Metabolite Extraction and Metabolite Profiling Analysis

Published: 2021-09-14 21:45:10
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Metabolites were extracted according to Lisec et al., (2006) with some modifications. Briefly, about 100 mg of each sample was taken and crushed in 100% methanol and the mixture was vortexed for 15 min. Subsequently, the tubes containing the homogenate were centrifuged at 14,000 g at 4 °C for 10 min. The supernatant was taken and mixed with 750 μL chloroform and 1400 μL of Milli Q. Afterwards, the mixture was centrifuged for 15 min at 2,200 g at 4 °C.
Finally, the supernatant was taken and dried in vacuum concentrator and stored at -80 °C till use. Prior to GC-MS analysis, the dried samples were dissolved and derivatized in a two-step procedure. First, 40 μL of methoximation mixture of methoxylamine hydrochloride dissolved in pyridine (20 mg mL−1) was added and placed in a thermomixer for 2 h at 37 °C. This was followed by trimethylsilylation with 40 μL of N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) at 37 °C for 30 min and cooled to room temperature before injection. A 1 μl aliquot of the analyte was injected in splitlessmode in an ISQ 7000 single quadrupole GC-MS system (Thermo Scientific, San José, CA, USA). Helium was used as the carrier gas, the front inlet purge flow was 3 ml_min−1, and the gas flow rate through the column was 1 mL_min−1. The initial temperature was kept at 90°C for 0.25 min, then raised to 180°C at a rate of 10°C_min−1, then raised to 240°C at a rate of 5°C_min−1, and finally to 285°C at a rate of 20°C_min−1 for 11.5 min. The injection, transfer line, and ion source temperatures were 280, 270 and 220°C, respectively. The energy was -70 eV in electron impact mode. The mass spectrometry data were acquired in full-scan mode 5. Analysis of metabolomics data to elucidate the PGPR induced metabolites and pathways responsible for nutrient stress tolerance Raw GC-MS data files obtained from acquisition were aligned and processed using XCMS online package ( Metabolites were identified by comparing the mass spectra and fragmentation pattern of individual metabolites with those of the NIST-Wiley Mass Spectra Library. Then, data normalization for each sample was done by the total sum of the signal integration area.
Next, a principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed by MetaboAnalyst online software ( using normalized data. Also, Student’s t test (P

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