SE142:/MS2

Sample Set Information

ID	TSE1243
Title	Physiological roles of the beta-substituted alanine synthase gene family in Arabidopsis.
Description	The beta-substituted alanine (Ala) synthase (Bsas) family in the large superfamily of pyridoxal 5'-phosphate-dependent enzymes comprises cysteine (Cys) synthase (CSase) [O-acetyl-serine (thiol) lyase] and beta-cyano-Ala synthase (CASase) in plants. Nine genomic sequences encode putative Bsas proteins in Arabidopsis thaliana. The physiological roles of these Bsas isoforms in vivo were investigated by the characterization of T-DNA insertion mutants. Analyses of gene expression, activities of CSase and CASase, and levels of Cys and glutathione in the bsas mutants indicated that cytosolic Bsas1;1, plastidic Bsas2;1, and mitochondrial Bsas2;2 play major roles in Cys biosynthesis. Cytosolic Bsas1;1 has the most dominant contribution both in leaf and root, and mitochondrial Bsas2;2 plays a significant role in root. Mitochondrial Bsas3;1 is a genuine CASase. Nontargeted metabolome analyses of knockout mutants were carried out by a combination of gas chromatography time-of-flight mass spectrometry and capillary electrophoresis time-of-flight mass spectrometry. The level of gamma-glutamyl-beta-cyano-Ala decreased in the mutant bsas3;1, indicating the crucial role of Bsas3;1 in beta-cyano-Ala metabolism in vivo.
Authors	Watanabe M, Kusano M, Oikawa A, Fukushima A, Noji M, Saito K.
Reference	Plant Physiol. 2008 Jan;146(1):310-20. Epub 2007 Nov 16.
Comment

PubMed

Analytical Method Details Information

ID	MS2
Title	CE-TOF/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
Description	Sixteen plants were planted on a single plate separated into fourths to minimize the differences in growth conditions. Four wild-type plants were planted on one-fourth, and four bsas mutant plants were planted on each of the remaining fourths. Five plates were replicated for each bsas mutant. Each sample was extracted with a concentration of 25 mg fresh weight of tissues per microliter of the extraction medium (methanol:chloroform:water [3:1:1; v/v/v]) by using a Retsh mixer mill MM 310 at a frequency of 30 Hz−1 for 3 min at 4°C. After centrifugation for 5 min at 15,100g, 400 μL of the supernatant of each plate were put together in accordance with each section of fourths. Four hundred microliters of the 2-mL supernatant were used for GC-TOF/MS analysis, and another 400 μL were used for CE-TOF/MS analysis. Analysis of metabolites by CE-TOF/MS was performed using an Agilent CE capillary electrophoresis system (Agilent Technologies), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies), an Agilent 1100 series binary HPLC pump, and the Agilent G1603A CE-MS adapter and Agilent G1607A CE-ESI-MS sprayer kit. Agilent G2201AA ChemStation software for CE and Analyst QS software for TOF/MS were used. For cationic compounds, separations were carried out using a fused silica capillary (50 μm i.d. × 100 cm total length) filled with 1 m formic acid as the electrolyte. The sample solutions were injected at 50 mbar for 15 s (15 nL). Prior to each run, the capillary was flushed with electrolyte for 5 min. The applied voltage was set at 30 kV. The capillary temperature was maintained at 20°C, and the sample tray was cooled below 4°C. Fifty percent (v/v) methanol-water containing 0.5 μm reserpine was delivered as the sheath liquid at 10 μL min−1. ESI-TOF/MS was conducted in the positive ion mode and the capillary voltage was set at 4 kV. A flow rate of heated dry nitrogen gas (heater temperature 300°C) was maintained at 10 psig. In TOF/MS, the fragmentor, skimmer, and Oct RFV voltage were set at 110, 50, and 160 V, respectively. In acquiring a fragment ion mass spectrum, the fragmentor voltage was increased to 210 V. Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The methanol dimer ion ([2M + H]+; m/z 65.0597) and reserpine ([M + H]+; m/z 609.2806) provided the lock mass for exact mass measurements. Exact mass data were acquired at a rate of 1.5 cycles s−1 over a 50 to 1,000 m/z range. Analysis of anionic compounds and nucleotides was carried out as described previously (Soga et al., 2002a, 2002b).
Comment_of_details