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Sample Set Information

ID TSE1236
Title Development of a Direct Headspace Collection Method from Arabidopsis Seedlings Using HS-SPME-GC-TOF-MS Analysis.
Description Plants produce various volatile organic compounds (VOCs), which are thought to be a crucial factor in their interactions with harmful insects, plants and animals. Composition of VOCs may differ when plants are grown under different nutrient conditions, i.e., macronutrient-deficient conditions. However, in plants, relationships between macronutrient assimilation and VOC composition remain unclear. In order to identify the kinds of VOCs that can be emitted when plants are grown under various environmental conditions, we established a conventional method for VOC profiling in Arabidopsis thaliana (Arabidopsis) involving headspace-solid-phase microextraction-gas chromatography-time-of-flight-mass spectrometry (HS-SPME-GC-TOF-MS). We grew Arabidopsis seedlings in an HS vial to directly perform HS analysis. To maximize the analytical performance of VOCs, we optimized the extraction method and the analytical conditions of HP-SPME-GC-TOF-MS. Using the optimized method, we conducted VOC profiling of Arabidopsis seedlings, which were grown under two different nutrition conditions, nutrition-rich and nutrition-deficient conditions. The VOC profiles clearly showed a distinct pattern with respect to each condition. This study suggests that HS-SPME-GC-TOF-MS analysis has immense potential to detect changes in the levels of VOCs in not only Arabidopsis, but other plants grown under various environmental conditions.
Authors Kusano M, Iizuka Y, Kobayashi M, Fukushima A, Saito K.
Reference Metabolites. 2013 Apr 9;3(2):223-42. doi: 10.3390/metabo3020223.

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Sample Information

Title Arabidopsis thaliana
Organism - Scientific Name Arabidopsis thaliana
Organism - ID NCBI taxonomy 3702
Compound - ID
Compound - Source
Preparation Wild-type Arabidopsis thaliana plants (accession Columbia (Col-0)) were used in this study. The plant seeds, which had been sterilized by sodium hypochlorite solution (Wako Pure Chemical Industries, Osaka, Japan), were stratified at 4 °C for 3 days. They were subsequently grown in MS medium (Wako Pure Chemical Industries) containing vitamins (Sigma-Aldrich, Tokyo, Japan; Lot, RNBB4051) with 0.8% agar and 1% sucrose at pH 5.8. Samples were exposed to fluorescent light under a 16-h light (51-μmol·m−2·s−1)/8-h dark cycle at 23 °C for 7 days in a growth chamber (MLR-350H; Sanyo, Osaka, Japan). Next, 20 seedlings were transferred into 20-ml HS vial (Supelco, Missouri, US) containing 5-mL of (i) sterile liquid culture of MS medium with 1% sucrose at pH 5.8 or (ii) sterilized Milli-Q water. The vials were closed with magnetic screw caps equipped with either Silicon/PTFE septa (AMR, Tokyo, Japan) or affixed with MilliSeals (EMD Millipore, Billerica, MA, USA). The seedlings were grown on a shaker (MMS-3010; Eyela, Tokyo, Japan) under a 16-h light 51-μmol·m−2·s−1)/8-h dark cycle at 23 °C for 7, 14 and 21 days. The shaker speed was set at 150 rpm during the cultivation. The HS vials were directly used for HS collection via SPME.
Sample Preparation Details ID SS1

Sample Preparation Details Information

Title Headspace Collection
Description We used two methods and assayed to collect the HS of Arabidopsis seedlings after a 7 day-incubation. To quench enzyme activity in Arabidopsis, we treated the seedlings in the following manner: (i) a set of HS vials wrapped in aluminum foil were incubated at 80 °C for 30 min in a windy oven (WFO-600 ND; Eyela, Tokyo, Japan) and (ii) 0.25-M EDTA-NaOH water solution (pH 7.5) was added into another set of vials to attain a final EDTA concentration of 50 mM. Solid CaCl2 was then immediately added to a final concentration of 5 M. Next, 10-μL of n-alkane standard solutions C8-20 (0.8 mg/l) was added to each vial as an internal standard. The vials were closed with magnetic screw-caps and then sonicated (US-108; NSD, Suwa, Japan) at a frequency of 38-Hz for 5 min. Vials without any treatment were prepared and used as controls.

Nine types of SPME fibers were purchased from Supelco (Supelco, PA, USA). A SPME fiber coated with a 100-μm-thick layer of polydimethylsiloxane (PDMS) metal alloy (100-μm PDMS) was finally chosen for VOC profiling (see Results and Discussion section). SPME fibers were coated with (i) 30-μm- or (ii) 7-μm-thick layers of PDMS-fused silica (FS) fiber/stainless steel (SS), (iii) 75-μm-thick layer of carboxen/polydimethylsiloxane (CAR/PDMS) FS/SS, (iv) 85-μm-thick layer of CAR/PDMS StableFlex (SF) fiber/SS, (v) 65–μm-thick layer of PDMS/divinylbenzene (DVB) SF/SS, (vi) 50/30-μm-thick layer of DVB/CAR on PDMS (DVB/CAR/PDMS) SF/SS, (vii) 85-μm-thick layer of polyacrylate (PA 85) or (viii) carbowax-polyethylene glycol (C-PEG) for HS collection. Before the analysis, all fibers were conditioned at the appropriate conditioning temperature and time as shown in Table 3. Each fiber was exposed to the vial headspace for 20 min at 60 °C with continuous agitation.


Analytical Method Information

Method Details ID MS1
Sample Amount

Analytical Method Details Information

Instrument GC:Agilent 6890N MS:LECO Pegasus 4D MS system
Instrument Type
Ionization EI
Ion Mode Positive
Description After HS collection, the volatiles were thermally desorbed in splitless mode on a CTC CombiPAL autosampler (CTC analytics, Zwingen, Switzerland) connected to an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmington, USA) for 0.1 min at the appropriate inlet temperature, as shown below. Each fiber was baked for 5 min by applying the appropriate conditioning temperature (Table 3).

The capillary column used for the analysis was a 30-m×0.25-mm inner diameter fused-silica capillary column with a chemically bound 0.25-μL film Rxi-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) with a tandem connection to a fused silica tube (1 m, 0.15 mm). A mass spectrometer column change interface (ms NoVent-J; SGE, Yokohama, Japan) was used to prevent air and water from entering the MS during column change over. Helium was used as the carrier gas at a constant flow rate of 1.0 ml/min. The temperature program started with a 2 min isothermal step at 50 °C, followed by temperature ramping at 15 °C to a final temperature of 260 °C, which was then maintained for 2 min. The transfer line to the mass spectrometer was set to 250 °C. The TOF mass spectrometer was a Pegasus 4D MS system (Leco, MI, USA) with an EI source set to 200 °C. The acceleration voltage was turned on after a solvent delay of 200 s. Mass spectra were monitored with an acquisition rate of 30 spectra/s and over a mass-to-charge ratio range of m/z = 30–550.


Table 3. Conditioning temperature and time for the SPME fibers used in the study.
SPME fiber Conditioning temperature(°C) Conditioning time(h) Inlet temperature(°C)
100-μm PDMS 250 0.5 220
30-μm PDMS 250 0.5 220
7-μm PDMS 320 0.5 220
85-μm polyacrylate 280 1 220
60-μm PEG 240 0.5 220
75-μm CAR/PDMS 300 1 280
85-μm CAR/PDMS 300 1 280
65-μm PDMS/DVB 250 0.5 250
50/30-μm DVB/CAR/PDMS 270 0.5 250

Data Analysis Information

Title Data Processing and Analysis
Data Analysis Details ID DS1
Recommended decimal places of m/z

Data Analysis Details Information

Title Data Processing and Analysis
Description Data Processing

Non-processed MS data from GC-TOF-MS analysis were exported in NetCDF format, which was generated by the chromatography processing and mass spectral deconvolution software (Leco ChromaTOF version 2.32; LECO, MI, USA), to MATLAB 7.0 and MATLAB2011b (Mathworks, MA, USA), where all data pretreatment procedures, such as smoothing, alignment, time-window setting and hierarchical multivariate curve resolution (H-MCR), were carried out. The resolved mass spectra were matched against the reference mass spectra by using the NIST mass spectral search program from the NIST/EPA/NIH mass spectral library (Data Version: NIST 08, Software Version 2.0), Terpenoid library, Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry (the 4th Edition) commercial library and VocBinbase. Our custom software was also used for peak annotation written in Java. The metabolites were defined as annotated metabolites by comparison with RI values calculated by alkane standard mixtures (C8–20) and mass spectra from these three libraries. Peaks that showed a match factor of ≥800 in the mass spectral library were selected for annotation. Data were normalized by dividing each peak area by the peak area of n-tridecane and metabolite identifiers were organized using MetMask.

Data Analysis

The two data matrices (observations: biological replicates, variables: annotated peaks) were used for statistical analyses, respectively (see Supplementary Tables S1–3). Multivariate analysis was performed using the SIMCA-P+13.0 software (Umetrics AB, Umeå, Sweden), with log10 transformation and unit variance scaling. Metabolite profile data were log2 transformed and then statistically analyzed using the LIMMA package, which includes false discovery rate (FDR) correction for multiple testing (Benjamini and Hochberg, 1995) in the R environment for statistical computing (version 2.14.2). Analysis of variance testing of cross-validated predictive residuals (CV-ANOVA) was used as the significance test for assessing the reliability of O2PLS-DA models (significant, p cv < 0.01).

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