Method details

The analytical methods applied uniformly to all samples are briefly described here. The details such as plant growth conditions are available at the Metabolonote website which you can access through the button at the bottom of the peak list page.
Ex) The details for arabidopsis are available at the bottom of the peak list page of arabidopsis

The conditions for LC-separation and MS1 scans are the same as those of Food Metabolome Repository. Therefore, the retention times of LC, as well as the accurate m/z values for the peaks, can be compared between the two repositories. Be careful, however, the retention time is not so stable as in Food Metabolome Repository because the samples have been analyzed across a much longer period. The differences of the methods between this site and Food Metabolome Repository are the variety of the MS/MS settings and the way of selection of the valid peaks.

Metabolite extraction

The samples were homogenized in liquid nitrogen to a fine powder using mortar and pestle. The liquid samples were used as they are. The sample was mixed with 3 times volume (w/v or v/v) of 100% methanol containing 25 uM of 7-hydroxy-5-methylflavone as internal standard (IS) in a 2 mL tube and homogenized using Mixer Mill MM 300 (QIAGEN) at 25 Hz, for 2 min, twice. The homogenate was centrifuged under 17,400 x g, 5 min at 4 C. A supernatant was passed through a Polytetrafluoroethylene (PTFE) filter (pore size 0.2 um, Millipore), and the filtrate was applied to a C18 silica column (MonoSpin C18, GL Science) to remove highly-hydrophobic contaminants. The filtrate passed through the C18 column was used for LC-MS analyzes.

The same extraction procedure with 75% methanol was performed without a sample to prepare mock samples and used as negative controls.

Liquid chromatography (LC)-mass spectrometry (MS) analysis

Agilent 1100 system (Agilent) and Finnigan LTQ-FT (Thermo Fisher Scientific) were used. Aliquots (5 uL) of the methanol extract were applied to a TSK-gel ODS-100V column (4.6 x 250 mm, 5 um, TOSO), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile containing 0.1%(v/v) formic acid (Solvent B). The gradient program was as follows: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min). The flow rate was set at 0.25 mL/min for 0-100 min, and 0.5 mL/min for 100.1-107 min.

The conditions of mass spectrometry were as below:

Method Name	Description
Method 1	This method is used for measuring the accurate mass of the peaks and obtaining MS/MS spectra. Full scan: resolution, 100,000 by FT; m/z range, 100-1500 MS2 scan: data-dependent scan by IT for the most intense 5 ions in the full scan. Dynamic exclusion setting for MS2 scan: repeat count, 3; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; exclusion duration, 20 s.
Method 2	Same as Method 1 except below: Dynamic exclusion setting for MS2 scan: exclusion duration, 30 s.
Method 3	Same as Method 1 except below: Dynamic exclusion setting for MS2 scan: repeat count, 2; exclusion duration, 30 s.
Method 4	Same as Method 1 except below: Dynamic exclusion setting for MS2 scan: off.
Method 5	This method is used for obtaining MS3 spectra. Full scan: resolution, 12,500 by FT; m/z range, 100-1500 MS2 scan: data-dependent scan by IT for the most intense 5 ions in the full scan Dynamic exclusion setting for MS2 scan: same as Method 1 MS3 scan: data-dependent scan by IT for the most intense 2 ions in the MS2 scan
Method 7	Same as Method 5 except below: Dynamic exclusion setting for MS2 scan: same as Method 2
ESI: electrospray ionization, FT: Fourier transformation ion cyclotron resonance type mass spectrometry, IT: ion trap type mass spectrometry.

The mass analyses with electrospray ionization (ESI) in positive or negative modes were performed. The combinations of the Methods 1-7 and ionization polarity are different between the samples, but, at least one method for accurate mass measurement (Method 1-4) was applied for each sample. The raw data were acquired using Xcalibur software (Thermo Fisher Scientific).

Data processing

The PowerGetBach software, which is good at detecting whole peaks including lower intensity ones and therefore good at predicting appropriate adducts, was used.

Peak detection and alignment

In the case of the sample with more than three biological or technical replications, a single parameter setting for high resolution (Method 1-4) was applied for peak detection. In the case of the sample without replications, a single raw datum was processed using three different parameter settings for high resolution (Method 1-4). The data obtained in low-resolution settings (Method 5 and 7) was processed with a single parameter setting. The detected peaks in the samples with high- and low-resolution analyses and in the mock samples were aligned using PowerGetBatch software for each positive and negative modes. The reproducibly detected peaks in the samples and absent in the mock samples were selected as valid peaks. The selection was performed manually using Microsoft Excel with consideration of the analytical replications of the sample.

Database searching

Compound database searching was performed using the accurate mass value and the estimated adduct. The following databases are used: KEGG, KNApSAcK, Human Metabolome Database (HMDB), LipidMAPS, flavonoid database in metabolomics.jp. The UC2 database in the MFSearcher web service was used for rapid cross-database searching and compiling the constitutional isomers in one record.

Please note that different stereoisomers are included in one record. The marks such as '[-1fr]' shown in the DB search results in the peak information page represents the states of the molecule in the original database as follows: The number, the charge of the molecule, 'f', a fragment in the molecule registered as multiple compartments (such as salts) was hit, 'r', the molecule was registered as a radical.

FlavonoidSearch

Prediction of flavonoid aglycones was conducted by FlavonoidSearch using MS2 and MS3 spectra in positive mode. A possibility of the peak being a flavonoid is high when a higher hit score, usually larger than 0.3, is observed. Candidates of known flavonoids are listed in the FlavonoidSearch results.

FlavonoidSearch calculates similarities of query spectra to those of virtual mass fragment database which is constructed based on knowledge and heuristics of CID. The virtual mass fragment data were constructed approximately 4500 flavonoids among approx. 7000 known flavonoids. No virtual mass fragment data were attached to the rest of the approx. 2500 flavonoids. If the precursor mass was hit to these no virtual fragment ones, FlavonoidSearch returns score zero. Therefore, results with score zero remained in the result table to show the possibility of them.