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SUPPLEMENTAL DATA
Direct Derivatization and Quantitation of Ultra-trace Gibberellins in
Sub-milligram Fresh Plant Organs
Dongmei Li1, Zhenpeng Guo1,*, and Yi Chen1,2,*
1Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences,
Beijing 100190, China
2Beijing National Laboratory for Molecular Sciences, Beijing 100190, China
*To whom correspondence should be addressed.
Prof. Yi Chen
E-mail: [email protected]
Tel.: +86 10 62618240, Fax: +86 10 62559373.
Dr. Zhenpeng Guo
E-mail: [email protected]
Tel.: +86 10 82615622, Fax: +86 10 62559373.
Supplemental Methods
Chemicals and reagents
Gibberellin (GA) and isotope deuterium-labeled GA (GAi and dGAi, i=1, 3, 4, 5, 6, 7, 8, 9, 15,
20, 29, 34, 44, 51) were all purchased from Olchemim Ltd. (Olomouc, Czech Republic).
Formic acid (FA) and HPLC grade methanol (MeOH), acetonitrile (ACN) and ethyl acetate
were from Fisher Scientific (Waltham, MA, USA). Analytical grade hydrochloric acid (HCl)
was obtained from Beijing Chemical Works (Beijing, China), and N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) from Sigma (St. Louis,
Mo, USA). C18 SPE cartridges (3 mL, 500 mg) were obtained from Thermo Scientific
(Bellefonte, PA, USA). Pure water produced by a Millipore Milli-Q academic system
(Billerica, MA, USA) was used throughout the study.
Preparation of solutions
Stock solutions of each GAi and dGAi were prepared in MeOH at the concentration of 10 μM,
and stored at -20 °C. To prepare a working mixture of all 14 GAs, each at 100 nM, combined
100 μL of each GAi stock solution and added 8.6 mL MeOH to form a 10 mL solution of
GAs. A working mixture of all 14 dGAs was prepared in the same way. The working solutions
of GAs and dGAs at different concentrations were then obtained by dilution of the 100 nM
solution with MeOH just before use. EDC solutions at different concentrations were prepared
by dissolving EDC powder in water which was acidified to pH 4.5 with 1 M HCl. For
investigating the effects of solvents on the labeling reaction, EDC solutions were prepared by
dissolving EDC powder in different solvents, including MeOH, ACN, methylene chloride,
water-MeOH and water-ACN mixtures.
Extraction of GAs
Frozen plant samples were placed in a 600 µL eppendorf tube and 2 fmol of each dGA were
added as internal standard (IS). The resultant was manually ground to fine powder using a
glass rod and extracted with 100 µL solvent composed of 75% MeOH, 20% H2O and 5% FA.
The mixture was shaken on a shaker (MS 3 digital, IKA®-Webke GmbH & Co.KG, Staufen,
Germany) at 4 °C and 500 rpm for 2h, plus 10 h unshaking extraction at 4 °C. After
centrifugation at 10000×g for 10 min at 4 °C (Allegra 64R Centrifuge, Beckman Coulter Inc.,
Brea, USA), the supernatant was collected, and the lower pellet was re-extracted twice with
50 µL MeOH for 10 min each. The combined supernatant was evaporated under a nitrogen
gas stream using a nitrogen evaporator (MD 200 Sample Concentrator, Allsheng Instruments
CO., Ltd., Hangzhou, China) and re-dissolved in 20 µL H2O which was acidified to pH 2.5
with 1 M HCl. It was further extracted with ethyl acetate (3×60 µL). The ethyl acetate phase
was combined, and dried under nitrogen gas for the further derivatization.
Labeling procedures
To label trace analytes, an aliquot of 10 μL GA standards was transferred to a 600 μL
eppendorf tube. After dried under a nitrogen stream, 50 μL of 20 mM EDC aqueous solution
was added and the tube was shaken at 35 °C and 750 rpm for 1.5 h. In case of labeling a
prepared plant sample, the resultant mixture was ultrasonicated for 5 min prior to further
shaking-based reaction. The resulting solutions, after centrifugation, were ready for liquid
chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) or
otherwise stored at -20 °C for later analysis.
To prepare EDC-GA3 for FT-IR characterization, GA3 was weighed at 20.8 mg (0.060 mmol)
and dissolved in 10 mL H2O at pH 4.5, followed by addition of 1.1082 g (5.8 mmol) EDC.
After reaction at 35 °C for 4 h, the solution was loaded onto a C18 SPE cartridge which was
pre-conditioned with 9 mL MeOH, 9 mL 80% MeOH and 9 mL H2O, and washed with 6 mL
H2O to remove the excessive EDC. The target product of EDC-GA3 was collected by elution
with 30 mL of 80% MeOH and dried first in a rotary evaporator (RE-52AA, Zhenjie
Instruments CO., Ltd., Shanghai, China) at room temperature and then in a freeze dryer (FD-
1D-50, Biocool Instruments CO., Ltd., Beijing, China). The resulting residue was collected
and ready for later use.
Characterization of EDC-GA3 by FT-IR
The characterization was performed on an FT-IR instrument, model Tensor 37 from Bruker
Optics (Ettlingen, Germany). The freeze-dried EDC-GA3 was mixed with dry KBr powder
and then pressed into a thin KBr disk for FT-IR measurement. Samples analyzing were
acquired at wavelength between 400–4000 cm-1 with a resolution of 4 cm-1 and the scanning
interval of 1.929 cm-1.
Separation-based quantification
All separation-based assays and identifications were performed on LCMS-8040 and LCMS-
8050 (Shimadzu, Kyoto, Japan). The latter has an LC unit consisting of CBM-20A system
controller, LC-30AD pump, DG U-20A5R degasser, SIL-30AC autosampler, and CTO-30A
column oven, coupled to a quadrupled type tandem mass spectrometer (LCMS-8050) via an
ESI interface. The former is much the same as the latter with one grade lowered components
such as LC-20AD pump, DGU-20A3R degasser, SIL-20A autosampler and CTO-20AC
column oven, coupled to a quadrupled type tandem mass spectrometer (LCMS-8040) via also
an ESI interface. The LCMS-8050 and LCMS-8040 system are both controlled by
LabSolutions LCMS Ver.5.6 (Shimadzu, Kyoto, Japan).
A sample (10 μL) was injected into a reversed-phase packed column (XR-ODS, 50 mm × 3.0
mm I.D., 2.2 μm, Shimadzu) installed for either LCMS-8050 or LCMS-8040. It was eluted at
a column temperature of 40 °C and flow rate of 0.3 mL/min, with a binary solvents of 0.1%
FA in water (A) and 0.1% FA in ACN (B) at programed gradient from 99:1 A:B (v/v) to 50:50
A:B (v/v) over 31 min, and positive ion species were detected by MS via MRM mode. The
MS parameters for LCMS-8040 were set as follows: nebulizing gas flow at 3 L/min, drying
gas flow at 15 L/min, DL temperature at 300 °C, heat block temperature at 450 °C and CID
gas at 230 kPa; while those for LCMS-8050 were nearly the same except for two new factors
of heating gas flow at 10 L/min and interface temperature at 300 °C.
Figure 1. MRM chromatograms (transition of 504.30 → 388.20) of EDC-labeled 1 pM GA1
(black line) and condensed EDC-labeled 0.1 pM GA1 (red line) obtained with LCMS-8050.
Investigation of the lowest labeling concentration of GAs was limited by the sensitivity of MS
instrument. By a moderately sensitive instrument of LCMS-8040 (Shimadzu Co, Kyoto,
Japan), the reaction could reach at least 10 pM GA1 standard. By using a more sensitive
instrument such as Shimadzu LCMS-8050, the direct labeling concentration could further be
lowered to 1 pM GA1. This looks not the lowest reactable concentration but an even advanced
instrument was required to directly prove it. At present, the deduction was confirmed by the
following experiment:
GA1 at 0.1 pM reacted first with EDC, then was frozen with liquid nitrogen and completely
dried in a freeze dryer (extremely low temperature stops the reaction), and finally re-dissolved
in proton-free solvent of acetonitrile (causing no reaction) to form 1 pM EDC-GA1 and
subjected to MS detection. The result shows that the peak intensity and retention time are
comparable with that from 1 pM GA1.
Supplemental Figure 2. Possible reaction pathways and bypass for labeling GA with EDC. 1
GA3; 2 EDC; 3 O-acylurea intermediate and O→N migration; 4 N-acylurea product; 5-7
isomers of GA3 under base or high temperature conditions; 8 amidation of GA3.
Supplemental Figure 3. Representative MS and MS/MS spectra and the cleavage mode of
EDC-GA3. The MS/MS spectrum shows fragmentation patterns for EDC-GA3.
The N-acylurea structures of EDC-GAs were confirmed through their fragmentation rules in
ESI-MS/MS. For example, [(EDC-GA3)+H]+ loses two fragments of m/z 71 and 45 attributing
to CH3CH2NHC=O and CH3NHCH3 from the tertiary amino unit of EDC, respectively. The
abundant fragment at m/z 129 is attributed to the N-acylurea unit. The same rules were
followed when GA3 was replaced by other GAs.
Supplemental Figure 4. Fourier Transform infrared spectra of GA3 (lower line) and EDC-
GA3 (upper line).
The stretching vibrations of hydroxyl and antisymmetric stretching of carboxyl in free GA3,
centered at 3449.06 and 1743.13 cm-1, respectively, disappear after reaction with EDC, with
two new bands appearing at 1652.82 and 1563.89 cm-1 corresponding to C=O (amide I)
stretching and N-H (amide II) vibrations, respectively.
Supplemental Figure 5. Effects of temperature (-4, 15, 25, 35, 50, 60, 70 and 90 °C) and
time (0–150 min) on the labeling reaction of GA3 with EDC. The data were obtained with
LCMS-8040. The reaction concentration of GA3 and EDC were 1 nM and 10 mM,
respectively.
(A) Reaction temperature (°C) at: (■) -4; (●) 15; (▲) 25; (▼) 35; (♦) 50.
(B) Reaction temperature (°C) at: (■) 60; (●) 70; (▲) 90. Solid lines refer to the target
product of EDC-GA3, while dash lines refer to the isomer of EDC-GA3.
The target peak area of EDC-GA3 changes in three formats: At and below 15 °C, the reaction
cannot reach its equilibration state even after 150 min, giving a continuously increasing plot
of the peak area vs. time. The plot slop becomes very small at -4 °C, and the target peak is not
more detectable if the temperature is further lowered, implying that temperature can serve as a
way to control the reaction in case required. At temperatures between 25 and 50 °C, the peak
area of EDC-GA3 increases first with time and then turns to a plateau with a turning point
shortened from ca. 90 min at 25 °C to 45 min at 50 °C. Further increase of the temperature
shows negligible effect on reducing the reaction time but results in a decrease of the target
peak area (solid lines) and emerging of an unexpected peak (dash lines). This phenomenon
should be caused by the isomerization of GA3 at a high temperature due to the lactone
rearrangement. The structural rearrangement of GA3 was further confirmed by first incubating
free GA3 at 90 °C and then reacting with EDC after cooled to 35 °C. The two ionic species
were indeed detected which was consistent with that of directly labeling GA3 at high
temperature.
Supplemental Table 1. Selected reaction monitoring conditions for EDC-GAs and EDC-
dGAs on LCMS-8050.
AnalytesQuasi-molecular
ionsFragment ions Q1 Pre Bias (V)
CE
(V)Q3 Pre Bias (V) RT (min)
EDC-GA1 504.30 388.20-26.0 -34.0 -18.0 12.3
EDC-dGA1 506.30 390.20
EDC-GA3 502.25 431.30-26.0 -24.0 -21.0 12.1
EDC-dGA3 504.25 433.30
EDC-GA4 488 .30 372.25-25.0 -35.0 -26.0 19.6
EDC-dGA4 490.30 374.25
EDC-GA5 486.30 370.20-18.0 -35.0 -26.0 16.1
EDC-dGA5 488.30 372.20
EDC-GA6 502.30 386.15-26.0 -36.0 -26.0 14.5
EDC-dGA6 504.30 388.15
EDC-GA7 486.30 370.20-25.0 -32.0 -25.0 19.4
EDC-dGA7 488.30 372.20
EDC-GA8 520.30 404.15-38.0 -37.0 -29.0 7.7
EDC-dGA8 522.30 406.15
EDC-GA9 472.30 356.15-24.0 -34.0 -24.0 21.9
EDC-dGA9 474.30 358.15
EDC-GA15 486.30 370.25-18.0 -35.0 -25.0 23.2
EDC-dGA15 488.30 372.25
EDC-GA20 488.30 372.15-25.0 -35.0 -26.0 16.4
EDC-dGA20 490.30 374.15
EDC-GA29 504.30 388.20-26.0 -35.0 -27.0 8.5
EDC-dGA29 506.30 390.20
EDC-GA34 504.30 388.20-26.0 -35.0 -27.0 17.5
EDC-dGA34 506.30 390.20
EDC-GA44 502.30 386.20-26.0 -36.0 -27.0 18.2
EDC-dGA44 504.30 388.20
EDC-GA51 488.30 372.15-18.0 -35.0 -25.0 17.7
EDC-dGA51 490.30 374.15
GA, gibberellins; EDC, N-(3-dimethylaminopropyl)-N’-ehtylcarbodiimide hydrochloride; EDC-GAi, EDC-labeled GAi; EDC-dGAi: EDC-
labeled deuterium-labeled GAi; CE, collision energy; RT, retention time.
By MRM mode, quasi-molecular ions [(EDC-GA)+H]+ and the intensive fragment ions were
selected to preform MS detection. The abundant fragment at m/z 129, which was detected
from all EDC-GAs, was not selected because of the serious interference at low m/z region. It
should be noted that some GAs, such as GA1, GA29 and GA34, form an equally weighed
product of EDC-GA, all giving a peak of [(EDC-GA)+H]+ at m/z 504.30 and fragment at m/z
388.20, respectively, but they can easily be differentiated by their different retention times.
Supplemental Table 2. LC gradient programs for both LCMS-8040 and LCMS-8050
systems.
Time (min) Gradient (percentage of mobile phase Ba by volume)
0.01 1
0.01-1.00 Increase linearly to 4
1.00-2.00 Increase linearly to 6
2.00-5.00 Increase linearly to 9
5.00-9.00 Increase linearly to 12
9.00-21.00 Increase linearly to 34
21.00-25.00 Increase linearly to 50
25.00-27.00 100
27.00-31.00 Decrease linearly to 1
LC, liquid chromatography.a Mobile phase A: distilled water with 0.1% formic acid; mobile phase B: acetonitrile with 0.1% formic acid.
LC resolution was simply adjusted by changing the gradient elution program to completely
resolve all the target EDC-GAs available at present. It is better to elute the excessive EDC
before EDC-GAs, which was achieved by a slow increase of the content of ACN or its
gradient during the initial phase of separation.
Supplemental Table 3. Linearity, LOD and LOQ of 14 GAs obtained with LCMS-8050.
Analytesy = ax + b Liner rangea
(fmol)
LODa
(fmol)
LOQa
(fmol)Slope a Intercept b R2
GA1 0.7319 0.009480 0.9989 0.01-10 0.00327 0.0109
GA3 1.129 0.01091 0.9991 0.05-10 0.0125 0.0417
GA4 1.458 0.02724 0.9957 0.01-10 0.00686 0.0229
GA5 0.7906 0.01125 0.9988 0.01-10 0.00984 0.0328
GA6 1.218 0.002130 0.9943 0.01-10 0.00950 0.0317
GA7 1.914 0.04769 0.9995 0.01-10 0.0112 0.0374
GA8 1.310 0.004700 0.9960 0.01-10 0.00727 0.0242
GA9 0.9919 0.006742 0.9993 0.05-10 0.0221 0.0735
GA15 0.6776 0.01639 0.9984 0.05-10 0.0270 0.0901
GA20 0.8206 -0.0009900 0.9984 0.01-10 0.00775 0.0258
GA29 1.241 -0.004950 0.9998 0.01-10 0.00665 0.0222
GA34 0.5386 0.06220 0.9968 0.05-10 0.0195 0.0650
GA44 0.9825 0.003390 0.9974 0.05-10 0.0142 0.0474
GA51 0.5509 0.02120 0.9976 0.01-10 0.0950 0.0317
LOD, limit of detection (S/N=3); LOQ, limit of quantification (S/N=10).a The injection volume is 10 μL.
To evaluate the linearity of the method, eight solutions containing various amounts of each
authentic GA standards (0, 1, 5, 10, 50, 100, 500 and 1000 pM) and a fixed amount of
corresponding dGA (50 pM) as internal standards were prepared, labeled with EDC and then
analyzed. The calibration curves were constructed between the adjusted peak area ratio (y) of
EDC-GA over EDC-dGA and the amount ratio (x) of GA over dGA as the following formula:
{ y=ax+b ¿{y=AGA
AdGA−AGA α d¿¿¿¿
(1)
where a is the slop and b the intersect; AGA, αd and AdGA are the measured peak area of EDC-
GA, its natural abundance with isotope deuterium (ca. 5.5%), and the peak area of EDC-dGA,
respectively; and nGA is the amount of GA and ndGA is that of dGA. Noticeably, the portion
corresponding to the natural deuterium of EDC-GA has to be subtracted from the measured
peak of EDC-dGA in order to increase the linearity of the working curves.
Supplemental Table 4. Precisions (intra- and inter-day) and recoveries for the determination
of 14 deuterium-labeled GAs spiked in Arabidopsis thaliana flowers.
Analytes
Low concentration
(Added: 1 fmol, n = 3)
Medium concentration
(Added: 3 fmol, n = 3)
High concentration
(Added: 10 fmol, n = 3)
RSDa
(%)
RSDb
(%)
Recoveryc
(%)
RSDa
(%)
RSDb
(%)
Recoveryc
(%)
RSDa
(%)
RSDb
(%)
Recoveryc
(%)
GA1 4.19 2.11 87.2 0.840 1.09 85.5 1.59 3.83 90.8
GA3 4.93 5.01 89.8 3.59 5.21 86.4 2.31 2.19 89.7
GA4 3.23 4.27 90.9 3.42 3.98 86.9 7.56 9.50 85.9
GA5 6.53 9.75 85.5 4.81 9.26 85.0 4.76 6.06 89.0
GA6 3.66 11.3 88.8 1.93 8.54 87.0 8.93 7.44 92.1
GA7 8.69 5.36 82.3 3.43 3.60 84.9 2.37 2.04 84.0
GA8 3.38 5.12 65.9 2.87 3.54 63.1 4.98 6.25 72.0
GA9 2.79 2.65 105 4.39 10.2 91.1 1.08 7.83 90.0
GA15 7.81 11.5 97.3 3.21 8.35 93.1 5.20 3.09 91.2
GA20 7.76 5.99 81.7 3.83 4.72 85.5 7.31 1.79 84.8
GA29 3.06 9.23 74.2 2.02 6.12 79.7 1.23 1.73 72.9
GA34 9.18 3.69 96.1 2.67 3.77 90.8 3.15 2.90 91.3
GA44 8.06 8.89 94.0 4.48 6.38 91.8 6.42 7.20 92.2
GA51 1.03 3.00 90.3 7.10 6.76 88.9 3.63 5.55 87.6
RSD, relative standard deviation.a The results for intra-day precisions at three concentration levels;b The results for inter-day precisions at three concentration levels;c The mean value of recoveries for both intra-day and inter-day.
To evaluate the analytical accuracy of the method, whole flower samples of A. thaliana were
spiked with three level of each dGA at 1, 3 and 10 fmol, extracted, labeled with EDC, and
then analyzed. The intra-day variation of peak area of EDC-dGA was determined by three
replicates of each level in one day, and the inter-day variation was determined by repeating
each level for three different days. The recovery of each dGA was calculated based on the
peak area ratio of EDC-dGA that obtained from the spiked sample extracts and the standard
solution containing the same amount of dGA after derivatization.