SE154:/S1/M1/D1

Sample Set Information

ID	TSE1310
Title	Metabolomics of a single vacuole reveals metabolic dynamism in an alga Chara australis.
Description	Metabolomics is the most reliable analytical method for understanding metabolic diversity in single organelles derived from single cells. Although metabolites such as phosphate compounds are believed to be localized in different organelles in a highly specific manner, the process of metabolite compartmentalization in the cell is not thoroughly understood. The analysis of metabolites in single organelles has consequently presented a significant challenge. In this study, we used a metabolomic method to elucidate the localization and dynamics of 125 known metabolites isolated from the vacuole and cytoplasm of a single cell of the alga Chara australis. The amount of metabolites in the vacuole and the cytoplasm fluctuated asynchronously under various stress conditions, suggesting that metabolites are spatially regulated within the cell. Metabolite transport across the vacuolar membrane can be directly detected using the microinjection technique, which may reveal a previously unknown function of the vacuole.
Authors	Oikawa A, Matsuda F, Kikuyama M, Mimura T, Saito K.
Reference	Plant Physiol. 2011 Oct;157(2):544-51. doi: 10.1104/pp.111.183772. Epub 2011 Aug 16.
Comment

PubMed

Sample Information

ID	S1
Title	Chara australis
Organism - Scientific Name	Chara australis
Organism - ID	NCBI taxonomy:31298
Compound - ID
Compound - Source
Preparation	Species: Chara australis Organ: Internodal cells Organ specification: Vacuole and cytoplasm (interior of the cell excluding the vacuole) Amount: A single vacuole (approximately 25–50 μL) and the cytoplasm of a single cell. Growth conditions: C. australis was cultured in a plastic bucket, containing tap water and leaf mold extract, under fluorescent lamps with a 14-h light/10-h dark cycle in an air-conditioned room (25°C) at Tsuruoka Metabolome Campus, RIKEN laboratory. Experimental conditions: For habituation of C. australis, samples were transferred to another plastic bucket containing artificial pond water (APW) consisting of 0.1 mM each of KCl, NaCl, CaCl2, and 1 mM of NaHCO3 under a 12-h light/12-h dark cycle in chambers maintained at 25°C for at least 1 week. Sampling: A sample consisted of a single cell. Five different cells were sampled at each time point. Therefore, 5 replications were performed for each sample. Different light conditions: Five internodal cells were isolated at 7 time points (0, 3, 6, 12, 15, 18, 24 h after lighting). Continuous light/dark conditions: Five internodal cells were isolated at 0, 24, and 48 h after changing light conditions to continuous light or dark. CO2 deficiency: One bucket was filled with APW (control and +CO2); a second bucket, with APW without NaHCO3 (-CO2). After 36 h, 5 internodal cells were isolated. Heat stress: One bucket was stored at 25°C; a second bucket, at 37°C. Five internodal cells were isolated at 0, 24, and 48 h after application of heat stress. Sampling date: 1–2 September 2009 (different light conditions), 2–4 September 2009 (continuous light/dark conditions), 22 June 2009 (CO2 deficiency), and 15–17 February 2010 (heat stress). Isolation of vacuole and cytoplasm: A single internodal cell was isolated from neighboring cells and exposed to air on a paraffin wax board. After loss of turgor pressure, both ends of the cell were cut. By inclining the board, the vacuolar solution was collected and used as the vacuolar fraction. After removing the vacuolar solution completely, the remaining biomaterial of the cell was used as the cytoplasmic fraction. Metabolism quenching method: Samples were immersed in liquid nitrogen within 30 s of sampling. After quenching, samples were stored in liquid nitrogen and extracted within 30 min.
Sample Preparation Details ID
Comment

Analytical Method Information

ID	M1
Title	CE-TOFMS
Method Details ID	MS1
Sample Amount
Comment

Analytical Method Details Information

ID	MS1
Title	CE-TOFMS
Instrument	CE:Agilent CE capillary electrophoresis system (Agilent Technologies) TOF-MS:Agilent G3250AA LC/MSD TOF system (Agilent Technologies) CE-MS:Agilent G1603A
Instrument Type
Ionization	ESI
Ion Mode	positive and negative
Description	Sample preparation Sample processing and extraction: Vacuole solution (200μL) was collected and diluted with water for the liquid-liquid distribution. Frozen cytoplasm was homogenized with zirconia beads using a Mixer Mill (Retsch, Haan, http://www.retsch.com) at 27 Hz for 2 min. The solution was suspended in 200μL of water and used as the cytoplasmic solution. Liquid-liquid distribution: To exclude proteins and hydrophobic compounds such as lipids, which can have a negative effect on the reproducibility of electrophoresis, extracts were separated by adding methanol and chloroform to samples. Chloroform (200 μL) and methanol (500μL), including 8μM of internal standards (methionine sulfone for cation analysis and camphor 10-sulfonic acid for anion analysis), were used for compensation of the peak area after CE-MS analysis. The solution was added to 200μL samples, and the mixture was homogenized at 27 Hz for 1 min. The sample solution was then centrifuged at 20,400 x g for 3 min at 4°C. For further purification of the solution and reduction in volume, the upper layer was evaporated for 30 min at 45°C with a centrifugal concentrator and then separated into 2 layers. Ultracentrifugation: Because high molecular weight compounds, such as oligo-sugars, may reduce CE performance, the upper layer was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9,100 x g for 120 min. Sample concentration: The filtrate was dried for 120 min with a centrifugal concentrator. The residue was dissolved in 20μL water containing 200μM of internal standards (3-aminopyrrolidine for cation analysis and trimesic acid for anion analysis) to compensate for migration time in the peak annotation step. This solution was used for CE-MS analyses. CE-TOFMS conditions CE-TOF MS instruments: All CE-TOFMS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, www.home.agilent.com), an Agilent G3250AA LC/MSD TOF System, an Agilent 1100 series binary HPLC pump, a G1603A Agilent CE-MS adapter, and a G1607A Agilent CE-ESI-MS sprayer kit. For systems control and data acquisition, we used the G2201AA Agilent ChemStation software for CE data and Analyst QS software for Agilent TOFMS. Separation column and electrolytes: Separations were carried out in a fused silica capillary (50μm i.d. x 100 cm total length) filled with 1 M formic acid as the electrolyte for cation analysis and 20 mM ammonium formate (pH 10.0) as the electrolyte for anion analyses. The capillary temperature was maintained at 20°C. Sample injection: The samples were injected with a pressure injection of 50 mbar for 15 s (15 nL). The sample tray was cooled below 4°C. Separation parameters: Prior to each run, the capillary was flushed with the electrolyte for 5 min. The applied voltage for separation was set at 30 kV. Methanol-water (50% v/v) containing 0.5μM reserpine was delivered as the sheath liquid at 10 μL/min. Ionization: ESI-TOFMS was conducted in the positive ion mode for cation analyses and in the negative ion mode for anion analyses, and the capillary voltage was set at 4 kV. Dry gas condition: A flow rate of heated dry nitrogen gas (heater temperature, 300°C) was maintained at 10 psig. Voltage settings in TOFMS: The fragmentor, skimmer, and Oct RFV voltage were set at 110 V, 50 V, and 160 V for cation analysis, and at 120 V, 60 V, and 220 V for anion analysis, respectively. Mass calibration: Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. For lock mass correction and subsequent exact mass measurement, the methanol dimer ion ([2M+H]+, m/z = 65.0597) and reserpine ([M+H]+, m/z = 609.2806) were used for cation analysis, and the formic acid dimer ion ([2M-H]-, m/z = 91.0037) and reserpine ([M-H]-, m/z = 607.2661) were used for anion analysis. Mass data acquirement: Exact mass data were acquired at a rate of 1.5 cycles/s over a 50–1000 m/z range. LC-MS conditions: LC-MS analysis was performed according to Matsuda et al., 2009. Quality control: We analyzed the standard compound mixture at the beginning and end of each CE-MS sequence analysis (maximum 36 samples). To confirm reproducible sensitivity, the detected peak area was compared with that of a standard compound mixture. The standard compound mixture was composed of major detectable metabolites, such as amino acids and organic acids, and a new mixture was prepared at least every 6 months. For all of the analyses in this study, no differences were observed in the sensitivity of the standard compound mixture.
Comment_of_details

Data Analysis Information

ID	D1
Title	Data Processing and Statistics
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS1
Title	Data Processing and Statistics
Description	Data processing Peak picking and alignment: Raw CE-MS data were analyzed with the proprietary software MasterHands (Sugimoto et al., 2010b; Sugimoto et al., 2010c). In brief, peaks were detected from sliced electropherograms (0.02 m/z width), and the accurate m/z value for each peak was calculated by Gaussian curve fitting. Migration times of the detected peaks were normalized by a dynamic time-warping method; numerical parameters were optimized using the simplex method and matching peaks across multiple data sets by dynamic programming (Sugimoto et al., 2010a). Peaks were picked and aligned using this software. Peak annotation: Metabolites in the standard compounds were assigned to the remaining features by matching their m/z values and normalized migration times using the software described in Peak picking and alignment. Quantification: For normalization, the area of the detected peak was divided by the area of the internal standard peak. Based on the of calibration curves for standard compounds, metabolite amounts were quantified. In C. australis internodal cells, the vacuolar solution accounts for approximately 95% of the whole cell volume (Sakano and Tazawa, 1984). Therefore, in the present study, the cytoplasmic fraction was estimated to occupy about 5% of the total cell volume; this value was used to calculate metabolite levels in the cytoplasmic fractions. Statistics Data transformation: Calculated amounts were standardized by subtracting the mean amount for each metabolite in a sequence experiment from the calculated amount of each sample and then dividing this value by the standard deviation of each metabolite (z-score). Levels of undetected metabolites were set at 0, and then standardized. Statistics: Standardized amounts were submitted for hierarchical clustering analysis (HCA) and processed with PermutMatrix software (http://www.atgc-montpellier.fr/permutmatrix) according to Euclidean distance and Ward’s method (Caraux and Pinloche, 2005; Ward, 1963).
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