SE155:/S1/M1/D1

Sample Set Information

ID	TSE1311
Title	Effects of freeze-drying of samples on metabolite levels in metabolome analyses.
Description	Freeze-drying (FD) is a useful technique for removing water from biological tissues, such as food samples. Cellular components freeze at once, and the ice sublimates under conditions of high vacuum and low temperatures. Because biological activity is restricted during FD, the degradation of cellular metabolites is often believed to be limited. However, the cellular structure is damaged by several factors, such as the increase in cell volume during freezing, and this has serious effects on the levels of some cellular metabolites. We studied these effects of FD on metabolite levels when using it as a sample preparation step in metabolome analysis. We observed significant decreases in the levels of some metabolites, such as succinate and choline, in Arabidopsis and pear, respectively. We also found that the effects of FD on certain metabolite levels differed between Arabidopsis plants and pear fruits. These results suggest that it is necessary to confirm the metabolite recovery in each sample species when FD is used for sample preparation.
Authors	Oikawa A, Otsuka T, Jikumaru Y, Yamaguchi S, Matsuda F, Nakabayashi R, Takashina T, Isuzugawa K, Saito K, Shiratake K.
Reference	J Sep Sci. 2011 Dec;34(24):3561-7. doi: 10.1002/jssc.201100466. Epub 2011 Aug 24.
Comment

PubMed

Sample Information

ID	S1
Title	Arabidopsis thaliana
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	Arabidopsis (Arabidopsis thaliana ecotype Col-0) plants were cultured on Murashige and Skoog medium containing 1% w/v sucrose (Wako, Osaka, Japan) in a growth chamber at 221C under continuous light for 2 wk. All plant parts used in the experiments were described below.
Sample Preparation Details ID	SS1
Comment

Sample Preparation Details Information

ID	SS1
Title	Freeze-drying
Description	Arabidopsis and pear were freeze-dried using a freeze-dryer (FDU-2200; EYELA, Tokyo, Japan). During FD, the pressure was reduced to 2.0 Pa. The temperature in the cold trap was -80°C, and the heating plate reached 381C. The yield rate of dryness (YRD) was alculated with the following equation: YRD(%) = ( FW - DW ) / FW x 100 where FW and DW represent the fresh weight and dry weight, i.e. sample weights before and after FD, respectively.
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Analytical Method Information

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

Analytical Method Details Information

ID	MS1
Title	Metabolome analysis using CE-MS
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 For frozen (F) samples, Arabidopsis plants and pear fruits were frozen in liquid nitrogen immediately after harvesting. They were homogenized using a mixer mill MM310 (Retsch, Haan, Germany) at a frequency of 27 Hz for 3 min at 4°C. Freeze-dried samples, approx. 50 mg of Arabidopsis plants and approx. 100 mg of pear fruits were homogenized to powder using the mixer mill mentioned above. We used three replicates for each sample. Twenty volumes (Arabidopsis) or five volumes (pear) of methanol were added to both F and FD samples (20 mL/mg or 5 mL/mg fresh weight), as well as 8 mM of internal standards (200 mM methionine sulfone for cation analyses, or 200 mM camphor 10-sulfonic acid for anion analyses). The internal standards were used for calibrating the peak area after CE-MS analyses. The samples were homogenized once more at 27 Hz, for 1 min. The sample solutions were then centrifuged at 20 400 x g for 3 min at 4°C. Subsequently, 500 mL chloroform and 200 mL water were added to a 500 mL sample of the supernatant. This mixture was vortexed for 3 min, and centrifuged at 20 400 x g for 3 min at 4°C. The upper layer was evaporated in a centrifugal concentrator for 30 min at 45°C, and then separated into two layers. The upper layer (100–200 mL) was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9100 x g for 150 min. The filtrate was dried in a centrifugal concentrator for 120 min. The residue was dissolved in 20 mL water and used for CE-MS analyses. Metabolome analysis using CE-MS Details of the conditions for CE-MS were as described in a previous report.
Comment_of_details

PubMed

Data Analysis Information

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

Data Analysis Details Information

ID	DS1
Title	Data processing and analysis (CE-MS)
Description	Raw metabolome data were analyzed using the proprietary software MasterHands. 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 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. Peaks were picked and aligned using this software. 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 above. Data analysis For normalization, the area of the peak from the CE-MS analysis was divided by the area of the internal standard peak. The relative peak areas were used for comparing the metabolite levels between F and FD samples. For principal component analysis (PCA), the relative areas of ionic metabolites and the amounts of plant hormones were standardized by subtracting the mean amount of metabolite (from a sequence of experiments) from the calculated amount for each sample. The resulting value was divided by the standard deviation of each metabolite (z-score). PCA was processed with the software DrDMASS1 (http://kanaya.naist.jp/DrDMASSplus/).
Comment_of_details