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					  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
HPLC-TOF-MS method
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 484.24m/z@11.4min 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
                                                                                                                                                                                                   A                                                              B
Introduction                                                                                                                                                                                                                                                                                                        regulation level, the maximum intensity in all
                                                                                                                                                                                                                                   7.0                  Height

                                                                                                                                                                                                                                                                                                                    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 484.24m/z@11.4min 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 [1] as potential metabolite being
pooled sample generated from all individual samples                                                                                                                                            QC Samples: QC1: Extraction buffer + ISTD; QC 2:                                                                     upregulated during herbivory. [1] 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
                   A                                                               B
                                                                                                                                                                                               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-
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         N                                            OH
                                                                                                                                                                                                                                                                                                                          HO                                                               N                                                                                       N
                                                                                                                                                                                                                                                                                                                                                                                           H                                                                                       H

            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
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Fragment formulae
            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.
   1.0                                                               1.0
                                                                                                                                                            5                                                                                                                                                                                                                                                                                                                                                                                                                                                 322.2112
                                                                                                                                                                   Statistics                                                                                                                                              0.5
   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

   0.0                                                               0.0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               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,

   -0.5                                                              -0.5
                                                                                                                                                                                                                                                                                                                                       C 7 H 17 N 2 , 0.17 mDa

   -1.0                                                              -1.0
            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
                                                                                                                                                                                                                                                                             m/z           [M+H]+
                                                                                                Int.                                                                         Int.
                                                                                                                                                                                                                                                                 11.4min-    484.2437 C26H34N3O6                    Fig. 4: A- SmartFormula 3D results, B- MS/MS and
                                                                                              x10 4
                                                                                                        o/o: 0.17                                                                   o/o: 0.21
                                                                                                                                                                                                                                                                 484.24m/                                           simulated spectrum with annotated formulae.
                                                                                                 4                                                                         4000
                                                                                                        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

                                                                                              x10 4
                                                                                                                                                                           x10 4
                                                                                                        o/o: 0.22

                                                                                                                                                                                    o/o: 0.17
                                                                                                                                                                                                                                                                 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

                                                                                                Int.                                                                         Int.
                                                                                                                                                                                                                                                                                                                  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

                                                                                                 0                                                                          2.5

                                                                                                               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
                                                                                                                                                                                                                                                                 163.12m/    3
                                                                                                                                                                                    ** 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.