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Sample Set Information

ID SE195
Title LC-MS analysis of LIPID REMODELLING REGULATOR 1 (LRL1) transgenic lines of Chlamydomonas reinhardtii
Description A metabolome analysis was conducted by LC-MS using a wild type Chlamydomonas reinhardtii (C9) and a paromomycin insertional LIPID REMODELLING REGULATOR 1 (LRL1) mutant line (lrl1-1) grown in phosphorus (P)-replete and P-deplete conditions for 2, 4 and 8 days.
Authors Nur Akmalia Hidayati, Yui Yamada-Oshima, Masako Iwai, Takashi Yamano, Masataka Kajikawa, Nozomu Sakurai, Kunihiro Suda, Kanami Sesoko, Koichi Hori, Takeshi Obayashi, Mie Shimojima, Hideya Fukuzawa, Hiroyuki Ohta
Reference LIPID REMODELLING REGULATOR 1 (LRL1) is differently involved in the phosphorus-depletion response from PSR1 in Chlamydomonas reinhardtii. The Plant Journal (in press)

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Sample Information

ID S2819
Title TAP-P, day8, lrl1-1, mix
Organism - Scientific Name Chlamydomonas reinhardtii
Organism - ID NCBI Taxonomy:3055
Compound - ID
Compound - Source
Preparation Mutant strain lrl1-1 was cultured in TAP-P medium for 8 days.
Sample Preparation Details ID SS1

Sample Preparation Details Information

Title C. reinhardtii strains and culture conditions
Description The C. reinhardtii strain C9 (CC-408, wild type mt-) and lrl1-1 were obtained from the Kyoto University C. reinhardtii mutant library. Screening of lrl1-1 from the Chlamydomonas mutant library was performed according to Gonzalez-Ballestar et al. 2014 . Briefly, several primers spanning the upstream region to the 3' end region of Cre03.g197100 were designed. The superpool library was used as the PCR template, and any amplified product from a primer pair that included a specific region of Cre03.g197100 and AphVIII were sequenced, and its positional tag was identified. Further DNA sequencing indicated that AphVIII-tag insertion was in the upstream region of Cre03.g197100, 115 bp upstream of the 5' untranslated region (UTR), which is predicted to be the promoter region. Liquid cultures were grown in an Erlenmeyer flask mixotrophically in Tris-acetate-phosphate (TAP) medium. For all cultures, cells were cultivated under continuous illumination at 20-40 umol m^-2 s^-1 and 100 rpm shaking at 25C. To induce P-starvation, mid-log phase cells (3-5 x10^6 cells/ml) were centrifuged at 2000 rpm for 5 min and washed twice in a P-free (TAP-P) medium. Potassium phosphate was replaced by 1.5 mM KCl in TAP-P medium. Cells were initially adjusted to 1x10^5 cells/ml on culture day 0. Cultured cells were collected by centrifugation, immediately frozen in liquid nitrogen, and stored at -80C until use.

Analytical Method Information

Title Method 5: ESI Positive, Data dependent MS2/MS3 scan (FT, IT, IT)
Method Details ID MS5
Sample Amount ~5 mg wet weight/ 20 uL injection

Analytical Method Details Information

Title LC-FT-MS, ESI, Positive (method 5)
Instrument Agilent1100 HPLC (Agilent), LTQ-FT (Thermo Fisher Scientific)
Instrument Type LC-FTICR-MS
Ionization ESI
Ion Mode Positive
Description - 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 5]

The method for MSn analysis named "Method 5" 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 applied as follows: repeat count, three; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; and exclusion duration, 20 s.

Comment_of_details [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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