Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA125425

Local Sample ID:	56_
Subject ID:	SU001563
Subject Type:	Plant
Subject Species:	Zea mays
Taxonomy ID:	4577

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Subject:

Subject ID:	SU001563
Subject Type:	Plant
Subject Species:	Zea mays
Taxonomy ID:	4577

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
56_	SA125425	FL015360	Control	Sample Type
56_	SA125425	FL015360	14 day	Age
56_	SA125425	FL015360	38 returned to 38	Growth Temperature

Collection:

Collection ID:	CO001558
Collection Summary:	Samples were collected and flash frozen in liquid N2
Sample Type:	Plant

Treatment:

Treatment ID:	TR001578
Treatment Summary:	For inoculations, single isolates of Cochliobolus heterostrophus were cultured on V8 agar for 3-4 weeks as described by (Christensen et al., 2018). Spot inoculations were performed on 14-d-old plants in which four 10 μl droplets of either C. heterostrophus suspension (1x106 spores mL-1) or control solution (sterile 0.1% Tween 20) were applied in a staggered fashion (Fig. 2a and Fig. 4a) onto the middle portion of the third leaf about 10cm from the distal tip. Following inoculation, both heat-stressed and non-heat-stressed plants were placed in a plastic humidity chamber and placed in the 28°C Percival. At 24 hours post-inoculation, infected and non-infected controls were removed from the humidity chambers and maintained at 28°C for the remainder of the experiment. At 72 hours post-inoculation, leaves (n=6) were photographed and immediately harvested into liquid N2. Lesions were digitally measured using ImageJ software (ImageJ 1.36b; Wayne Rasband, NIH, Bethesda, MD, USA).

Treatment ID:

TR001578

Treatment Summary:

For inoculations, single isolates of Cochliobolus heterostrophus were cultured on V8 agar for 3-4 weeks as described by (Christensen et al., 2018). Spot inoculations were performed on 14-d-old plants in which four 10 μl droplets of either C. heterostrophus suspension (1x106 spores mL-1) or control solution (sterile 0.1% Tween 20) were applied in a staggered fashion (Fig. 2a and Fig. 4a) onto the middle portion of the third leaf about 10cm from the distal tip. Following inoculation, both heat-stressed and non-heat-stressed plants were placed in a plastic humidity chamber and placed in the 28°C Percival. At 24 hours post-inoculation, infected and non-infected controls were removed from the humidity chambers and maintained at 28°C for the remainder of the experiment. At 72 hours post-inoculation, leaves (n=6) were photographed and immediately harvested into liquid N2. Lesions were digitally measured using ImageJ software (ImageJ 1.36b; Wayne Rasband, NIH, Bethesda, MD, USA).

Sample Preparation:

Sampleprep ID:	SP001571
Sampleprep Summary:	LCMS grade reagents (Thermo Fisher Scientific) were used throughout the extraction. Frozen 1.5-mL fast prep tubes with Zirmil® beads (1.1mm; SEPR Ceramic Beads and Powders, Mountainside, NJ, USA) containing 50 mg of ground and frozen tissue were carefully thawed to 4°C then transferred to ice. An internal standard mix of 12 μL containing caffeine, D6-Abscisic acid, D5-Jasmonic acid, D5-Cinnamic acid, D5-Indole-3-acetic acid, 13C-alpha linolenic acid, and nicotine at 8.33 μg/mL was added to each sample along with 750 μL of 10 mM of ammonium acetate and 750 μL of methanol (MeOH). Samples were mixed by vortex then homogenized at 6000 RPM for 30 seconds in a FastPrep® FP 120 tissue homogenizer (Qbiogene) and then sonicated in an ambient water bath for 15 min. Samples were centrifuged for 10 minutes at 20,000 RCF at ambient temperature, then 1 mL of supernatant was transferred to individual 4 mL glass vials. The supernatant was dried under a nitrogen stream at 30°C, reconstituted in 200 μL of 0.1% formic acid in water, and vortexed for 30 seconds. Vial contents were transferred to a 1.5-mL snap-cap tube, placed on ice for 10 minutes, then centrifuged at 20,000 RCF for 10 minutes at ambient temperature. 150 μL of supernatant was transferred to glass LC vials for UHPLC-HRMS analysis.

Sampleprep ID:

SP001571

Sampleprep Summary:

LCMS grade reagents (Thermo Fisher Scientific) were used throughout the extraction. Frozen 1.5-mL fast prep tubes with Zirmil® beads (1.1mm; SEPR Ceramic Beads and Powders, Mountainside, NJ, USA) containing 50 mg of ground and frozen tissue were carefully thawed to 4°C then transferred to ice. An internal standard mix of 12 μL containing caffeine, D6-Abscisic acid, D5-Jasmonic acid, D5-Cinnamic acid, D5-Indole-3-acetic acid, 13C-alpha linolenic acid, and nicotine at 8.33 μg/mL was added to each sample along with 750 μL of 10 mM of ammonium acetate and 750 μL of methanol (MeOH). Samples were mixed by vortex then homogenized at 6000 RPM for 30 seconds in a FastPrep® FP 120 tissue homogenizer (Qbiogene) and then sonicated in an ambient water bath for 15 min. Samples were centrifuged for 10 minutes at 20,000 RCF at ambient temperature, then 1 mL of supernatant was transferred to individual 4 mL glass vials. The supernatant was dried under a nitrogen stream at 30°C, reconstituted in 200 μL of 0.1% formic acid in water, and vortexed for 30 seconds. Vial contents were transferred to a 1.5-mL snap-cap tube, placed on ice for 10 minutes, then centrifuged at 20,000 RCF for 10 minutes at ambient temperature. 150 μL of supernatant was transferred to glass LC vials for UHPLC-HRMS analysis.

Combined analysis:

Analysis ID	AN002466	AN002467
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)	ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	peak intensity	peak intensity

Chromatography:

Chromatography ID:	CH001808
Instrument Name:	Thermo Vanquish
Column Name:	ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002286
Analysis ID:	AN002466
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The raw acquisition data were processed using a similar workflow described in previous work (Chamberlain et al., 2019a, b), which we detail here. Raw data files were converted from .raw to .mzxml format using RawConverter (He et al., 2015). MZmine 2 was used for processing the raw data including detecting masses, building chromatograms, grouping isotopic peaks, removing duplicate peaks, and aligning features (Pluskal et al., 2010). Identification was assigned to features by m/z (≤5 ppm) and retention time (0.2 min) (level 1 – identified compounds according to Metabolomics Standards Initiative standards (Sumner et al., 2007)) matching tousing our method-specific metabolite library produced from pure standards previously analyzed using this the above-mentioned chromatographic gradient. Processed data were exported from MZmine as a feature list containing the signal intensity for each feature in each sample. A small value (half the minimum value in the dataset) was used to replace zeros (no detection). The data were filtered to remove sample features with  10% signal contribution from their corresponding features in the extraction blanks. From this point, the data were further processed, normalized, and filtered using MetaboAnalyst 4.0 (Chong et al., 2018). For whole-metabolome comparative analyses, the data were normalized to total ion signal and feature intensities were auto-scaled to facilitate statistical comparisons (van den Berg et al., 2006). Statistical significance, defined as p  0.05, was determined using the two-tailed student’s t-test, and values for significance were adjusted for the false discovery rate with the Bonferroni-Holm method (HOLM, 1979).
Ion Mode:	POSITIVE

MS ID:	MS002287
Analysis ID:	AN002467
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The raw acquisition data were processed using a similar workflow described in previous work (Chamberlain et al., 2019a, b), which we detail here. Raw data files were converted from .raw to .mzxml format using RawConverter (He et al., 2015). MZmine 2 was used for processing the raw data including detecting masses, building chromatograms, grouping isotopic peaks, removing duplicate peaks, and aligning features (Pluskal et al., 2010). Identification was assigned to features by m/z (≤5 ppm) and retention time (0.2 min) (level 1 – identified compounds according to Metabolomics Standards Initiative standards (Sumner et al., 2007)) matching tousing our method-specific metabolite library produced from pure standards previously analyzed using this the above-mentioned chromatographic gradient. Processed data were exported from MZmine as a feature list containing the signal intensity for each feature in each sample. A small value (half the minimum value in the dataset) was used to replace zeros (no detection). The data were filtered to remove sample features with  10% signal contribution from their corresponding features in the extraction blanks. From this point, the data were further processed, normalized, and filtered using MetaboAnalyst 4.0 (Chong et al., 2018). For whole-metabolome comparative analyses, the data were normalized to total ion signal and feature intensities were auto-scaled to facilitate statistical comparisons (van den Berg et al., 2006). Statistical significance, defined as p  0.05, was determined using the two-tailed student’s t-test, and values for significance were adjusted for the false discovery rate with the Bonferroni-Holm method (HOLM, 1979).
Ion Mode:	NEGATIVE