TPJ_4635_sm_AppendixS2

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Appendix S2. Mass spectrometry analysis of IAA and GAs
Prior analysis by LC/MS the samples were derivatised according to the MS-probe concept
Kojima et al. (2009). Each sample was dissolved in 75µl of isopropanol and 20µl H2O.
Thereafter 4µl 500mM bromocholine (Sigma-Aldrich) in 70% acetonitrile and 0.8µl triethylamine
were added to the sample, and kept at 80°C for 2h. After evaporation in a speed-vac
concentrator each sample was dissolved in 15-30µl 0.1% HCOOH, and thereafter further
analysed by LC/MS in multiple-reaction-monitoring (MRM) mode.
Chromatography was performed on a Agilent 1200 HPLC system, equipped with column oven,
coupled to a Agilent 6460 triple quadrupole mass spectrometer equipped with a jet stream
electrospray source operating in positive ion mode. The jet-stream gas temperature was 200ºC
with a gas flow of 6 L hr-1, and a sheath gas temperature equal 400 ºC and flow of 12L/hr;
nebulizer pressure was set to 25 psi. The ion spray voltage was set at 3.5 kV for positive ion
mode, nozzle voltage was set to 1500V. The fragmentor voltages, transition stages and collision
energies were set according to Table S1. N2 was used as collision gas, the dwell time was 100200 ms and the resolution was set to unit resolution.
A 5 µL aliquot of sample (4˚C) was injected onto a 2.1 x 50 mm, 1.7 µm HPLC column (Kinetex,
Phenomenex) held at 50˚C in a column oven. The gradient elution buffers were A (H2O, 0.1%
formic acid) and B (acetonitrile, 0.1% formic acid), and the flow-rate was 300 µl min-1. The
mobile phase was first held at 0.9% B for 1 min, and then between 1 and 5 min a linear gradient
from 1 to 30% B, between 5 and 12 min from 30 to 60% and then in one min up to 95%B, and
held for 1 min. All samples including standards were analysed in randomised order.
The LC/MS analysis was done by the MassHunter™ software v 2.00 and data processing by the
MassHunter™ Quantitation software v 4.00 (Agilent, USA).
Table S1. Summary of retention time and precursor-to-product ion transitions used for the
quantification of derivatised IAA and GAs (including internal standards) using LC-ESI-MS/MS.
1
n.d.=not possible to use qualifier ions due to low intensity.
Plant
hormone
RT (min)
Fragmentor
(V)
IAA
13
C6-IAA
GA1
2
H2-GA1
GA4
2
H2-GA4
GA8
2
H2-GA8
GA9
2
H2-GA9
4.2
4.2
4.47
4.45
6.20
6.22
3.55
3.53
6.94
6.93
124
124
180
180
185
185
172
172
157
157
Transition
stage
quantifier
261.1→130.0
267.1→136.0
434.3→375.1
436.3→377.1
418.3→359.1
420.3→361.1
450.3→391.2
452.3→393.2
402.3→343.1
404.3→345.1
Transition
stage
qualifier
261.1→202.0
267.1→208.0
n.d.1
n.d.1
418.3→225.1
420.3→227.1
n.d.1
n.d.1
402.3→225.1
404.3→277.1
Collision energy
quantifier/qualifier
(eV)
30/14
30/14
30
30
26/45
26/45
30
30
26/38
26/38
GA19
2
H2-GA19
GA20
2
H2-GA20
GA24
2
H2-GA24
GA29
2
H2-GA29
GA34
2
H2-GA34
GA53
2
H2-GA53
5.36
5.34
5.50
5.49
6.84
6.83
3.80
3.79
4.99
4.99
6.05; 6.40
6.04; 6.38
162
162
155
155
157
157
170
170
155
155
152
152
448.3→389.1
450.3→391.1
418.3→359.1
420.3→361.1
434.3→375.1
436.3→377.1
434.3→375.1
436.3→377.1
434.3→375.1
436.3→377.1
434.3→375.1
436.3→377.1
448.3→299.1
450.3→301.1
418.3→241.1
420.3→243.1
434.3→285.0
436.3→287.1
n.d.1
n.d.1
n.d.1
n.d.1
434.3→285.1
436.3→287.1
22/26
22/26
26/45
26/45
22/30
22/30
26
26
30
30
26/34
26/34
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