Fig. 1 provides an outline of this protocol. Procedures in four of the sections (I. Extraction; II. Preparation of fatty acid methyl esters; III. Derivatization; and IV. Chromatography and mass spectrometry) are briefly described in our previous paper6. Details for each section are described below.
1． Place 50 mg brown rice powder and 300 mg quartz sand into a 2-mL tube.
2． Add 1 mL of a methanol–chloroform–2% acetic acid mixture (5:2:1, v/v/v) containing 5 μg mL−1 ribitol and 20 μg mL−1 testosterone (as internal standards) into the tube.
3． Shake the tube in a mixer mill for 2 min at a frequency of 30 Hz.
4． Centrifuge the tube at 12,000 × g for 10 min.
5． Collect the supernatant into a new 2-mL tube, and re-extract the precipitate using the same procedure.
6． Combine the supernatants obtained after the first and second extractions.
7． Transfer the supernatant (800 μL) into a new 2-mL tube.
8． Add 500 μL of ultrapure water and 400 μL of chloroform into the tube and mix by vortex mixer for 1 min.
9． Centrifuge the tube at 12,000 × g for 2 min.
10． Separate the upper phase (polar fraction) from the lower phase with the inter-phase (non-polar fraction).
11． Lyophilize the fractions in the tubes in a centrifugal concentrator for 6 h (polar fraction) or 1 h (non-polar fraction).
12． Lyophilize the dried fractions in the tubes in a freeze dryer for 16 h.
II. Preparation of fatty acid methyl esters (FAMEs)
Transesterification of esterified fatty acids to FAMEs was performed using a base-catalyzed method.
1． Add sodium methoxide (500 μL, approximately 30% in methanol) into the tube (dried non-polar fraction).
2． Incubate the tube at 55°C for 90 min with shaking (1,000 rpm).
3． Add 1 mL of 1% acetic acid (to neutralize the NaOH) and 400 μL chloroform into the tube, mix by vortex mixer, and centrifuge the tube at 12,000 × g for 2 min.
4． Remove the upper phase, add 1 mL of ultrapure water into the tube, mix by vortex mixer, and centrifuge the tube again as above.
5． Remove the upper phase, and lyophilize the remaining lower phase in the tubes in the centrifugal concentrator for 1 h.
6． Lyophilize the dried fractions in the tubes in a freeze dryer for 16 h.
Metabolites were derivatized using a combination of methoxamine hydrochloride and MSTFA.
1． Add methoxamine hydrochloride solution (20 μL; 40 mg mL−1 in pyridine) into the dried polar and non-polar fractions (esterified fatty acids are converted into FAMEs) and incubate the tube at 30°C for 90 min with shaking (1,000 rpm).
2． Add 80 μL of MSTFA into the tube and incubate at 37°C for 30 min with shaking as above.
3． Centrifuge the tube at 12,000 × g for 3 min.
4． Filter the supernatant through a hydrophilic PTFE membrane filter unit, and collect the flow through into a vial with a grass micro insert.
5． Prepare n-alkane mixture in another vial with a grass micro insert by mixing alkane standard solutions (C8–C20 and C21–C40, 25 μL each) with 50 μL of pyridine.
IV. Chromatography and Mass spectrometry
GC–MS analysis was performed using a Micromass GCT Premier Mass Spectrometer connected to an Agilent 6890 gas chromatograph and an PAL GC-xt autosampler. MassLynx 4.0 software was used to control the GC–MS system. Derivatized samples and an n-alkane mixture were independently analyzed in the same batch.
・ Column: HP-5ms capillary column (length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 μm)
・ Carrier gas: helium (> 99.999% purity)
・ Carrier gas flow rate: 1.0 mL min−1
・ Oven temperature program: 70°C for 1 min then increase the temperature by 1°C min−1 to 76°C, followed by another temperature increase by 6°C min−1 to 350°C, with a final hold time of 3 min
・ Injection port temperature: 230°C
・ Transfer line temperature: 250°C
・ Injection volume: 1 μL
Mass spectrometry parameters:
・ Ionization mode: electron ionization (EI) mode
・ Electron energy: 70 eV
・ Trap current: 50 μA
・ Filament current: 3.6 A
・ Emission current: 100 μA
・ Ion source temperature: 250°C
・ Detector voltage: 2,700 V
・ Scan frequency: 10 spectra/second
・ Mass range: 50–650 m/z
Maintenance for the GC–MS system:
・ Instrument tuning: Perform every month
・ Calibration: Perform every day
・ Reference compound (heptacosa): Replace or add as needed
・ Syringe wash solvent (acetonitrile): Replace every day
・ Injection port liner: Replace every 2–4 weeks (depending on the frequency of use)
・ Injection port septa: Replace every 1–2 weeks (depending on the frequency of use)
・ Ion source: Clean every month
・ Worn out parts (filament, column, syringe, etc.): Replace as needed
V. Data transformation
The raw data file (.raw) was converted to netCDF data file (.cdf) using the Databridge interface of the MassLynx file converter. From the set of netCDF data files, the data matrix was generated using "MetAlign":https://www.wur.nl/en/show/MetAlign-1.htm (version 041012)7. The parameters for the baseline correction and ion peak alignment by MetAlign were as follows:
Part A (Program configuration, Data selection and Baseline correction):
・ peak slope factor (× noise) = 4
・ peak threshold factor (× noise) = 8
・ peak threshold (absolute value) = 500
・ average peak width at half height (scans) = 4
Part B (Scaling and aligning data sets):
・ Data scaling: no scaling
・ Internal peak search criteria: beginning of the first region = 0 (max. shift = 5)
・ End of the first region = 3,500 (max. shift = 5)
・ Tuning alignment option and criteria: pre-align processing, iterative
・ Calculation criteria for chromatography shift profiles: max. shift per 100 scans = 35
・ Min. factor (× noise) at the first and last iterations = 7 each
・ Min. number of masses at the first and last iterations = 10 and 5, respectively
VI. Metabolite identification
"AIoutput2":http://prime.psc.riken.jp/Metabolomics_Software/AIoutput/index.html (version 1.30)8 was used to read the MetAlign processed data file (.csv) for the deconvolution of overlapped ion peaks based on their retention times to list metabolite-candidate peaks. Retention index values for individual metabolite-candidate peaks were obtained by normalizing their retention times referring to those of n-alkane species (C8–C40) from total ion current chromatograms. A custom reference mass spectral library was built using a software "AMDIS":http://www.amdis.net/ (Automated Mass Spectral Deconvolution and Identification System). Identification of metabolites by the AIoutput2 was based on the Pearson’s product-moment correlation coefficient (PPMCC) considering the retention times and weighted mass spectra of the compounds in the reference mass spectral library.
Data analysis steps for AIoutput2 is as follows:
Part 1 (create a Peak table):
1． Export the processed data (MetAlign) as a text file (.csv)
2． Read the file (.csv) by AIoutput2 software version 1.30
3． Fill in the parameters (Height threshold, and RT bining)
4． Click ‘Make’ to make a MZTable sheet
Part 2 (Peak identification and annotation):
1． Specify the Available Index (Retention time or Retention index)
2． Specify the Analysis Type (Non target or Target)
3． Fill in the parameters (RI tolerance, and Match threshold)
4． Click ‘Search’ to make PeakTable and IdentificationTable sheets. In the IdentificationTable sheet, the reference mass spectral library compound names are assigned to each metabolite-candidate peak and the values of the identification score, Delta-RI, and PPMCC can be obtained. Delta-RI is a value obtained by subtracting the RI value of a metabolite-candidate peak from that of an assigned reference library compound.
Part 3 (Filtering):
1． Select the PeakTable sheet and fill in the class row with numbers (1, 2, 3, ...). Replicates should be assigned the same number.
2． Select filtering type (Accurate, Mild, or Rough)
3． Fill in the parameters [Height filter, and RSD(CV) filter]
4． Click ‘Done’ to make a PeakTableUpDate sheet
*We used the following parameters in the data analysis steps for AIoutput2 in the current study4:
・ Height threshold = 500
・ RT bining = 1
・ Available index: retention index
・ Analysis type: non-targeted
・ RI tolerance = 20
・ Match threshold (identification score) = 0.6
・ Filtering: accurate
・ Height filter = 3,000
・ RSD(CV) filter = 30