Supporting Information
A comprehensive high-resolution mass spectrometry approach for
characterization of metabolites combining ambient ionization, liquid
chromatography and imaging methods
Arton Berisha1, Sebastian Dold1, Sabine Guenther1,, Nicolas Desbenoit1, Zoltan Takats1,2,
Bernhard Spengler1 and Andreas Römpp1
1
Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen,
Schubertstrasse 60, 35392 Giessen, Germany
2
Computational and Systems Medicine, Department of Surgery and Cancer, Faculty of
Medicine, Imperial College London, Sir Alexander Fleming Building, London SW7 2AZ,
United Kingdom
*Correspondence to: A. Römpp, Institute of Inorganic and Analytical Chemistry, Justus
Liebig University Giessen, Schubertstrasse 60, 35392 Giessen, Germany.
E-mail: andreas.roempp@anorg.chemie.uni-giessen.de
1
SUPPORTING INFORMATION 1:
Structure of aglycons of anthocyanins
Figure S 1
Basic structure of anthocyanins and the substitution pattern of the main aglycones occuring in
nature. (J. Fleschhut, Untersuchungen zum Metabolismus, zur Bioverfuegbarkeit und zur
antioxidativen Wirkung von Anthocyanen, Doctoral Thesis, Karlsruhe, 2004).
Aglycons are derivatives of the flavylium cation. Anthocyanins are formed by glycosylation
of aglycones, mainly in position 3 or 5. Glycosylation has an important effect for the stability
of anthocyanins that are more stable than their aglycones. (OM. Andersen. Anthocyanins,
Encyclopedia of life sciences, MacMillan Publishers Ltd., London, 2002, p.597-605).
2
SUPPORTING INFORMATION 2:
Differentiation of grape types by LD-ESI MS screening and PCA analysis
Figure S 2
LD-ESI spectra of three different types of grapes (a) Dunkelfelder, (b) Dakapo, (c) Accent
performed on a LTQFT Ultra, shown are the average spectra of ten single MS measurements.
(d) Statistical differentiation via principal component analysis as 2-D score plot.
Data analysis PCA
For PCA analysis the software package The Unscrambler X (v10.1; Camo Software AS.,
Oslo, Norway) was used. The data of the LD-ESI measurements was binned with a homebuilt software package XRawBrowser V1.42 to a window size of Δm/z = 0.01 (Alfons Hester
Build_1.4.2.28; 2007-2011).
10 grapes were measured per variety by LD-ESI. The 2-D scores plot shows that it is possible
to differentiate between the used grape varieties.
3
SUPPORTING INFORMATION 3:
MALDI Imaging of Accent grape using Exactive MS
Figure S 3 MALDI imaging measurements of various anthocyanins
4
MALDI Imaging of DUNKELFELDER grape using Exactive MS
Figure S 4
a) MALDI imaging measurement of Dunkelfelder grape using Exactive MS. b) optical image.
The thickness of the cryo-section of the sample was 40 µm. Total image size of 280x210
pixels, with a pixel size of 20 µm. c) Mass spectrum of a single 20 µm pixel of the exocarp.
5
MALDI Imaging of DAKAPO grape using LTQ Orbitrap MS
Figure S 5
MALDI imaging measurement of Dakapo grape using LTQ Orbitrap MS. a) Optical image of
the 100 µm thick cryo-section. b) MALDI imaging of the area of endosperm and mesocarp,
and c) MALDI imaging of exocarp. d) The mass spectrum of a single 20 µm pixel of exocarp.
6
SUPPORTING INFORMATION 4:
Extract preparation for HPLC-ESI
Grape peel extracts of Accent grapes were obtained using an extraction method described by
[9, 70]
. In brief: The grape was peeled off using tweezers, shock-frozen in liquid nitrogen and
ground to a powder. Aliquots of 100 mg were extracted in a solution of 1 mL methanol:water
(50:50 v/v) for 20 min with sonification. The extracts were centrifuged for 10 min (at 13,000
x g) and 200 µL of the supernatant was collected. Before measurements, the samples were
diluted to 1:25 (v/v) using 0.7 % formic acid in water. After filtration with a syringe filter (RC
membrane, 0.2 µm pore size; Phenomenex, Aschaffenburg, Germany) the sample was
transferred into a HPLC vial and 10 µL of sample solution was injected in each HPLC run
(for sensitivity analysis 20, 40 and 80 µL were injected additionally).
Calibration
For quantification, a calibration curve of malvidin-3-O-glucoside in a Dakapo grape extract was used
(provided by the Geisenheim Research Center, Department for Wine Analysis and Beverage
Research, Geisenheim, Germany). The use of the most abundant species (malvidinmonoglucoside in our case) is a common approach for the analysis of anthocyanins, e.g. in
the following studies:Vitaglione, P., Donnarumma, G., Napolitano, A., Galvano, F.,
Gallo, A., Scalfi, L. and Fogliano, V. (2007) Protocatechuic acid is the major human
metabolite of cyanidin-glucosides, Journal of Nutrition 137 (9), 2043-2048.
Nurmi, T., Mursu, J., Heinonen, M., Nurmi, A., Hiltunen, R. and Voutilainen, S. (2009)
Metabolism of Berry Anthocyanins to Phenolic Acids in Humans, Journal of
Agricultural and Food Chemistry 57 (6), 2274-2281.
Novotny, J. A., Clevidence, B. A. and Kurilich, A. C. (2012) Anthocyanin kinetics are
dependent on anthocyanin structure, British Journal of Nutrition 107 (4), 504-509.
7
MS of Accent extract
HPLC-ESI MS (FT ICR): Selectivity example compound m/z 611
IV
m/z = 611.14
RT: 0.00 - 22.04
NL: 4.94E6
m/z= 611.13-611.18
F: FTMS + p ESI Full
ms [150.00-850.00]
MS
C0278_FT_Acc2_80u
L_AB
12.28
100
III
90
m/z = 611.14
Relative Abundance
80
70
60
50
II
40
m/z = 611.14
30
I
20
m/z = 611.16
7.33
10
11.81
12.73
0
4
6
I
80
60
287.1
40
20
0
Relative Abundance
Relative Abundance
C0278_FT_Acc2_80uL_AB #478 RT: 7.23
T: ITMS + c ESI d Full ms2 611.16@cid20.0 ...
449.1
100
213.2
431.2
200
400
m/z
16
18
20
III
C0278_FT_Acc2_80uL_AB #830 RT: 12.01
T: ITMS + c ESI d Full ms2 611.14@cid20.0 ...
287.1
100
40
20
cyanidin-diglucoside
14
II
60
600
10
12
Time (min)
C0278_FT_Acc2_80uL_AB #818 RT: 11.83
T: ITMS + c ESI d Full ms2 611.14@cid20.0 ...
303.1
100
80
549.3 611.1
8
0
287.1
200
80
60
40
413.2 474.9 565.1
400
m/z
303.1
20
600
delphinidin-coumaroyl-glucoside
0
213.2
200
22
IV
Relative Abundance
2
Relative Abundance
0
80
60
40
20
413.3 474.8 565.0
400
m/z
C0278_FT_Acc2_80uL_AB #846 RT: 12.25
T: ITMS + c ESI d Full ms2 611.14@cid20.0 ...
303.1
100
600
cyanidin-caffeoyl-glucoside
0
257.1 304.1 449.2
200
400
m/z
611.1
600
delphinidin-coumaroyl-glucoside
Figure S 6
HPLC-ESI MS measurement of the isomeric compounds cyanidin-caffeoyl-monoglucoside
and delphinidin-coumaroyl-monoglucoside with m/z 611.14.
8
SUPPORTING INFORMATION 5:
Summary identified compounds
m/z
Compound
Modification / Adduct
147.07640
Glutamine
[M+H]+
147.11279
Lysine
[M+H]+
150.05832
Methionine
HPLC-ESI
LD-ESI
MALDI
ppm
ppm
ppm
0.53
0.41
0.27
1.22
[M+H]
+
0.40
+
0.38
0.13
156.07674
Histidine
[M+H]
173.04205
Pentose
[M+Na]+
0.17
175.11893
Arginine
[M+H]+
0.40
0.29
182.08116
Tyrosine
[M+H]
+
0.22
0.16
166.08625
Phenylalanine
[M+H]+
0.30
203.05261
Hexose
[M+Na]+
205.09714
283.10236
271.06010
Tryptophan
Di-Pentose
Pelargonidin
0.05
0.00
0.05
[M+H]
0.20
0.01
(mesocarp)
[M+H]
+
0.64
0.40
-0.56
[M+NH4]+
[M+K+H2O]+
aglycone / [M]+
0.15
0.18
-0.22
+
0.07
0.24
-0.31
Cyanidin
aglycone / [M]
291.08632
(Epi-)Catechin
[M+H]+
Peonidin
0.51
+
287.05502
301.07067
(endosperm)
-0.07
0.27
aglycone / [M]
+
-0.03
0.10
-0.47
+
-0.13
0.07
-0.36
303.04993
Delphinidin
aglycone / [M]
317.06558
Petunidin
aglycone / [M]+
0.03
0.13
-0.32
Malvidin
+
-0.03
0.03
-0.51
-0.15
-0.30
0.06
331.08123
335.09487
Pentose-Hexose
aglycone / [M]
[M+Na]
+
[M+K]+
365.10544
Di-Hexose
[M+Na]+
-0.25
0.16
0.08
+
-0.46
0.28
0.97
433.11293
Pelargonidin
monoglucoside / [M]
449.10784
Cyanidin
monoglucoside / [M]+
-1.69
0.24
0.27
463.12349
Peonidin
monoglucoside / [M]+
-1.38
0.30
0.11
+
-1.89
0.00
0.56
-0.57
-0.55
0.08
-1.90
0.06
0.44
-0.81
0.22
0.53
-1.66
-0.02
0.32
-0.81
0.24
0.28
465.10276
Delphinidin
monoglucoside / [M]
+
475.12294
Pelargonidin
acetyl glucoside / [M]
479.11841
Petunidin
monoglucoside / [M]+
491.11841
Cyanidin
acetyl monoglucoside / [M]
+
+
493.13406
Malvidin
monoglucoside / [M]
505.13406
Peonidin
acetyl monoglucoside / [M]+
9
507.11332
521.12897
Delphinidin
Petunidin
acetyl monoglucoside / [M]+
-1.36
0.20
0.59
acetyl monoglucoside / [M]
+
-1.29
0.08
0.59
+
-1.07
0.32
0.77
0.55
0.55
535.14462
Malvidin
acetyl monoglucoside / [M]
579.14970
Procyanidin Bx
[M+H]+
579.14970
Pelargonidin
-0.28
coumaroyl monoglucoside / [M]
+
-0.16
+
-1.08
595.14462
Cyanidin
coumaroyl monoglucoside / [M]
595.16519
Pelargonidin
diglucoside / [M]+
609.16027
Peonidin
-1.14
coumaroyl monoglucoside / [M]
+
+
611.13953
Delphinidin
coumaroyl monoglucoside / [M]
611.13953
Cyanidin
caffeoyl monoglucoside / [M]+
611.16066
Cyanidin
diglucoside / [M]+
+
-1.18
0.54
0.57
1.22
-1.70
0.59
a
0.95 a
-0.69
0.59 a
0.95 a
-0.77
-0.54
0.46
-1.12
1.04
a
1.34 a
625.15518
Petunidin
coumaroyl monoglucoside / [M]
625.15518
Peonidin
caffeoyl monoglucoside / [M]+
-0.40
1.04 a
1.34 a
625.17631
Peonidin
diglucoside / [M]+
-0.80
0.58
0.32
+
627.13445
Delphinidin
caffeoyl monoglucoside / [M]
627.15558
Delphinidin
diglucoside / [M]+
639.17083
Malvidin
coumaroyl monoglucoside / [M]+
641.15010
Petunidin
caffeoyl monoglucoside / [M]
+
+
641.17123
Petunidin
diglucoside / [M]
655.16560
Malvidin
caffeoyl monoglucoside / [M]+
655.18688
Malvidin
diglucoside / [M]
-1.79
-0.45
0.26
0.51
-0.81
0.44
0.55
-0.69
-0.62
+
1.58
0.55
0.09
0.50
-0.63
-0.85
0.35
0.41
+
-0.82
1.60
-0.33
757.19744
Cyanidin
coumaroyl diglucoside / [M]
771.21309
Peonidin
coumaroyl diglucoside / [M]+
-1.23
0.78
0.21
coumaroyl diglucoside / [M]
+
-1.13
0.79
0.19
+
0.42
1.02
0.17
-0.37
1.62
0.47
773.19236
Delphinidin
787.20801
Petunidin
coumaroyl diglucoside / [M]
801.22366
Malvidin
coumaroyl diglucoside / [M]+
Table S 1
Identified compounds in Accent grape exocarp.
a
Can not be separated by LD-ESI and MALDI, because compounds are isomers.
10
SUPPORTING INFORMATION 6
MS/MS measurements of wheat seed on QExactive (positive ion mode)
Wheat seed samples of the variety Florence Aurore infected with Fusarium graminearum
isolate IFA 65 (Department of Agrobiotechnology, IFA, Tulln, Austria) were used. Inoculum
production, plant material and inoculation were done according to methods previously
described by (Gottwald et al. BMC Genomics 2012, 13:369). Infected and mock (healthy
wheat seeds treated with water instead of inoculation) seeds were used for LD-ESI without
further sample preparation.
Wheat seed extraction:
For HPLC-MS/MS measurements infected wheat seeds, total weight 24.5 mg were extracted
without homogenization in 1 mL MeOH:H2O 50/50, v/v. For extraction the solution with
seeds was sonicated for 10 min, stirred for further 20 min, and then filtered using 0.2 µm
syringe filters. The supernatant was collected after centrifugation and diluted 1:2 with H2O
and injected in HPLC. As DON standard a solution of 0.5 µg/mL deoxynivalenol (Romer
Labs Diagnostics GmbH, Tulln, Austria) in MeOH:H2O 50/50, v/v, was used.
11
Figure S 7
a) LD-ESI MS/MS of ion m/z 297 (positive ion mode) of the Fusarium gram. (isolate IFA 65)
infected wheat sample. b) The HPLC-ESI MS/MS measurement of the infected seed extract.
c) MS/MS of DON from literature (Kappenstein et al., Mycotoxin Res.; 2002, 18 Suppl 1:525.)
12
Figure S 8
HPLC-MS extracted ion chromatograms (positive ion mode) of DON standard solution,
wheat seed extract infected, and wheat seed extract of healthy mock control.
13
SUPPORTING INFORMATION 7
Mouse brain tissue
Mouse brain sections of C57Bl6/N mice (age 12–20 weeks) were prepared according to the
procedure previously described by Guenther et al. 2011. In brief, mouse tissue sections were
cut in sections of 20 μm thickness with a cryotome (HM500, Microm, Walldorf, Germany) at
−20 °C and thaw-mounted on glass slides. The mounted samples were stored at −80 °C until
analysis. For measurements, tissue sections were brought to room temperature in a desiccator
(30 min). A solution of the matrix 2,5-dihydroxybenzoic acid (DHB; 98% purity, Aldrich,
Germany) in a concentration of 30 mg/mL was prepared in acetone/water (0.1% TFA) 1:1
v/v. Matrix solution was applied with a specially designed pneumatic sprayer and samples
were analyzed by MALDI imaging immediately afterwards..
14