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MB Sample ID: SA229660
| Local Sample ID: | C18-RN5-neg |
| Subject ID: | SU002417 |
| Subject Type: | Plant |
| Subject Species: | Brassica napus |
| Taxonomy ID: | 3708 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002417 |
| Subject Type: | Plant |
| Subject Species: | Brassica napus |
| Taxonomy ID: | 3708 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| C18-RN5-neg | SA229660 | FL029030 | day 1 | Developmental stage (DPI) |
| C18-RN5-neg | SA229660 | FL029030 | Resistant | Genotype |
| C18-RN5-neg | SA229660 | FL029030 | Treated with Plasmodiophora brassicae | Treatment |
Collection:
| Collection ID: | CO002410 |
| Collection Summary: | A total of 36 samples comprised two types of genotypes [CR (5 individuals pooled in each biological replicate) and CS (5 individuals pooled in each biological replicate)], two treatments (inoculated and uninoculated) and three biological replicates generated from three independent experiments and collected at 1-, 4-, and 7–DPI were used. Immediately after collection, all root samples were flash-frozen in liquid nitrogen and stored at -80 °C. |
| Collection Protocol Filename: | Collection_protocol-canola |
| Sample Type: | Plant |
| Collection Location: | Alberta, Canada |
| Storage Conditions: | -80℃ |
| Collection Vials: | 2 mL round-bottom Eppendorf tubes |
| Storage Vials: | 2 mL round-bottom Eppendorf tubes |
Treatment:
| Treatment ID: | TR002429 |
| Treatment Summary: | A single spore-derived isolate of P. brassicae pathotype 3H (P3H) was used in this study. To assess the disease progression post-inoculation, histology, and scanning electron microscopy (SEM) was conducted on P. brassicae inoculated and uninoculated root tissues. Samples were collected by Adhikary as follows: seedlings were randomly selected, gently uprooted from the soil, the roots were washed thoroughly and collected in triplicates at after 24 hours of inoculation [hereinafter indicated as 1-day post-inoculation (DPI)], 4-, and 7-DPI. |
Sample Preparation:
| Sampleprep ID: | SP002423 |
| Sampleprep Summary: | Approximately 100 mg of fresh weight of root tissue samples (n=3) were aliquoted from finely homogenized frozen root tissues, placed into 2 mL safe-lock centrifuge tubes with two zirconium oxide beads, flash frozen in liquid nitrogen and stored at − 80 °C until further processing. To accommodate for different extraction procedures, two sample sets were prepared from the same bulk to independently analyze the content of primary metabolites (free amino acids, sugars and sugar phosphates, organic acids), and secondary metabolites (glucosinolates) using High Performance Liquid Chromatography – High-Resolution Accurate Mass – Full Scan Mass Spectrometry (HPLC-(HRAM)-MS). Samples for extraction of primary and secondary metabolites were spiked with 10ng of two labeled aromatic CKs (13C5-oT and 2H7-BAR) and extracted with ice-cold methanol (methanol-water [8:2, v/v]) following procedures of Chen et al 3 with modifications. Each filtered extract was split in half and each 500 µL transferred to a new 2 mL tube. Divided extracts were evaporated to dryness at ambient temperature in a speed vac concentrator. Sample residues designed for the analysis of primary metabolites were redissolved in 500 µl of 90% acetonitrile (acetonitrile: water, v/v) and samples for the analysis of glucosinolates were redissolved in 500 µL of 5% acetonitrile with 0.1% formic acid (acetonitrile: formic acid: water, v/v/v). Additionally, sample mixtures composed of 10uL of each sample extract were prepared separately for primary and secondary metabolite analysis and used to generate MS/MS data for compound identification. All samples were filtered using 0.2 µm PVDF spin filter with 2 mL receiver tubes (InnoSep Spin, Canadian Life Sciences, Peterborough, Canada) and transferred to insert-equipped 2 mL HPLC vials. A volume of 25 μL of each sample was injected into a Dionex UltiMate 3000 HPLC coupled to a QExactive Orbitrap mass spectrometer. |
| Sampleprep Protocol Filename: | Protocol_methods-canola |
| Extraction Method: | 80% MeOH |
| Extract Cleanup: | 0.2 µm PVDF spin filter with 2 mL receiver tubes |
| Extract Storage: | -20℃ |
| Sample Resuspension: | Primary metabolites - 90% ACN, glucosinolates - 5% ACN |
| Sample Spiking: | two labeled aromatic CKs (13C5-oT and 2H7-BAR) |
Combined analysis:
| Analysis ID | AN003801 | AN003802 | AN003803 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm,2.7um) | Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm,2.7um) | Phenomonex Kinetex C18 (50 x 2.1mm,2.6um) |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | NEGATIVE |
| Units | normalized relative level | normalized relative level | normalized relative level |
Chromatography:
| Chromatography ID: | CH002812 |
| Chromatography Summary: | Primary metabolites were resolved with an InfinityLab Poroshell 120 HILIC-Z column (2.1 × 100 mm, 2.7 µm; Agilent, Santa Clara, CA, US), using a flow rate of 0.2 mL min-1 with a mobile phase of 10 mM ammonium bicarbonate in water (A) and 10 mM ammonium bicarbonate in 95% acetonitrile (B). The following gradient was used to elute the analytes: mobile phase 100% B decreased to 90% over 2.5 min and to 50% over the next 5 min and returned to 100% over 0.5 min for 12 min of column re-equilibration. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Agilent InfinityLab Poroshell 120 HILIC-Z (100 x 2.1mm,2.7um) |
| Column Temperature: | RT |
| Flow Rate: | 0.2 ml/min |
| Injection Temperature: | 4 |
| Solvent A: | 100% water; 10 mM ammonium bicarbonate |
| Solvent B: | 95% acetonitrile/5% water; 10 mM ammonium bicarbonate |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002813 |
| Chromatography Summary: | Glucosinolates were resolved with a Kinetex C18 column (2.1 × 50 mm, 2.6 μm) using a flow rate of 0.3 mL min −1 with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The following gradient was used to elute the analytes: mobile phase B was held at 0% for 1.25 min before increasing to 50% over 2.75 min and to 100% over the next 0.5 min. Solvent B was then held at 100% for 2 min before returning to 0% over 0.5 min for 4 min of column re-equilibration. |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Phenomonex Kinetex C18 (50 x 2.1mm,2.6um) |
| Column Temperature: | RT |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4 |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003543 |
| Analysis ID: | AN003801 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The Orbitrap QExactive was operated with a heated electrospray ionization (HESI) probe in positive mode. Temperatures of the HESI-II auxiliary gas heater and capillary were 450 and 300 °C, respectively, and the spray voltage was 3.9 kV. Sheath, auxiliary, and sweep gases were operated at 30, 8, and 0 (arbitrary units), respectively, and the S-lens RF level was 60. Each sample was analyzed using a mass range of m/z 70−900, and data were acquired at 70,000 resolution, automatic gain control (AGC) target of 1e6, and maximum injection time (IT) of 100 ms. Additionally, the top 10 data-dependent acquisition experiments were performed for the mixed sample from each group (HILIC and C18) to obtain compound MS/MS spectra. Processing of all full scan and ddMS2 data was conducted using the Xcalibur 4.1 software. Metabolites were identified by accurate mass, comparison of retention times to authentic standards or by accurate mass and comparison of fragmentation patterns to MS/MS databases (METLIN, PubChem). Metabolite relative concentration was normalized based on the average recoveries of 13C5-oT and 2H7-BAR as the internal standards |
| Ion Mode: | POSITIVE |
| MS ID: | MS003544 |
| Analysis ID: | AN003802 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The Orbitrap QExactive was operated with a heated electrospray ionization (HESI) probe in negative mode. Temperatures of the HESI-II auxiliary gas heater and capillary were 450 and 300 °C, respectively, and the spray voltage was 3.9 kV. Sheath, auxiliary, and sweep gases were operated at 30, 8, and 0 (arbitrary units), respectively, and the S-lens RF level was 60. Each sample was analyzed using a mass range of m/z 70−900, and data were acquired at 70,000 resolution, automatic gain control (AGC) target of 1e6, and maximum injection time (IT) of 100 ms. Additionally, the top 10 data-dependent acquisition experiments were performed for the mixed sample from each group (HILIC and C18) to obtain compound MS/MS spectra. Processing of all full scan and ddMS2 data was conducted using the Xcalibur 4.1 software. Metabolites were identified by accurate mass, comparison of retention times to authentic standards or by accurate mass and comparison of fragmentation patterns to MS/MS databases (METLIN, PubChem). Metabolite relative concentration was normalized based on the average recoveries of 13C5-oT and 2H7-BAR as the internal standards |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003545 |
| Analysis ID: | AN003803 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The Orbitrap QExactive was operated with a heated electrospray ionization (HESI) probe in negative mode. Temperatures of the HESI-II auxiliary gas heater and capillary were 450 and 300 °C, respectively, and the spray voltage was 3.9 kV. Sheath, auxiliary, and sweep gases were operated at 30, 8, and 0 (arbitrary units), respectively, and the S-lens RF level was 60. Each sample was analyzed using a mass range of m/z 70−900, and data were acquired at 70,000 resolution, automatic gain control (AGC) target of 1e6, and maximum injection time (IT) of 100 ms. Additionally, the top 10 data-dependent acquisition experiments were performed for the mixed sample from each group (HILIC and C18) to obtain compound MS/MS spectra. Processing of all full scan and ddMS2 data was conducted using the Xcalibur 4.1 software. Metabolites were identified by accurate mass, comparison of retention times to authentic standards or by accurate mass and comparison of fragmentation patterns to MS/MS databases (METLIN, PubChem). Metabolite relative concentration was normalized based on the average recoveries of 13C5-oT and 2H7-BAR as the internal standards |
| Ion Mode: | NEGATIVE |
