PresentationPDF Available

Compound Discoverer as a tool for bioprospecting and detection of defense compounds during fungal infection of Spruce wood

Authors:

Abstract

Our presentation on how we used Compound Discoverer (ver. 2.1) as a tool for detecting defense compounds in fungal infected Spruce wood, held at the "8th International Proteome Discoverer User's Meeting and 4th Compound Discoverer User's Meeting" (December 2019), can be downloaded from here!



Marit Almvik, Nina Elisabeth Nagy, Hans Ragnar Norli, Ari
Hietala, Sven-Roar Odenmarck, Monica Fongen, Anas Kamleh
NIBIO & Thermo Fisher Scienti$c
marit.almvik@nibio.no
1/6/20 2
Thermo Scienti$c Q Exactive
()
Thermo Scienti$c Q Exactive
()Photo: Erling Fløistad
(NIBIO)
1/6/20 3

 !"#$
%
&'"
%&%#$
-A research institute for
agriculture, forestry and
the environment
-700 employees
Photos: Erling Fløistad
(NIBIO)
Pesticides and natural
products
Pesticides and natural
products Photo: Erling Fløistad
(NIBIO)
Why Norway Spruce (()?
1/6/20 6
BERGE
N
¼ of our trees are infected with root rot
fungi
Spruce
(()
1/6/20 8
Heartwo
od Anili
n
wood
Reaction
zone
Sapwood
What
defense
compound
s are
produced
by the tree
in the
reaction
zone?
Could these
compounds
be exploited
as e.g.
fungicides?
Photo: Magnar Sandland
(NIBIO)
Spruce
(()
1/6/20 9
Heartwo
od Anili
n
wood
Reaction
zone
Sapwood
What
defense
compound
s are
produced
by the tree
in the
reaction
zone?
Photo: Magnar Sandland
(NIBIO)
Kickstart with Compound Discoverer software:
Got a 2-visits contract with Compound Discoverer expert Dr.
Anas Kamleh at Thermo Fisher Scienti$c Europe (Sweden)!
)"*
1/6/20 10
+,)##)
"-
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6,7  %

2 88 9:
Optimized:
-The dilution factor of extract (1:10 in AcN)
-Injection volume (1 µl)
-The mobile phase gradient
-Scan range
-ddMS2-settings: AGC targets, IT and Apex trigger
Optimized:
-The dilution factor of extract (1:10 in AcN)
-Injection volume (1 µl)
-The mobile phase gradient
-Scan range
-ddMS2-settings: AGC targets, IT and Apex trigger
3.!9:;
"","
,4
/"6,<,4"
5"

4=
.
9:2 <  <+ 
+ <88
2+< <>+> <
5 <?
/"6,<,4"
5"

4=
.
9:2 <  <+ 
+ <88
2+< <>+> <
5 <?
In order to create good quality MS2
spectrums:
In order to create good quality MS2
spectrums:
* Wood samples extracted with 80%
methanol in water
* 72 spruce wood extracts analyzed with
FMS
* pQC-samples analyzed with FMS-ddMS2
(pool of all samples, pool of reaction zone
and pool of anilin zone)
* Wood samples extracted with 80%
methanol in water
* 72 spruce wood extracts analyzed with
FMS
* pQC-samples analyzed with FMS-ddMS2
(pool of all samples, pool of reaction zone
and pool of anilin zone)
Spruce
(()
1/6/20 16
Heartwo
od Anili
n
wood
Reaction
zone
Sapwood
Sampling spots
Fungal infected:
SW = Sapwood
RZ = Reaction
zone
ANW = Anilin
wood
HW =
Heartwood
Healthy:
Healthy HW
Healthy SW
Sampling spots
Fungal infected:
SW = Sapwood
RZ = Reaction
zone
ANW = Anilin
wood
HW =
Heartwood
Healthy:
Healthy HW
Healthy SW
Wood
Type
Wood
Type
Tree
height
Tree
height
Replicate
s
Replicate
s
CD version
2.1
CD version
2.1
The PCA
loadings plot
aided in $nding
those
compounds
that created
the diJerence
between the
wood zones
1900
features/compounds
upregulated in the
reaction zone as
compared to the
healthy sapwood
1900
features/compounds
upregulated in the
reaction zone as
compared to the
healthy sapwood
I wish I could
change the
order of the
samples..
I wish I could
change the
order of the
samples..
1/6/20 24
CD reports 4 adducts of alfa-Conidendrin
[+FA, +Cl, 2M-H, 2M+FA] as separate
compounds with separate formulas, even
though they belong to the same
compound with identical retention time.
CD reports 4 adducts of alfa-Conidendrin
[+FA, +Cl, 2M-H, 2M+FA] as separate
compounds with separate formulas, even
though they belong to the same
compound with identical retention time.
1/6/20 25
MS1 of alfa-
Conidendrin
MS1 of alfa-
Conidendrin
Another MS1 of alfa-Conidendrin (a
scan 0.002 minutes before)
m/z 757.251 is [2M+FA-H]- of m/z
355.119 and not [M-H]-
Another MS1 of alfa-Conidendrin (a
scan 0.002 minutes before)
m/z 757.251 is [2M+FA-H]- of m/z
355.119 and not [M-H]-
1/6/20 27
CD reports many lines for the same
compound
CD reports many lines for the same
compound
1/6/20 28
1. Increase the weight of M-H to 90, this will favour M-H when
confusion arises,
2. Add the ion “M-FA-H”; then when the software is confused
of which adduct to be reported, it will report two lines for
the compound. One of them will be your correct compound,
Might be $xed by optimizing Ion
De$nition?
I was adviced to:
Might be $xed by optimizing Ion
De$nition?
I was adviced to:
But it didn’t really solve the
problem..
But it didn’t really solve the
problem..
The top predicted
formula was most often
correct!
The top predicted
formula was most often
correct!
ChemSpider suggestions can be confusing..
Turns out that a-Conidendrin is the same as
'8'8$<7<"3<8<><"3<<
"3"$<5<"3<>88<"<2)<
:@A@4A:<<*
ChemSpider suggestions can be confusing..
Turns out that a-Conidendrin is the same as
'8'8$<7<"3<8<><"3<<
"3"$<5<"3<>88<"<2)<
:@A@4A:<<*
'
--0"&%B,(
".99-C--94 2?9 49"<<4<2<-$
'
--0"&%B,(
".99-C--94 2?9 49"<<4<2<-$
1/6/20 31
FISh Coverage of
Hydroxymatairesinol
FISh Coverage of
Hydroxymatairesinol
Score
10%
Score
10%
In-house mzVault
MS2-spectrum
match
Hydroxymataires
inol (with
reference
standard)
In-house mzVault
MS2-spectrum
match
Hydroxymataires
inol (with
reference
standard)
Score
97%
Score
97%
Conidendric
acid
Conidendric
acid
Secoisolaricires
inol
Secoisolaricires
inol
HMR-
GGE
HMR-
GGE
Isolariciresinol-
GGE
Isolariciresinol-
GGE
Dehydroabietic
acid
Dehydroabietic
acid
Unknown
C12H8O7
Unknown
C12H8O7
1/6/20 33
Compound Formula MolW. Max.
Area
Hydroxymatairesinol* C20H22O7374.139
62
243 799
143
Lignan
α-Conidendrin* C20H20O6356.126
07
80 253
037
Lignan
Conidendric acidGC-MS C20H22O7374.136
67
51 276
089
Lignan
TodolocatolGC-MS C20H24O7376.152
44
28 976
934
Lignan
OH-LariciresinolGC-MS C20H20O7376.152
61
19 490
474
Lignan
Isorhapontin* C21H24O9420.142
53
16 697
837
Stilbene
glucoside
Conidendrin
guaiacylglyceryl ether
C30H32O10 552.199
79
14 878
644
Lignan
HMR GGE C30H34O11 570.210
07
10 092
664
Lignan
HMR Dimer C40H42O14 746.257
82
7 589 185 Lignan
Secoisolariciresinol* C20H26O6362.173
07
6 171 855 Lignan
Oxo-Dehydroabietic acid C20H26O3314.188
50
5 998 380 Diterpenic resin
acid
OH-Dehydroabietic acid C20H28O3316.203
76
5 882 887 Diterpenic resin
acid
Resin acid 1 C20H30O2302.224
45
272 926 Diterpenic resin
acid
Selected
identi$ed
and
tentativel
y
identi$ed
compoun
ds
Selected
identi$ed
and
tentativel
y
identi$ed
compoun
ds
Many of the compounds have been reported in low amounts in healthy Spruce before, with
GC-MS after derivatization, HPLC or LC-MS, but they have never been analysed together in
one analysis as here. Concentrations are much higher in infected Spruce.
Many of the compounds have been reported in low amounts in healthy Spruce before, with
GC-MS after derivatization, HPLC or LC-MS, but they have never been analysed together in
one analysis as here. Concentrations are much higher in infected Spruce.
Bioassay testing
We are now testing bioactivity of crude extracts and
fractionated extracts from the Spruce reaction zone on
various bacteria, fungi and cancer cell lines
1/6/20 34
Pros and wishes
Extremely useful with diJerential analysis and statistic evaluations on
the Yy (Trend lines, PCA; Vulcano plot)
The predicted formulas are almost always correct
A dangerous tool for the curious mind..
FISh score not so enlightening do to lack of fragments in ESI negative
library (?)
Working with plant extracts: ChemSpider searches generates many
hits - with complex compund names ;"--*$
mzCloud match is very useful, but need to add more compounds into
mzCloud
Will it be possible to download MS2 spectrums from mzCloud into in-
house mzVault MS2 database?
Use CD with FMS-DIA $les? 1/6/20 35
Thank you for your attention
marit.almvik@nibio.no
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