Method details

The LC-MS apparatus and the analytical conditions were different from those in the sister websites Food Metabolome Repository (FMR) and Plant Metabolome Repository (PMR). Please be careful the following things when you browse and analyze the data.

• A given mass tolerance 20 ppm is recommended for precursor mass search.
  The mass detection accuracy is lower than those in FMR and PMR. Although the mass accuracy is less than 5 ppm in usual cases, however, it may become lower (approx. less than 16 ppm) in the peaks with lower intensity and lower m/z value (< 200).
• The retention time of LC is different because of the use of different column and elution conditions.
• Only MS/MS spectra were available.
• The MS/MS spectrum in this website may be different from those obtained in FMR and PMR even for the same compound.
• Inadequate fragmentations are often observed in the peaks with lower m/z value (< 300) detected in ESI negative mode.

Metabolite extraction

The samples were immediately frozen in liquid nitrogen, and stored at -80 degree C. In the case of water-containing solid sample, it was ground to a fine powder under liquid nitrogen using mortar and pestle and stored at -80 degree C until use. The liquid samples were used as they are. Compounds in the sample were extracted by approximately 75%(v/v) methanol. In the case of liquid samples and samples containing abundant water such as plant leaves, 3 times volume (v/v or w/v) of 100% methanol containing 1 uM 7-hydroxy-5-methylflavone as an internal control (IS) was added. In the case of liquid samples that do not mix with 75% methanol, such as essential oils, 4 times volume (v/v) of 75% methanol containing 1 uM IS was added. In the case of samples with low water content such as granules of herbal medicine, 40 times of 100 times volume (w/v) of 75% methanol containing 1 uM IS was added. The sample in methanol in a 2 mL tube was homogenized using a zirconia bead (5 mm diameter) and Mixer Mill MM 400 (Verder Scientific, Co., Ltd.) at 25 Hz for 2 min, twice. A homogenate was centrifuged at 17,400 x g, 5 min at 4 degree C. A supernatant, or if separated into two liquid layers, the methanol layer, was applied to a C 18 silica column (MonoSpin C18, GL Sciences Inc.) to remove highly hydrophobic contaminants. The filtrate was passed through a polytetrafluoroethylene (PTFE) filter (pore size 0.2 um, Millipore) and used for LC-MS analyses.

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

Nexera X2 system (Shimadzu Corporation) and Compact system (Bruker Japan K.K.) were used. An aliquot (2 uL) of the methanol extract was applied to an InertSustain AQ-C18 column (3 um x 2.1 mm x 150 mm, GL Sciences) connected after a guard column (InertSustain AQ-C18 Cartridge Guard Column E, GL Sciences), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile (Solvent B). The gradient program was as follows: 2% B (0 min), 2% B (3 min), 98% B (30 min), 98% B (35 min), 2% B (35.01 min), and 2% B (42 min). The flow rate was set at 0.2 mL/min. The column oven temperature was set to 40 degree C.

The compounds separated by the LC were detected using the mass spectrometer under the conditions below:

ESI positive mode

MS settings

Ionization:  Electrospray Ionization (ESI),  Polarity  Positive,  Data aqcuisition:  Profile mode,  Scan rate:  1 Hz,  Mass scan range:  50-1200,  End plate offset:  500 V,  Capillary voltage:  4000 V,  Nebulizer bas (N2) pressure:  2.5 Bar,  Dry gas (N2) flow:  8.0 L/min,  Dry gas temperature:  200℃,  Transfer Funnel1 RF:  200.0 Vpp,  Funnel2 RF:  200.0 Vpp,  In-source CID Energy:  0.0 eV,  Hexapole RF:  50.0 Vpp,  Quadrupole Ion Energy:  3.0 eV,  Low Mass m/z:  55.00,  Collision energy:  10.0 eV,  Collision RF:  450.0 Vpp,  Transfer Time:  80.0 us,  PrePulse Storage:  3.0 us 

MS/MS settings

Auto MS/MS:  on,  Precursor ion number:  5,  Isolation width:  3 - 15 Da,  Collision energy:  35 eV,  Active Exclusion:  on,  Exclude:  after 3 Spectra,  Release:  after 0.3 min,  Reconsider Precursor:  on,  if Current Intens. / Previous Intens.:  2.0 

ESI negative mode

The same settings as ESI positive mode were applied except follows:
Polarity:  Negative,  MS/MS Collision energy:  30 eV, 

For mass calibration, 1 mM sodium formate in 50% (v/v) 2-propanol was injected directly into the MS at 38.50–40.50 min of LC separation with a flow rate 0.1 mL/min. The eluent at 0-3 min was wasted. The signals detected at 0-3 min are those from calibrants automatically inhalated from the line. The raw data were obtained by Hystar software (ver.3.2 SR4, Bruker Daltonik, GmbH).

Data analysis

The parameter setting files for PowerGetBatch tool (described below) were available here.

Mass calibration and conversion to mzXML format

Mass values were calibrated using the signals of sodium formate detected at 39-40 min using Compass DataAnalysis software (ver. 4.2, Bruker Daltonik, GmbH). The calibrated mass chromatogram was converted to an mzXML-formatted file using the MSConvert function of the ProteoWizard software (ver. 3.0, http://proteowizard.sourceforge.net/, Kessner et al., Bioinformatics 24: 2534-2536, 2008).

Peak detection

The compound peaks were detected and characterized using the mzXML files and PowerGetBatch software (ver. 0.5.4). Three different sets of peak detection parameters were applied for each sample and a single set of parameters was used for the mock sample. The peaks detected with all three parameters for a sample and not detected in two mock samples obtained in the same measurement batch were selected as valid peaks. The peaks with a retention time less than 3 min or greater than 32 min were omitted. The most intense and major pattern of the MS/MS spectrum was selected among the alignment results for each peak.

Database searching

Compound database searching was performed using the accurate mass value and the estimated adduct. A 20 ppm mass tolerance was applied. 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.

Prediction of flavonoid aglycones

For the peaks with MS/MS spectra detected in the positive mode, possibility of flavonoid aglycones was estimated using FlavonoidSearch system.