| Description
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- Sample Extraction
Frozen chlamydomonas cells were homogenized with mortar and pestle in liquid nitrogen. The chlamydomonas cell powder or culture medium was extracted with three times-volume (w/v or v/v) of methanol containing 25 µM of 7-hydroxy-5-methylflavone as an internal standard. After homogenizing the samples by mixing twice for 2 min each time using a Mixer Mill MM 300 (QIAGEN K.K., Tokyo, Japan) and a zirconia bead (5 mm diameter in 2 mL tube) at 25 Hz, the homogenates were centrifuged (17,400 xg for 5 min at 4C). After recovery of the supernatant from each homogenate, it was filtered through a 0.2-µm polytetrafluoroethylene (PTFE) membrane (Merck Millipore). Hydrophobic compounds in the filtrate were removed by absorption to a C18 silica column (MonoSpin C18, GL Science, Tokyo, Japan). The eluate was used for liquid chromatography (LC)-Fourier transform ion cyclotron resonance (FT-ICR)-mass spectrometry (MS) analysis. A mock sample was prepared as above with water instead of the cell powder. A single extraction for each cell culture and mock sample, and triplicated extractions for medium were performed. An equal volume of three extracts which were prepared from the triplicated culture of a single treatment were mixed (hereafter referred as to ‘mixed sample’) and analyzed with the methods 5 and 7 for obtaining MS3 spectra.
-LC-MS analysis
The samples were analyzed using an Agilent 1100 system (Agilent) coupled to a Finnigan LTQ-FT (Thermo Fisher Scientific). Twenty microliters of extract was applied to a TSK-gel column ODS-100V (4.6 x 250 mm, 5 µm; TOSOH). Water (LC/MS grade; solvent A) and acetonitrile (LC/MS grade; solvent B) were used as the mobile phase with 0.1% v/v formic acid added to both solvents. 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 to 0.25 mL/min (0–100 min) and 0.5 mL/min (100.1–107 min). The column oven temperature was set to 40C. Compounds were detected in electrospray ionization (ESI)-positive mode over the m/z range 100–1500. Multistage MSn analyses were carried out using collision-induced dissociation in a linear ion trap detector with a normalized collision energy of 35.0% and an isolation width of 4.0 (m/z). The ESI settings were a spray voltage of 4.0 kV and capillary temperature of 300C. The nitrogen sheath gas and auxiliary gas were set at 40 and 15 arbitrary units, respectively. To monitor the HPLC eluate, a photodiode array detector was used with a wavelength range of 200–650 nm. Data were acquired and browsed using Xcalibur software version 2.0.7 (Thermo Fisher Scientific).
- MSn analysis [Method 7]
The method for MSn analysis was as below. Full mass scan with FT-ICR at a resolution of 12,500, MS2 scans for the most intense five ions of the full mass scan with IT, and MS3 scans for the most intense two ions of the MS2 scan with IT. A dynamic exclusion setting was as follows: repeat count, three; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; and exclusion duration, 30 s.
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| Comment_of_details
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[column] TSK-gel ODS-100V (4.6 x 250 mm, 5 micrometer; TOSOH)
[gradient] Solvent A: water containing 0.1% v/v formic acid; Solvent B: acetonitrile containing 0.1% v/v formic acid; Gradient: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min)
[total separation time] 107 min
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