Short Legends for Supporting Information (words 1572)
Supporting Figure S1: Survival yield (SY) of ammonium and sodium adducts
To assess the precursor ion stabilty, we compared the survival yield curves of ammoniated and
sodiated precursor ions of attenoside, lyciumoside II, lyciumoside IV, nicotianoside I,
nicotianoside II and nicotianoside IV in positive ion mode. We determined the survival yield
curves, starting at 5 eV and increasing the unit increments until the precursor was no longer
detected. The spectrum was analyzed and the experimental survival yield was calculated as the
ratio of the intensity of the precursor ion to the sum of intensities of the precursor and fragment
ions (Kertesz et al. 2009). Analyzing the survival yield curves reveals that neither the
ammonium nor the sodium adducts break under default (see experimental procedures)
conditions, indicating that the fragmentation purely arises from the protonated precursor ions.
Calculating the CE50 values (the point where 50% of the precursor ions remain intact) for all six
HGL-DTGs revealed that the sodiated precursor is more stable than the ammonium adduct
(Table S3). Therefore, 2-4 times higher CID voltages should be applied for analyzing the
fragmentation pattern of sodiated precursors.
Supporting Figure S2: Example for sugar cluster
Shown is the ESI-MS/MS spectrum in positive ion mode of attenoside at 17.5 eV. Hereby the
ammonium adducts is used as the precursor ion (dark grey). Here the fragment m/z 471.1708
emerges as a very abundant signal (grey). Analyzing the pseudoMS/MS/MS spectrum of the
fragment m/z 471.1708 indicates (sum formula = C18H31O14+) that this is a cluster of three
sugars (2× Glc and 1× Rha) with one waterloss. This and other sugar fragments can be typically
seen for higher molecular weight HGL-DTGs.
Supporting Figure S3: Fingerprint of the aglycone fragment
Shown is the ESI-pseudoMS/MS/MS spectrum in positive ion mode of the aglycone fragment
m/z 271.2420 and its typical fragmentation which can be used as a fingerprint (Sodhi et al.
2014) to distinguish HGL-DTGs from other compound classes.
Supporting Figure S4: Diagnostic fragmentation for rhamnose-containing HGL-DTGs
Shown are ESI-MS/MS spectra in positive ion mode of the rhamnose-containing HGL-DTGs
lyciumoside IV, attenoside and nicotianoside III as well as of lyciumoside I and lyciumoside II,
which don’t contain rhamnose. These two classes of compounds can be distinguished by the
presence of m/z 417.2999 (sum formula C26H41O4+) and m/z 435.3105 (sum formula C26H43O5+)
which indicate the rhamnose (rha) moiety and can’t be seen in the only glucose-containing
compounds.
Supporting Figure S5: Validation of MS-based identification
Shown is a) the IS-CID MS spectrum deconvoluted with Dissect of the compound b) RGHGL (3O-(α-rhamnopyranosyl-(1->4)-β-glucopyranosyl)-17-hydroxygeranyllinalool) recently identified
by Poreddy et al. 2015 via NMR in frass of M. sexta. The frass material was collected from
caterpillars of the 2nd instar fed with leaf material of N. attenuata and extracted with 80%
methanol. Base peak of the Dissect cluster is the fragment m/z 271.2420 which represents the
17-HGL-aglycone. The typical adduct pattern between [M+NH4]+ and [M+Na]+ can be observed
as well as neutral losses of Hex (hexose 162.0528u) and Dhex (deoxyhexose 146.0579u). In
addition the fragmentation of the sugars can be identified with the fragments m/z 309.1180
which represents a Hex+Dhex-H2O and m/z 147.0651 which represents Dhex. Further
diagnostic fragments m/z 417.2999 and m/z 435.3105 indicate the presence of rhamnose
moieties which is in agreement with the presence of rhamnose-containing HGL-DTG in the N.
attenuata leaves used to feed the caterpillars. The overall prediction solely based on IS-CID MS
indicates a HGL-DTG with on hexose (glucose) and one deoxyhexose (rhamnose) moiety.
Supporting Figure S6: Novel diterpene glycosides in N. cavicola
Positive full-scan MS spectra of novel diterpene glycosides in N. cavicola and their MS/MS
spectra in positive ion mode. Shown are the MS/MS spectra of [M+H]+, [M+NH4]+, [M+Na]+ (a)
from DTG752, [M+H]+ (b) from DTG898 and the [M+H]+ (c) from DTG812. Analyzing the fullscan MS spectra reveals the difference in the ratio between the adducts of those novel
diterpene glycosides compared to the HGL-DTGs. Hereby, the fragment m/z 289.2525 is the
base peak and a novel aglycone fragment m/z 307.2632 appears, indicating an additional
hydroxyl group at the diterpene skeleton.
Supporting Figure S7: Induction of tryptophan accumulations in N. attenuata, N.
obtusifolia and N. cavicola leaves after MeJA treatment
Elongated N. attenuata, N. cavicola and N. obtusifolia plants were left unelicited (Control), or
one leaf per plant was elicited with 150 mg methyl jasmonate (MeJA) in 20 µl of lanolin paste to
supplement jasmonate production or with 20 µl of lanolin paste (Lan) as a control for Lan +
MeJA treatment (N=5). In this figure, we compare the levels of tryptophan among different
Nicotiana species (a – c) to verify the efficiency of the elicitation treatment with MeJA. Asterisks
indicate significant differences from WT in one-way ANOVAs (Control, Lan, Lan+MeJA) within
each treatment: *P ≤ 0.05, ** P < 0.01, *** P < 0.001 in Bonferroni-corrected tests. Bars
represent relative standard errors.
Supporting Figure S8: MeJA-elicited changes in the accumulations of HGL-DTGs in N.
attenuata, N. obtusifolia and N. cavicola leaves
Elongated N. attenuata, N. cavicola and N. obtusifolia plants were left unelicited (Control), or
one leaf per plant was elicited with 150 mg methyl jasmonate (MeJA) in 20 µl of lanolin paste to
supplement jasmonate production or with 20 µl of lanolin paste (Lan) as a control for Lan +
MeJA treatment (N=5). Shown are the levels of the dereplicated and novel putatively annotated
HGL-DTGs and their proposed biosynthetic capacity in all three plant species. An overall
induction of HGL-DTGs could be detected in N. attenuata. In N. obtusifolia, levels of several
HGL-DTGs were increased (nicotianoside III, nicotianoside IV, DTG 1128 and DTG 1214) and
those of smaller molecular weight HGL-DTGs like lyciumoside I and DTG 810 and their
malonylated forms were reduced. Others remain unchanged, indicating a completely different
regulation than in N. attenuata. The treatment with lanolin seems to repress HGL-DTG
accumulation. In N. cavicola no HGL-DTGs (besides nicotianoside II) was affected by the
treatment. Asterisks indicate significant differences from WT in one-way ANOVAs (Control, Lan,
Lan+MeJA) within each treatment: *P ≤ 0.05, ** P < 0.01, *** P < 0.001 in Bonferroni-corrected
tests. Bars represent the relative standard error.
Supporting Table S1: Validation of Find Dissect
Shown is the comparison of 31 Find Dissect clusters and their MS/MS spectra to validate the
efficiency of the clustering algorythm. The overlap and percentage of features explained and
identified via the MS/MS library are given for all 31 clusters.
Supporting Table S2: Compound table for 46 HGL-DTGs in N. attenuata
Shown are the characterized and newly assigned HGL-DTGs in N. attenuata.
Supporting Table S3: CE50 values
Shown are the CE50 values of the ammonium and sodium adducts of lyciumoside II, IV,
attenoside, nicotianoside I, II and IV.
Supporting Table S4: Annotation of HGL-DTG fragments
Shown are the non-redundant fragments and their predicted composition.
Supporting Table S5: Dereplication of HGL-DTGs in N. obtusifolia
Dereplication and annotation results for Find Dissect clusters according to the proposed rules in
N. obtusifolia. The green colour indicates when a compound is in agreement with all proposed
“rules”.
Supporting Table S6: Compound Identification table for all solanaceous species
Shown are the characterized and newly assigned HGL-DTGs in all measured solanaceous
species.
Supporting Table S7: Dereplication of HGL-DTGs in N. cavicola
Dereplication and annotation results for Find Dissect clusters according to the proposed rules in
N. cavicola. The green colour indicates when a compound is in agreement with all proposed
“rules”. The orange colour denotes for the novel Di-HGL-DTGs.
Supporting Table S8: Statistics for Figure S8 and Figure S9
Shown are the results of ANOVA comparing individual HGL-DTGS in N. attenuata (a), N.
obtusifolia (b) and N. cavicola (c) after methyl jasmonate (MeJA) treatment. Supporting Table
S8 (d) shows the results of ANOVA comparing Tryptophan in N. attenuata, N. obtusifolia and N.
cavicola after methyl jasmonate (MeJA) treatment.
Supporting Data S1: Data for the validation of Find Dissect
Shown is the comparison of the Dissect clusters and their MS/MS spectra at a collision induced
dissociation voltage of 22.5 eV. To validate the efficiency of Find Dissect, we examined 31
Dissect clusters from N. alata, N. cavicola and N. obtusifolia. The Dissect clusters were
annotated via the MS/MS spectrum of their particular precursor ion and by the extensive MS/MS
library. Molecular formulae were identified via SmartFormula.
Supporting Data S2: MS/MS spectra for the characterization of HGL-DTGs in N. attenuata
Shown are the high resolution AutoMS/MS spectra measured via the Impact II – Q-TOF for the
identified HGL-DTGs in N. attenuata. Molecular formulae were determined via SmartFormula.
Supporting Data S3: MS/MS library in N. attenuata
Shown are the high resolution MS/MS, AutoMS/MS and direct injection MS/MS spectra at
different CID voltages that were used for the MS/MS library and the formation of the “rules” for
the identification of HGL-DTGs.
Supporting Data S4: Compound spectra Output of Find Dissect in N. obtusifolia
Shown is the Dissect Output after manual and automatic annotation via the MS/MS fragment
library of N. obtusifolia leaf material at an retention time of 19-28 min.
Supporting Data S5: MS/MS library in N. obtusifolia
Shown are the supporting MS/MS spectra for the identification and annotation of HGL-DTGs in
N. obtusifolia.
Supporting Data S6: Compound spectra Output of Find Dissect in N. cavicola
Shown is the Dissect Output after manual and automatic annotation via the MS/MS fragment
library of N. cavicola leaf material at an retention time of 19-28 min.
Supporting Data S7: MS/MSlibrary in N. cavicola
Shown are the supporting MS/MS spectra for the identification and annotation of HGL-DTGs in
N. cavicola.
Supporting experimental procedures Method S1: Purification of HGL-DTGs from N.
attenuata
Supporting Appendix S1: Logic of Find Dissect’s peak identification algorithms