SE151:/MS1

Sample Set Information

ID	TSE1306
Title	Plant lipidomics based on hydrophilic interaction chromatography coupled to ion trap time-of-flight mass spectrometry.
Description	Plants synthesize a wide range of hydrophobic compounds, generally known as lipids. Here, we report an application of liquid chromatography ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS) for plant lipidomics. Using hydrophilic interaction chromatography (HILIC) for class separation, typical membrane lipids including glycerolipids, steryl glucosides and glucosylceramides, and hydrophobic plant secondary metabolites such as saponins were analyzed simultaneously. By this method, we annotated approximately 100 molecules from Arabidopsis thaliana. To demonstrate the application of this method to biological study, we analyzed Arabidopsis mutant trigalactosyldiacylglycerol3 (tgd3), which has a complex metabolic phenotype including the accumulation of unusual forms of galactolipids. Lipid profiling by LC-MS revealed that tgd3 accumulated an unusual form of digalactosyldiacylglycerol, annotated as Gal(β1 → 6)βGalDG. The compositional difference between normal and unusual forms of digalactosyldiacylglycerol was detected by this method. In addition, we analyzed well-known Arabidopsis mutants ats1-1, fad6-1, and fad7-2, which are also disrupted in lipid metabolic genes. Untargeted lipidome analysis coupled with multivariate analysis clearly discriminated the mutants and their distinctive metabolites. These results indicated that HILIC-MS is an efficient method for plant lipidomics.
Authors	Okazaki Y, Kamide Y, Hirai MY, Saito K.
Reference	Metabolomics. 2013 Mar;9(Suppl 1):121-131. Epub 2011 May 31.
Comment

PubMed

Analytical Method Details Information

ID	MS1
Title	LCMS-IT-TOF
Instrument	LC, Shimadzu LC-20AD system; MS, Shimadzu LCMS-IT-TOF
Instrument Type
Ionization	ESI
Ion Mode	negative
Description	Sample preparation Total lipids were extracted according to the method of Bligh and Dyer (Bligh and Dyer 1959) with slight modifications. The frozen plant material was milled on a Mixer Mill MM300 (Retsch, Haan, Germany) for 2 min at 15 Hz with a zirconium bead. The milling process was carried out under cryogenic conditions (the grinding jars were cooled with liquid nitrogen before each milling process to ensure a low milling temperature to keep the plants frozen). The frozen powdered plant material was kept in liquid nitrogen and a 20-fold volume of extraction solvent (CHCl3–MeOH–H2O = 50:100:31.45, v/v/v) was added with 1 μM 10:0/10:0 PC as the internal standard, followed by vigorous stirring on a vortex mixer. After dark incubation for 5 min at room temperature, the homogenate was centrifuged at 3,000×g for 3 min at room temperature. Two hundred microliters of supernatant was mixed with CHCl3 and H2O (52.6 μl each), stirred on a vortex mixer, and incubated on ice for 15 min in darkness. The mixture was centrifuged at 1,000×g for 3 min, and 85 μl of the lower layer was collected. The organic phase was dried by a centrifugal concentrator at room temperature and the residue was dissolved in 81 μl of CHCl3 for LC-MS analysis. metadata (from Supplementary material 1) Chromatography instrument description: Shimadzu, LC-20AD HPLC system, operated by LCMSsolution v.3.6. Auto-injector: Shimadzu, SIL-20AC, LCMSsolution v.3.6., 1microL injection, 1 wash per injection (0.2 mL MeCN) Separation column and pre/guard column: Shimadzu, Atlantis HILIC Silica (pore size: 3 microm) 2.1 by 100 mm Separation parameter: A two-solvent system was used to generate the mobile phase: solvent A, methanol-water (95:5, v/v) containing 0.2% ammonium formate (pH 5.9); solvent B, acetonitrile-methanol-water (95:2:3, v/v/v) containing 0.2% ammonium formate (pH 5.9). The pH of both solvents A and B was adjusted by adding 30% NH4OH to the mixtures of solvents containing 0.2% (v/v) formic acid.; elution program: 100% solvent B (0-3.33 min), 100~65% solvent B (3.33-10 min), 65~30% (10-11.33 min), 30% solvent B (11.33-14.66 min), 100% solvent B (14.66-40 min). The flow rate was held at 0.2 mL/min, and it was then increased to 0.4 mL/min at 14.66 min after the beginning of the gradient, maintained for 13.33 min, and then decreased to 0.2 mL/min. Total elution time was 40 min. Instrument description: Shimadzu, LCMS-IT-TOF, operated by LCMSsolution v.3.6. Sample introduction and delivery: From LC Ionization source: ESI negative. interface voltage, 4.5 V; curved desolvation line temperature, 200C; heat block temperature, 200C; ion accumulation time, 10 msec; nebulizer gas, N2 (15 L/min). Mass analyzer description and acquisition mode: ion trap-time-of-flight operated at scan mode Data acquisition parameters: detection mode: scan (m/z 150-1600; event time: 0.2 s; profile). Data file format: Original data file (.lcd)
Comment_of_details