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MB Sample ID: SA194014
| Local Sample ID: | Mature_RPLC_Pos_3 |
| Subject ID: | SU002142 |
| Subject Type: | Plant |
| Subject Species: | Arabidopsis thaliana |
| Taxonomy ID: | 3702 |
| Genotype Strain: | Col-0 |
| Age Or Age Range: | Mature pollen, Pollen hydration Stage (45 min), Pollen germination stage (4 hours) |
| Species Group: | Pollen |
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Sample Preparation:
| Sampleprep ID: | SP002148 |
| Sampleprep Summary: | Total metabolites from pollen were extracted using a phase separation method previously described (Kambhampati et al., 2021) with slight modifications. Briefly, 20-30 mg pollen tissue, collected in Eppendorf tubes, was extracted using 700 µL of chilled 7:3 (v/v) methanol: chloroform spiked with 50 µM each of 1.4-piperazinediethanesulfonic acid (PEPES), ribitol, and norvaline as internal standards. After two metal beads were also added to the samples, they were homogenized using a Tissue-Lyser for 5 min at 30 Hz. The samples were incubated on a rotary shaker at 4°C for 2 hours after which 300 µL ddH2O was added. The samples were then centrifuged at 14,000 rpm for 10 min to achieve phase separation and the upper aqueous phase, as well as the lower organic phase, were collected separately. The aqueous phases containing polar and nonpolar metabolites were split into two equal parts and dried using a speed vacuum centrifuge (Labconco®, Kansas City, USA). The two dried parts were re-suspended in 50 µL 80% (v/v) methanol, and 30 % (v/v) methanol for metabolomics analyses using hydrophilic interaction (HILIC) and reverse phase chromatography (RPLC), respectively. The organic phase was also dried using a speed vacuum centrifuge and re-suspended in 50 µL of 49:49:2 (v/v/v) mixture of acetonitrile: methanol: chloroform. All samples were filtered using a 0.8 µM PES membrane centrifuge filter (Sartorius, Goettingen, Germany) and transferred to a glass vial for injection into an LC-MS/MS system. |
| Sampleprep Protocol Filename: | Total_Metabolite_Extraction.docx |
| Processing Storage Conditions: | -80℃ |
