Computational protocol: Galacturonosyltransferase 4 silencing alters pectin composition and carbon partitioning in tomato

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Protocol publication

[…] For metabolic profiling, frozen leaves (~100mg) were extracted in 1.4ml of methanol, as described by ) with the modifications proposed by ); 60 μl of internal standard (0.2mg ml–1 of ribitol) was added for quantification. In the case of ripe fruits (~250mg), the reagent volumes were adjusted using 2ml of methanol and 120 μl of ribitol, as described by ). In both cases, the mixture was extracted for 15min at 70 °C, mixed vigorously with 1 volume of water, centrifuged at 2200g, and subsequently vacuum dried. The residue was redissolved and derivatized for 120min at 37 °C (in 60 μl of 30mg ml–1 methoxyamine hydrochloride in pyridine), followed by a 30min treatment at 37 °C with 120 μl of N-methyl-N-[trimethylsilyl]trifluoroacetamide. Sample volumes of 1 μl were then injected in splitless and split modes, using a hot-needle technique. The GC-MS system was composed of an AS 2000 autosampler, a GC 6890N gas chromatographer (Agilent Technologies, USA), and a Pegasus III time-of-flight mass spectrometer (LECO Instruments, USA). Chromatography was performed with an MDN-35 capillary column of 30 m length, with a 0.32mm inner diameter and 0.25 μm film thickness (Macherey-Nagel). Samples were injected at 230 ºC in splitless mode with helium carrier gas flow set at 2ml min–1. The flow rate was kept constant with electronic pressure control enabled. The temperature program was isothermal for 2min at 85 ºC, followed by a 15 ºC min–1 ramp to 300 ºC. The transfer line temperature was set at 250 ºC. For detection, the temperature of the ion source was set at 250 ºC (match transfer line condition). Mass spectra were recorded at 20 scans s–1 with a scanning range of 70–660 m/z. The remaining monitored chromatography time proceeded with the filament turned off. The filament bias current was –70V and the detector voltage was 1525V. Both chromatograms and mass spectra were evaluated using ChromaTOF chromatography processing and mass spectral deconvolution software, version 3.00 (LECO Instruments, USA). Identification and quantification were performed with TagFinder 4.0 software and the mass spectra were cross-referenced with those in the Golm Metabolome Database (; ). Three to six biological replicates were used for this analysis. […]

Pipeline specifications

Software tools ChromaTOF, TagFinder
Databases GMD
Applications MS-based targeted metabolomics, Metabolic profiles analysis
Organisms Solanum lycopersicum, Arabidopsis thaliana
Chemicals Carbon