GC-MS analyses
A detailed, step-by-step protocol is provided as a Supplementary
Material. This section summarizes how exact mass GC-MS analyses and data
extraction with TraceFinder® were performed. GC-MS analyses were carried
out using a GC-MS-Orbitrap Q Exactive (Thermo Scientific, Courtaboeuf,
France). Fifty five (55) µL of each standard solution (50 µM), or 4 µL
from the plant leaf extract, were poured into a vial (with insert) and
spin-dried at 39°C. Samples were derivatized (automatically with a
preparative robot) with 20 µL methoxyamine (20 mg mL-1in pyridine; 90 min at 37°C) and 30 µL
N-methyl-N(trimethylsilyl)-trifluoroacetamide (MSTFA) for 30 min at
37°C. Prior to injection, 5 µL of alkane mix (14 alkanes from
C9 to C36, 3 µg µL-1,
Connecticut n-Hydrocarbon Mix, Supelco) were added in each sample to
compute the retention index. Analyses were performed by injecting 1 µL
in splitless mode at 230°C (injector temperature) in a TG-5 SILMS column
(30 m x 0.25 mm x 0.25 µm; Thermo Scientific) set in a Trace 1300 Series
GC (Thermo Scientific). Helium was used as gas carrier with a constant
flow of 1 mL min-1. After one minute at initial GC
oven temperature (70°C), temperature was raised to 325°C at 15°C
min-1 and finally kept at 325°C for 4 min. MS analyses
were operated in positive polarity in full MS scan mode with the
following source settings: mass scan range 50-750 m/z, resolution
60,000, AGC target 1E6, MS transfer line 300°C and filament delay 4.12
min. Ionisation by electron impact (70 eV) was performed at 250°C ion
source temperature. Amino acids were identified automatically using
TraceFinder® (Thermo Scientific) using retention time, major
characteristic fragment (m/z ion) and a confirmation fragment, with a
maximum tolerance of 0.00007 Da via targeted screening.