MPI Chemical Ecology & Bruker Daltonics HPLC 2009, ENv34-Tu
Challenges in the investigation of the metabolic changes in
Nicotiana attenuata during insect herbivory using an improved
Matthias Schöttner1; Eva Rothe1; Quality Control Results
Emmanuel Gaquerel1; Beatrice All wildtype (WT) samples were investigated first to
The reproducibilty of the method was checked by identify changes related to time and treatment only
Berger1; Jörg Sauer2; Birgit means of the ISTDs reserpine & atropine. The RSD using principle component analysis (PCA) and a
Schneider2; Gabriela Zurek2; Aiko values of peak areas & heights were well below 10% Student‘s t-test. The late timepoints of the treated
Barsch2; Ian T. Baldwin1 for the entire sequence (Fig. 2A). A pooled sample samples are clearly separated in the PCA scores plot
QC2 representing a ‚chemical average‘ of all samples (Fig. 3A). The bucket statistics plots for the selected
nicely clusters in the middle of a PCA scores plot of loading at firstname.lastname@example.org shows the same
1Max Planck Institute for Chemical Ecology
all measured samples. The QC data is used to verify trend. A t-test of the treated and non-treated WT
07745 Jena – Germany the analytical performance and enables valid samples after 120h was applied to narrow down the
2Bruker Daltonik GmbH, 28359 Bremen - interpretation in the complex real samples. number of compounds to be investigated for further
Germany identification. The t-test result is returned for each
8.0 PC 2 bucket with the additional information about the
Introduction regulation level, the maximum intensity in all
analyses and the number of detected compounds per
group (Fig. 3B). The first six bucket statistics plot
The target of this study is the investigation of the 5.0
RSD Values [%]
are given as examples (Fig. 3B). In total 59
metabolic changes in leaf extracts of Nicotiana 4.0
compounds with p-values < 0.05 and a minimum of
attenuata after simulated insect herbivory (see Fig. 1) 3.0
2-fold upregulation in the treated samples were
by mechani-cally wounding and applying oral
found. This list of compounds was used as the basis
secretions of Manduca sexta (W+OS) using HPLC-ESI- 1.0
for a scheduled precursor list for a targeted
TOF-MS. Responses of wild-type (WT) plants are 0.0 -0.2
AutoMS/MS experiment. Molecular formulae of the
primarily compared before investigating the influence
target compounds have been generated based on
of a genetic modification in which the hydroxyproline- -0.2 0.0 0.2 0.4 PC 1
the accurate MS and MS/MS data (Fig. 3C).
rich glycopeptide systemin precursor (ppHypSys) is
After statistical analysis, the major challenge is the
either overexpressed (OV) or down-regulated (IR). Fig. 2: A- Reproducibility of reserpine & atropine in QC1 (buffer)
identification of the compounds involved in
The challenges in this type of study and strategies for and QC2 (pooled sample), N=16; B- PCA scores plot of all samples
and QC2. regulation. The accurate MS/MS data of the
quality control are discussed.
compound email@example.com has been processed
Preliminary results obtained from a first data set of with SmartFormula 3D (Fig. 4A), leading to an
the same sample origin (7 time-points with three MS/MS spectrum assigned with fragment formulae
biological replicates per time-point, treatment and Methods and the corresponding neutral losses (Fig. 4B). With
genotype analyzed with a 17min gradient) were used the molecular formula containing 3 nitrogen atoms,
to design the experiment presented here. The number Treated and untreated leaves (control) from WT, OV, a spermidine derivative can be hypothesized. The
of biological replicates was increased from three to IR (5 biological replicates) were harvested 1, 14, 86, fragment at 163m/z is typically observed for caffeic
five to access the biological variation. The and 120 h after treatment & flash frozen. acid derivatives, 177m/z for ferulic acid derivatives.
chromatographic separation was optimized in order to Extraction: MeOH/acetic acid buffer 40:60 v/v The combined information leads to a caffeoyl-
minimize suppression effects due to coelutions. A Internal Standards (ISTD): Atropine, reserpine. feruloyl-spermidine  as potential metabolite being
pooled sample generated from all individual samples QC Samples: QC1: Extraction buffer + ISTD; QC 2: upregulated during herbivory.  Youhnovsky, N. et.
was spiked with internal standards (reserpine, pooled sample of all extracts + ISTD. N= 16 al. Helvetica Chimica Acta 1998 81(9) 1654-1671.
atropine) served as quality control sample to monitor injections.
instrumental variation. Eluent A: water +0.1%AcN+ 0.05% HCOOH
Eluent B: AcN + 0.05% HCOOH Identification
Gradient: 0min-5%B; 2min- 5%B; 2-30min-5-80%B;
35min-80%B; 40min- 5%B; 45min-stop Flow rate: A
Column: Phenomenex GeminiNX 150x2mm
MS: Bruker micrOTOF & micrOTOF-Q
Ionization: ESI/ positive mode
Scan range: 50-1600 m/z; 1Hz acquisition rate MS peak list MS/MS peak list
Calibration: external with NaFormate clusters
Software: ProfileAnalysis for PCA and t-test O
Bucketing: time range: 0.5-30min mass range: 50-
HO N N
Fig. 1 (A) Nicotiana attenuata leaf attacked by 1600m/z Advanced bucketing; Normalization: None;
Manduca sexta. (B) Simluated insect herbivory by Scaling: Pareto. Structural Hypothesis for C26H33N3O6
wounding leaves with a fabric pattern wheel and Compounds need to be present in at least 85% of all
applying insect’s oral secretions.
+MS2(484.2428), 35-35eV, 11.43-11.84min #677-#701
B 2.0 C3H7N C 4 H 12 N 2 C 10 H 8 O 3
C3H7N C 4 H 12 N 2 C9H6O3
C: Example for SmartFormula
PC 2 PC 2 Int. 220.0963
x104 11.4min : 484.24m/z
Scores Loadings result and summary of formulae 1.0
for buckets from B.
0.5 0.5 4 0.0
SmartFormula3D spectrum: C 26 H 34 N 3 O 6
C17 H25 N2 O3 , 0.55 mDa
C16 H 23 N2 O3 , 3.2 mDa
C 10 H 9 O 3 , -0.11 mDa
C 12 H 14 N 1 O 3 , 0.49 mDa
C 13 H 16 N 1 O 3 , 0.52 mDa
C 9 H 5 O 2 , -0.95 mDa
C16 H26 N3 O3 , 1.2 mDa
C 9 H 7 O 3 , 0.0053 mDa
C 26 H 34 N 3 O 6 , 2 mDa
C17 H28 N3 O3 , 1.3 mDa
C 12 H 17 N 2 O 3 , 1.1 mDa,
C 14 H 18 N 1 O 3 , 1.6 mDa
C 8 H 18 N 3 O 1 , 1.4 mDa,
C 7 H 17 N 2 , 0.17 mDa
Late timepoints W+OS 0
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 PC 1 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 PC 1 0 10 20 30 Analysis
A: PCA (pareto scaling) of all WT samples, explained variance PC1 vs PC2: 48.4%. Bucket Exact Formula 0.0
150 200 250 300 350 400 450 m/z
11.4min- 484.2437 C26H34N3O6 Fig. 4: A- SmartFormula 3D results, B- MS/MS and
o/o: 0.17 o/o: 0.21
484.24m/ simulated spectrum with annotated formulae.
o: WT Control
o: WT+OS 11.4min : 484.24m/z 9.6min : 488.24m/z
14.2min- 598.3083 C26H45O14
2 4 6 8 Analysis 2 4 6 8 Analysis
5.1min- 251.1381 C13H19N2O3 • By means of internal standards and a pooled
14.2min : 598.31m/z
11.7min : 484.24m/z
251.14m/ quality control sample, the reproducibility of the
analytical method was monitored.
9.6min- 488.2380 C25H34N3O7
• PCA and t-test were successfully applied to filter
2 4 6 8 Analysis 2 4 6 8 Analysis
out metabolic changes.
x10 4 x10 5
o/o: 0.11 o/o: 0.64
6 4.0 z
5.1min : 251.14m/z 3.5
2.1min : 163.12m/z 11.7min- 484.2427 C26H34N3O6 • The identification of regulated metabolites can be
achieved based on molecular formulae derived from
2 4 6 8 Analysis 2 4 6 8 Analysis
accurate mass spectra plus chemical knowledge.
B: Excerpt of t-test result table and selected bucket statistics plots** for WT 120h samples. 2.1min- 163.12 C10H15N2
Fig. 3: PCA and t-test results for all wildtype samples and molecular formulae. • The same procedure will be applied to investigate
** negative regulation values indicate z the influence of the genetic modification.
upregulation in red group.
Fig. 3: PCA and t-test results for all wildtype samples and molecular formulae.
For research use only. Not for use in diagnostic procedures.