardt and
coworkers. Note that the compound with m/z 1014 also coelutes in peak 5. This mass
indicates the presence of the corresponding keto derivative of hydroxy
hexadecadienoyl-CoA and was found to a much higher content in assays containing
glyoxysomes from sunflower.
12:0-CoA
[M-H]948
100
90
90
(A-H2O)
408
80
70
60
50
40
hydroxy18:2-CoA
[M-H]1044
30
20
10
0
700
(Y-H2O )
601
100
relative abundance [%]
relative abundance [%]
80
hydroxy16:2-CoA
[M-H]1016
70
60
(A)
426
S
CoA
O
50
40
30
[M-H-PO3H]- 12:0-CoA
868 [M-H]
948
(Z)
521
20
10
750
800
850
900
m/z
950
1000
1050
1100
0
400
500
600
700
800
900
1000
1100
m/z
Supplemental Figure S9: ESI-MS spectra for peak 6 (RT 100 – 100.7 min). Precursor
ion analysis focusing on CoA-esters (m/z 408, left) and the respective MS/MS
spectrum from product ion analysis of dodecanoyl-CoA (m/z 948, right) from a βoxidation assay with glyoxysomes from etiolated cucumber and 13-HOD after 1 h
incubation. Note that dodecanoyl-CoA constitutes the intermediate with the longest
chain length that lacks the complete hydroxy diene system. The compound with m/z
1044 corresponds to traces of 13-HOD-CoA.