SE155:/S2/M2

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	S2
Title	Pear fruits
Organism - Scientific Name
Organism - ID
Compound - ID
Compound - Source
Preparation
Sample Preparation Details ID	SS1
Comment	Species is not written in the paper.

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.
Comment_of_details

Analytical Method Information

ID	M2
Title	LC-MS/MS
Method Details ID	MS2
Sample Amount
Comment

Analytical Method Details Information

ID	MS2
Title	Quantification of plant hormone levels (LC-MS/MS)
Instrument	LC: Agilent 1200 series, MS: Agilent 6410 Triple Quad LC-MS
Instrument Type
Ionization	ESI
Ion Mode
Description	Plant hormone levels were quantified as described in a previous report, with slight modifications. We used a 6410 Triple Quad LC-MS (Agilent Technologies, Palo Alto, CA), which includes an Agilent 1200 series rapid resolution LC system fitted with a ZORBAX Eclipse XDB-C18 column (1.8 mm, 2.1 x 50 mm). The F and FD samples were extracted with 5 volumes of 80% v/v acetonitrile containing 1% v/v acetic acid and internal standards. Extracts were centrifuged at 14 000 x g for 10 min at 4°C, and the supernatant was collected. This procedure was repeated once, and acetonitrile was removed in a SpeedVac (Thermo Fisher Scientific, Waltham, MA). Acidic water extracts were loaded onto an Oasis HLB extraction cartridge (30 mg, 1 mL; Waters, Milford, MA), and washed with 1mL of water containing 1% acetic acid to remove highly polar impurities. Phytohormones were eluted with 2mL of 80% acetonitrile containing 1% acetic acid, and the acetonitrile in this eluent was removed in a SpeedVac. Acidic water extracts were loaded onto an Oasis MCX extraction cartridge (30 mg, 1 mL; Waters, Milford, MA). After washing with 1mL water containing 1% acetic acid, acidic and neutral compounds were eluted with 2mL of acetonitrile (acetonitrile fraction). After washing with water containing 5% v/v ammonia, basic compounds were eluted with 2mL of 60% v/v acetonitrile containing 5% v/v ammonia. After drying these basic fractions, 20 mL water containing 1% acetic acid was added, and the basic hormones (trans-zeatin and isopentenyladenine) in these fractions were analyzed by LC-MS/MS. Subfractions (10%) of the acetonitrile fractions were collected and prepared as basic fractions to analyze for salicylic acid (SA). The remaining 90% of the acetonitrile fractions was concentrated in the SpeedVac to remove acetonitrile and loaded onto Oasis WAX extraction cartridges (30 mg, 1 mL; Waters, Milford, MA). After washing with 1mL of water containing 1% acetic acid, neutral compounds were eluted with 2mL of acetonitrile, and acidic compounds were eluted with 2mL of 80% acetonitrile containing 1% acetic acid. After drying these acidic fractions, 20 mL water containing 1% acetic acid was added and the acidic hormones (indole acetic acid, abscisic acid (ABA), jasmonic acid, jasmonic acid isoleucine conjugate, gibberellic acid 1, and gibberellic acid 4) in these fractions were analyzed by LC-MS/MS.
Comment_of_details

PubMed