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MB Sample ID: SA193948
| Local Sample ID: | Germinated_HILIC_Neg_2 |
| 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 |
Select appropriate tab below to view additional metadata details:
Subject:
| 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 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| Germinated_HILIC_Neg_2 | SA193948 | FL023749 | Germination | Stage |
| Germinated_HILIC_Neg_2 | SA193948 | FL023749 | HILIC | Chromatography |
| Germinated_HILIC_Neg_2 | SA193948 | FL023749 | Negative | Polarity |
Collection:
| Collection ID: | CO002135 |
| Collection Summary: | The Arabidopsis Col-0 plants were grown under controlled temperature (22°C) with a 16-h light (100-150 µmol m-2 s-1)/ 8-h dark photoperiod. Mature pollen grains from the fully opened flowers were collected from more than 1000 plants using a vacuum cleaner method (Johnson-Brousseau and McCormick, 2004) at around 5 hours into the light period. For mature pollen samples, collected pollen grains were resuspended in 2 ml of ice-cold Pollen Isolation Buffer (PIB, composed of 100 mM NaPO4, pH 7.5, 1 mM EDTA, and 0.1% (v/v) Triton X-100) right after collection followed by centrifuging at 15,000 g for 1 min (4°C). For hydrated pollen and germinated pollen samples, mature pollen grains were germinated in vitro according to a previously described pollen transcriptome study (Wang et al., 2008). In brief, pollen pellets were washed with 1 ml of liquid Pollen Germination Medium (PGM, composed of 15% (w/v) sucrose, 1.5 mM boric acid, 0.8 mM MgSO4, 1 mM KCl, 5 mM MES, 0.05% (w/v) lactalbumin hydrolysate, 10 µM myo-inositol, 5 mM CaCl2) before they were resuspended in 30 µl of liquid PGM and subsequently cultured in Petri dishes (35 mm in diameter). A 70 µm mesh was used to cover the pollen droplet to create a thin layer for optimal germination for each Petri dish. The Petri dishes were covered and placed in the dark for 45 min or 4 h and collected as hydrated pollen or germinated pollen, respectively. All pollen samples were washed by 1 ml ice-cold ddH2O three times before being stored in a -80 °C freezer. |
| Collection Protocol Filename: | PlantGrowth_PollenCollection.docx |
| Sample Type: | Plant |
| Collection Method: | Flash frozen in Liquid N2 |
| Collection Location: | University of Illinois, Urbana-Champain |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002154 |
| Treatment Summary: | For hydrated pollen and germinated pollen samples, mature pollen grains were germinated in vitro according to a previously described pollen transcriptome study (Wang et al., 2008). In brief, pollen pellets were washed with 1 ml of liquid Pollen Germination Medium (PGM, composed of 15% (w/v) sucrose, 1.5 mM boric acid, 0.8 mM MgSO4, 1 mM KCl, 5 mM MES, 0.05% (w/v) lactalbumin hydrolysate, 10 µM myo-inositol, 5 mM CaCl2) before they were resuspended in 30 µl of liquid PGM and subsequently cultured in Petri dishes (35 mm in diameter). A 70 µm mesh was used to cover the pollen droplet to create a thin layer for optimal germination for each Petri dish. The Petri dishes were covered and placed in the dark for 45 min or 4 h and collected as hydrated pollen or germinated pollen, respectively. |
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℃ |
Combined analysis:
| Analysis ID | AN003354 | AN003355 | AN003356 | AN003357 | AN003358 | AN003359 |
|---|---|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase | Reversed phase | Reversed phase | Reversed phase |
| Chromatography system | Eksigent Ekspert microLC 200 | Eksigent Ekspert microLC 200 | Eksigent Ekspert microLC 200 | Eksigent Ekspert microLC 200 | Eksigent Ekspert microLC 200 | Eksigent Ekspert microLC 200 |
| Column | SeQuant Zic-HILIC (150 x 0.5mm,5um) | SeQuant Zic-HILIC (150 x 0.5mm,5um) | Higgins Analytica Targa C18 (150 x 0.3mm,3.5um) | Higgins Analytica Targa C18 (150 x 0.3mm,3.5um) | Machery-Nagel Nucleodur C8 (150 x 0.5mm,5um) | Machery-Nagel Nucleodur C8 (150 x 0.5mm,5um) |
| MS Type | ESI | ESI | ESI | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE | POSITIVE | NEGATIVE |
| Units | Intensity | Intensity | Intensity | Intensity | Intensity | Intensity |
Chromatography:
| Chromatography ID: | CH002483 |
| Methods Filename: | Chromatography_PollentMetabolomics.docx |
| Instrument Name: | Eksigent Ekspert microLC 200 |
| Column Name: | SeQuant Zic-HILIC (150 x 0.5mm,5um) |
| Column Temperature: | 35 |
| Flow Rate: | 0.015 |
| Solvent A: | 100% water; 10 mM ammonium bicarbonate |
| Solvent B: | 95% acetonitrile/5% water; 10 mM ammonium bicarbonate |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002484 |
| Methods Filename: | Chromatography_PollentMetabolomics.docx |
| Instrument Name: | Eksigent Ekspert microLC 200 |
| Column Name: | Higgins Analytica Targa C18 (150 x 0.3mm,3.5um) |
| Column Temperature: | 35 |
| Flow Rate: | 0.015 |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002485 |
| Methods Filename: | Chromatography_PollenMetabolomics |
| Instrument Name: | Eksigent Ekspert microLC 200 |
| Column Name: | Machery-Nagel Nucleodur C8 (150 x 0.5mm,5um) |
| Column Temperature: | 35 |
| Flow Rate: | 0.04 |
| Solvent A: | 100% water; 0.1% acetic acid; 10 mM ammonium acetate |
| Solvent B: | 70% acetonitrile/30% isopropanol; 0.1% acetic acid; 10 mM ammonium acetate |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003123 |
| Analysis ID: | AN003354 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All parameters listed in the attached document |
| Ion Mode: | POSITIVE |
| Acquisition Parameters File: | MS_DataAcquisition.docx |
| MS ID: | MS003124 |
| Analysis ID: | AN003355 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All parameters listed in the attached document |
| Ion Mode: | NEGATIVE |
| Acquisition Parameters File: | MS_DataAcquisition.docx |
| MS ID: | MS003125 |
| Analysis ID: | AN003356 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All parameters listed in the attached document |
| Ion Mode: | POSITIVE |
| Acquisition Parameters File: | MS_DataAcquisition.docx |
| MS ID: | MS003126 |
| Analysis ID: | AN003357 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All parameters listed in the attached document |
| Ion Mode: | NEGATIVE |
| Acquisition Parameters File: | MS_DataAcquisition.docx |
| MS ID: | MS003127 |
| Analysis ID: | AN003358 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All parameters listed in the attached document |
| Ion Mode: | POSITIVE |
| Acquisition Parameters File: | MS_DataAcquisition.docx |
| MS ID: | MS003128 |
| Analysis ID: | AN003359 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | All parameters listed in the attached document |
| Ion Mode: | NEGATIVE |
| Acquisition Parameters File: | MS_DataAcquisition.docx |
