Summary of Study ST001196

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000807. The data can be accessed directly via it's Project DOI: 10.21228/M8CD73 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study ID	ST001196
Study Title	Non-targeted GC-MS Analysis of Insoluble Metabolites (part-II)
Study Summary	Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking yet has implications for improved yield from plants, algae, and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites—including carbohydrates, lipids, amino acids, pigments, co-factors, nucleic acids and polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A custom photobioreactor and novel multi-platform mass spectrometry workflow enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal sinusoidal LD (“sinLD”) cycle peaking at 1,600 mol photons m 2 s-1. We report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of the identified polar/semi-polar, non-polar, and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation, and cell division phases of growth. During the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially-relevant strain design.
Institute	Colorado State University
Department	Chemical and Biological Engineering
Last Name	Peebles
First Name	Christie
Address	700 Meridian Ave, Fort Collins, CO 80523
Email	christie.peebles@colostate.edu
Phone	970-491-6779
Submit Date	2019-03-02
Raw Data Available	Yes
Raw Data File Type(s)	cdf
Analysis Type Detail	GC-MS
Release Date	2019-07-17
Release Version	1

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Project:

Project ID:	PR000807
Project DOI:	doi: 10.21228/M8CD73
Project Title:	A comprehensive time-course metabolite profiling of the model cyanobacterium Synechocystis sp. PCC 6803 under diurnal light:dark cycles
Project Summary:	Cyanobacteria are a model photoautotroph and a chassis for the sustainable production of fuels and chemicals. Yet, knowledge of photoautotrophic metabolism in the natural environment of day/night cycles is lacking yet has implications for improved yield from plants, algae, and cyanobacteria. Here, a thorough approach to characterizing diverse metabolites—including carbohydrates, lipids, amino acids, pigments, co-factors, nucleic acids and polysaccharides—in the model cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) under sinusoidal diurnal light-dark cycles was developed and applied. A custom photobioreactor and novel multi-platform mass spectrometry workflow enabled metabolite profiling every 30-120 minutes across a 24-hour diurnal sinusoidal LD (“sinLD”) cycle peaking at 1,600 mol photons m 2 s-1. We report widespread oscillations across the sinLD cycle with 90%, 94%, and 40% of the identified polar/semi-polar, non-polar, and polymeric metabolites displaying statistically significant oscillations, respectively. Microbial growth displayed distinct lag, biomass accumulation, and cell division phases of growth. During the lag phase, amino acids (AA) and nucleic acids (NA) accumulated to high levels per cell followed by decreased levels during the biomass accumulation phase, presumably due to protein and DNA synthesis. Insoluble carbohydrates displayed sharp oscillations per cell at the day-to-night transition. Potential bottlenecks in central carbon metabolism are highlighted. Together, this report provides a comprehensive view of photosynthetic metabolite behavior with high temporal resolution, offering insight into the impact of growth synchronization to light cycles via circadian rhythms. Incorporation into computational modeling and metabolic engineering efforts promises to improve industrially-relevant strain design.
Institute:	Colorado State University
Department:	Chemical and Biological Engineering
Last Name:	Peebles
First Name:	Christie
Address:	700 Meridian Ave, Fort Collins, CO 80523 USA
Email:	wernerajz@gmail.com
Phone:	2699981811

Subject:

Subject ID:	SU001263
Subject Type:	Bacteria
Subject Species:	Synechocystis sp. PCC 6803
Taxonomy ID:	1148
Genotype Strain:	NCBI:txid1148
Cell Biosource Or Supplier:	ATCC

Factors:

Subject type: Bacteria; Subject species: Synechocystis sp. PCC 6803 (Factor headings shown in green)

mb_sample_id	local_sample_id	time
SA083177	11-Synechocystis_6803-cell-7a-2	-
SA083178	10-Synechocystis_6803-cell-7a-1	-
SA083179	12-Synechocystis_6803-cell-7a-3	-
SA083186	9-Synechocystis_6803-cell-630a-3	-0.5
SA083187	7-Synechocystis_6803-cell-630a-1	-0.5
SA083188	8-Synechocystis_6803-cell-630a-2	-0.5
SA083189	15-Synechocystis_6803-cell-730a-3	0.5
SA083190	14-Synechocystis_6803-cell-730a-2	0.5
SA083191	13-Synechocystis_6803-cell-730a-1	0.5
SA083183	4-Synechocystis_6803-cell-6a-1	-1
SA083184	5-Synechocystis_6803-cell-6a-2	-1
SA083185	6-Synechocystis_6803-cell-6a-3	-1
SA083192	16-Synechocystis_6803-cell-8a-1	1
SA083193	18-Synechocystis_6803-cell-8a-3	1
SA083194	17-Synechocystis_6803-cell-8a-2	1
SA083207	33-Synechocystis_6803-cell-5p-3	10
SA083208	32-Synechocystis_6803-cell-5p-2	10
SA083209	31-Synechocystis_6803-cell-5p-1	10
SA083210	36-Synechocystis_6803-cell-6p-3	11
SA083211	34-Synechocystis_6803-cell-6p-1	11
SA083212	35-Synechocystis_6803-cell-6p-2	11
SA083213	37-Synechocystis_6803-cell-630p-1	11.5
SA083214	39-Synechocystis_6803-cell-630p-3	11.5
SA083215	38-Synechocystis_6803-cell-630p-2	11.5
SA083216	42-Synechocystis_6803-cell-7p-3	12
SA083217	40-Synechocystis_6803-cell-7p-1	12
SA083218	41-Synechocystis_6803-cell-7p-2	12
SA083219	45-Synechocystis_6803-cell-730p-3	12.5
SA083220	43-Synechocystis_6803-cell-730p-1	12.5
SA083221	44-Synechocystis_6803-cell-730p-2	12.5
SA083222	48-Synechocystis_6803-cell-8p-3	13
SA083223	47-Synechocystis_6803-cell-8p-2	13
SA083224	46-Synechocystis_6803-cell-8p-1	13
SA083225	51-Synechocystis_6803-cell-9p-3	14
SA083226	50-Synechocystis_6803-cell-9p-2	14
SA083227	49-Synechocystis_6803-cell-9p-1	14
SA083228	54-Synechocystis_6803-cell-11p-3	16
SA083229	53-Synechocystis_6803-cell-11p-2	16
SA083230	52-Synechocystis_6803-cell-11p-1	16
SA083231	57-Synechocystis_6803-cell-1a-3	18
SA083232	55-Synechocystis_6803-cell-1a-1	18
SA083233	56-Synechocystis_6803-cell-1a-2	18
SA083180	3-Synechocystis_6803-cell-5a-3	-2
SA083181	1-Synechocystis_6803-cell-5a-1	-2
SA083182	2-Synechocystis_6803-cell-5a-2	-2
SA083195	21-Synechocystis_6803-cell-9a-3	2
SA083196	19-Synechocystis_6803-cell-9a-1	2
SA083197	20-Synechocystis_6803-cell-9a-2	2
SA083234	58-Synechocystis_6803-cell-3a-1	20
SA083235	60-Synechocystis_6803-cell-3a-3	20
SA083236	59-Synechocystis_6803-cell-3a-2	20
SA083237	63-Synechocystis_6803-cell-5a_day2-3	22
SA083238	61-Synechocystis_6803-cell-5a_day2-1	22
SA083239	62-Synechocystis_6803-cell-5a_day2-2	22
SA083240	66-Synechocystis_6803-cell-6a_day4-3	23
SA083241	65-Synechocystis_6803-cell-6a_day3-2	23
SA083242	64-Synechocystis_6803-cell-6a_day2-1	23
SA083243	67-Synechocystis_6803-cell-630a_day2-1	23.5
SA083244	68-Synechocystis_6803-cell-630a_day3-2	23.5
SA083245	69-Synechocystis_6803-cell-630a_day4-3	23.5
SA083246	72-Synechocystis_6803-cell-7a_day2-3	24
SA083247	70-Synechocystis_6803-cell-7a_day2-1	24
SA083248	71-Synechocystis_6803-cell-7a_day2-2	24
SA083249	QC-10	26
SA083250	QC-9	26
SA083251	QC-11	26
SA083252	QC-12	26
SA083253	QC-8	26
SA083254	QC-4	26
SA083255	QC-2	26
SA083256	QC-1	26
SA083257	QC-3	26
SA083258	QC-5	26
SA083259	QC-6	26
SA083260	QC-7	26
SA083198	24-Synechocystis_6803-cell-11a-3	4
SA083199	22-Synechocystis_6803-cell-11a-1	4
SA083200	23-Synechocystis_6803-cell-11a-2	4
SA083201	27-Synechocystis_6803-cell-1p-3	6
SA083202	26-Synechocystis_6803-cell-1p-2	6
SA083203	25-Synechocystis_6803-cell-1p-1	6
SA083204	30-Synechocystis_6803-cell-3p-3	8
SA083205	29-Synechocystis_6803-cell-3p-2	8
SA083206	28-Synechocystis_6803-cell-3p-1	8

Showing results 1 to 84 of 84

Showing results 1 to 84 of 84

Collection:

Collection ID:	CO001257
Collection Summary:	For each metabolomics time-point, a 10 mL culture were rapidly sampled via sterile on-reactor syringes into a pre-weighed centrifuge tube, quenched in -4°C 1X PBS, spun at 3,000g for 5 min., decanted, frozen in liquid nitrogen, and lyophilized at -50°C. The workflow from sampling to centrifugation took < 2 minutes; lyophilized samples were stored at -80°C for < 1 month prior to extraction. A biphasic extraction from lyophilized cell pellets was performed via a 2:1:1.6 MTBE:MeOH:H2O biphasic extraction, modified from the protocol developed by Salem et al. (Salem et al., 2016) resulting in a top layer of MTBE with non-polar soluble metabolites, a lower layer of MeOH:H2O with polar and semi-polar soluble metabolites, and an insoluble pellet. Each liquid layer was transferred to a fresh glass vial and dried under nitrogen gas overnight. The MTBE layer was resuspended in 1:1 toluene:MeOH and analyzed via Q-TOF-MS with a UPLC Phenyl Hexyl column (“RP-MS”). The MeOH:H2O layer was resuspended in 1:1 H2O:MeOH, split evenly and subjected to either i) derivatization in methoxyamine HCl and MSTFA followed by GC-MS analysis, or ii) targeted SRM analysis on a tandem quadrupole-MS equipped with a HILIC column. The insoluble pellet was hydrolyzed with a hydrochloric acid (HCl) based on previously published protocols (Fountoulakis and Lahm, 1998) (Huang, Kaiser and Benner, 2012) to analyze individual amino acids, nucleoside, and carbohydrate content of the insoluble polymers utilizing MTBSTFA derivatization for insoluble amino acids. Of the soluble phases, 10 µL were removed from each sample and pooled to create a QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR001278
Treatment Summary:	Synechocystis sp. PCC 6803 [N-1] (ATCC 27184, NCBI Taxonomy ID: 1080229) was utilized for all experiments. A light-emitting diode photobioreactor (LED PBR) was engineered to provide a rectified sinusoidal waveform light profile which (results in the negative half-cycle being set to zero) via two custom 4000K White LED panels (Reliance Laboratories, Port Townsend WA) arranged opposite a water bath facing inwards, 5% CO2 at 200 mL min-1 via in-house gas mixing and custom aerators to provide sufficient mixing, 27-30°C temperature control via a Huber Ministat and custom water bath (Midwest Custom Aquarium, Starbuck MN), and improved light penetration at high volumes via custom flat-panel reactors (FPRs) built in a circular geometry to maximize mixing (Allen Scientific Glass, Boulder CO) (Figure S1). At the peak, 1,600 mol photons m-2s-1 (E) was provided as measured by LightScout Quantum Meter (Model: 3415FXSE). . A single LED-PBR was inoculated and entrained to sinLD cycles for two days; this entrained culture was then use inoculated three biological triplicate FPRs in the LED PBR (Figure S2). Reactors were cultivated under the sinLD cycle profile for an additional day of entrainment prior to sampling (total of 3 days of entrainment).

Treatment ID:

TR001278

Treatment Summary:

Synechocystis sp. PCC 6803 [N-1] (ATCC 27184, NCBI Taxonomy ID: 1080229) was utilized for all experiments. A light-emitting diode photobioreactor (LED PBR) was engineered to provide a rectified sinusoidal waveform light profile which (results in the negative half-cycle being set to zero) via two custom 4000K White LED panels (Reliance Laboratories, Port Townsend WA) arranged opposite a water bath facing inwards, 5% CO2 at 200 mL min-1 via in-house gas mixing and custom aerators to provide sufficient mixing, 27-30°C temperature control via a Huber Ministat and custom water bath (Midwest Custom Aquarium, Starbuck MN), and improved light penetration at high volumes via custom flat-panel reactors (FPRs) built in a circular geometry to maximize mixing (Allen Scientific Glass, Boulder CO) (Figure S1). At the peak, 1,600 mol photons m-2s-1 (E) was provided as measured by LightScout Quantum Meter (Model: 3415FXSE). . A single LED-PBR was inoculated and entrained to sinLD cycles for two days; this entrained culture was then use inoculated three biological triplicate FPRs in the LED PBR (Figure S2). Reactors were cultivated under the sinLD cycle profile for an additional day of entrainment prior to sampling (total of 3 days of entrainment).

Sample Preparation:

Sampleprep ID:	SP001271
Sampleprep Summary:	Briefly, 6 mL of 75% methanol (MeOH) was added to pellets, vortexed, and transferred to glass vials. 9 mL of 100% methyl tert-butyl ether (MTBE) was added, vortexed for 30 seconds, placed on automatic shaker for 1.5 hours at 4 ºC, and sonicated for 15 minutes. 3.75 mL of water was added, each extraction was vortexed by hand for 1 minute, and centrifuged for 10 minutes at 3,270g at 4ºC. A biphasic solution with a pellet formed: the top, green MTBE layer and the bottom, clear MeOH:H2O layer were separated into separate tubes and dried under N2,gas overnight. The pellet was stored at -80 ºC. After drying, the MTBE layer was resuspended in 100 uL 1:1 toluene:MeOH, transferred to a LC-MS vial insert, and stored at -80C for <1 month prior to MS analysis. The MeOH:H2O layer was resuspended in 1 mL of 1:1 H2O:MeOH, transferred to a 1.7 mL centrifuge tube and spun at 15,000g for 2 minutes at 4 ºC. The supernatant was split into two 465 µL aliquots—one for GCMS and one for LC(HILIC)MS—in glass vials and dried under N2,gas. The protocol outlined above is suitable for filter-quenched cyanobacteria samples and centrifuged cell pellets. The polar methanol/water fraction resulting from the biphasic extraction was processed for analysis by hydrophilic interaction liquid chromatography (HILIC) LC-MS. Dried samples were resuspended in 100 µL 1:1 H2O:MeOH and 10 µL were aliquoted into a pooled QC sample. Samples were stored at -80 ºC until analysis. The pooled QC sample was mixed and aliquoted into twelve vials. A QC injection was run every tenth injection. The dried polar fraction for analysis by GC-MS was stored at -80 ºC until derivatization, immediately prior to MS analysis. Samples were derivatized in 30 uL methoxyamine HCl and 30 uL MSTFA, as specified in the following section. Ten microliters were removed from each sample to create a pooled QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection. The non-polar MTBE phase was processed for non-targeted LC-MS analysis. Twenty microliters from each sample were pooled, mixed, and aliquoted into thirteen pooled QC samples. QC injections were placed after every sixth injection. An acid hydrolysis protocol was developed for identification of amino acids, nucleosides, and carbohydrates bound in insoluble pellet of protein, DNA/RNA, and polysaccharides, respectively. Pellets remaining from the biphasic extraction were removed from storage at -80 degrees C and residual solvent was evaporated under nitrogen gas. Pellets were re-suspended in 3 mL of 6 M hydrochloric acid (HCl) using vigorous vortexing and pipette re-suspension. The resulting suspension was a bright teal. The suspension was transferred equally two three separate glass vials for hydrolysis of the separate polymer constituents. Hydrolysis of proteins to amino acids was completed with a hydrochloric acid (HCl) hydrolysis, based on previously published protocols (Fountoulakis and Lahm 1998). Briefly, vials were incubated at 110 degrees C for with a loose cap seal. After 4 hours, the acid in each vial was entirely evaporated; 1 mL of 6 M HCl was added to each vial, vortexed, sealed tightly, and returned to 110 degrees C. After a total of 24 hours, vials were removed, and remaining acid was evaporated under nitrogen gas. Samples were resuspended in 150 µL of 1:1 MeOH:H2O, 20 uL was removed to create a pooled QC sample, the pooled QC was aliquoted into fourteen vials, and the solvent was evaporated under nitrogen gas. Amino acid samples were derivatized in 30 uL of methoxyamine HCl in pyridine and 30 uL of MTBSTFSA. The peak integration of each amino acid in each sample was manually checked and curated in the software Chromeleon™. Aspartic acid with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Threonine with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Polysaccharides and nucleic acid polymers were hydrolyzed to nucleosides using a modified protocol from Huang et al. (2012) (Huang, Kaiser, and Benner 2012). Briefly, vials were incubated at 130 degrees C for 10 minutes, removed and allowed to cool, and 100 uL was transferred to a glass teardrop vial. The remaining pellet was incubated at 160 degrees C for 40 minutes, removed and allowed to cool, re-suspended in 100 uL LC-MS grade water, vortexed for 15 seconds, centrifuged at 1,500g for 2 minutes, and the supernatant was transferred to the glass teardrop vial which contained purines from the 130 degree C incubation. The acid was evaporated under nitrogen gas. Samples were removed after the 10 minute 130ºC incubation to preserve purines (guanine and adenine) from degradation during the 40 minute 160ºC incubation.

Sampleprep ID:

SP001271

Sampleprep Summary:

Briefly, 6 mL of 75% methanol (MeOH) was added to pellets, vortexed, and transferred to glass vials. 9 mL of 100% methyl tert-butyl ether (MTBE) was added, vortexed for 30 seconds, placed on automatic shaker for 1.5 hours at 4 ºC, and sonicated for 15 minutes. 3.75 mL of water was added, each extraction was vortexed by hand for 1 minute, and centrifuged for 10 minutes at 3,270g at 4ºC. A biphasic solution with a pellet formed: the top, green MTBE layer and the bottom, clear MeOH:H2O layer were separated into separate tubes and dried under N2,gas overnight. The pellet was stored at -80 ºC. After drying, the MTBE layer was resuspended in 100 uL 1:1 toluene:MeOH, transferred to a LC-MS vial insert, and stored at -80C for <1 month prior to MS analysis. The MeOH:H2O layer was resuspended in 1 mL of 1:1 H2O:MeOH, transferred to a 1.7 mL centrifuge tube and spun at 15,000g for 2 minutes at 4 ºC. The supernatant was split into two 465 µL aliquots—one for GCMS and one for LC(HILIC)MS—in glass vials and dried under N2,gas. The protocol outlined above is suitable for filter-quenched cyanobacteria samples and centrifuged cell pellets. The polar methanol/water fraction resulting from the biphasic extraction was processed for analysis by hydrophilic interaction liquid chromatography (HILIC) LC-MS. Dried samples were resuspended in 100 µL 1:1 H2O:MeOH and 10 µL were aliquoted into a pooled QC sample. Samples were stored at -80 ºC until analysis. The pooled QC sample was mixed and aliquoted into twelve vials. A QC injection was run every tenth injection. The dried polar fraction for analysis by GC-MS was stored at -80 ºC until derivatization, immediately prior to MS analysis. Samples were derivatized in 30 uL methoxyamine HCl and 30 uL MSTFA, as specified in the following section. Ten microliters were removed from each sample to create a pooled QC sample, mixed, and aliquoted into thirteen vials. A QC sample was run after every sixth injection. The non-polar MTBE phase was processed for non-targeted LC-MS analysis. Twenty microliters from each sample were pooled, mixed, and aliquoted into thirteen pooled QC samples. QC injections were placed after every sixth injection. An acid hydrolysis protocol was developed for identification of amino acids, nucleosides, and carbohydrates bound in insoluble pellet of protein, DNA/RNA, and polysaccharides, respectively. Pellets remaining from the biphasic extraction were removed from storage at -80 degrees C and residual solvent was evaporated under nitrogen gas. Pellets were re-suspended in 3 mL of 6 M hydrochloric acid (HCl) using vigorous vortexing and pipette re-suspension. The resulting suspension was a bright teal. The suspension was transferred equally two three separate glass vials for hydrolysis of the separate polymer constituents. Hydrolysis of proteins to amino acids was completed with a hydrochloric acid (HCl) hydrolysis, based on previously published protocols (Fountoulakis and Lahm 1998). Briefly, vials were incubated at 110 degrees C for with a loose cap seal. After 4 hours, the acid in each vial was entirely evaporated; 1 mL of 6 M HCl was added to each vial, vortexed, sealed tightly, and returned to 110 degrees C. After a total of 24 hours, vials were removed, and remaining acid was evaporated under nitrogen gas. Samples were resuspended in 150 µL of 1:1 MeOH:H2O, 20 uL was removed to create a pooled QC sample, the pooled QC was aliquoted into fourteen vials, and the solvent was evaporated under nitrogen gas. Amino acid samples were derivatized in 30 uL of methoxyamine HCl in pyridine and 30 uL of MTBSTFSA. The peak integration of each amino acid in each sample was manually checked and curated in the software Chromeleon™. Aspartic acid with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Threonine with 2- and 3-derivitization agent modifications were detected and summed for the total spectral abundance. Polysaccharides and nucleic acid polymers were hydrolyzed to nucleosides using a modified protocol from Huang et al. (2012) (Huang, Kaiser, and Benner 2012). Briefly, vials were incubated at 130 degrees C for 10 minutes, removed and allowed to cool, and 100 uL was transferred to a glass teardrop vial. The remaining pellet was incubated at 160 degrees C for 40 minutes, removed and allowed to cool, re-suspended in 100 uL LC-MS grade water, vortexed for 15 seconds, centrifuged at 1,500g for 2 minutes, and the supernatant was transferred to the glass teardrop vial which contained purines from the 130 degree C incubation. The acid was evaporated under nitrogen gas. Samples were removed after the 10 minute 130ºC incubation to preserve purines (guanine and adenine) from degradation during the 40 minute 160ºC incubation.

Combined analysis:

Analysis ID	AN001992
Analysis type	MS
Chromatography type	GC
Chromatography system	Thermo ISQ
Column	Trace 1310 GC
MS Type	EI
MS instrument type	GC-TOF
MS instrument name	Thermo ISQ
Ion Mode	POSITIVE
Units	spectral abundance per cell

Chromatography:

Chromatography ID:	CH001440
Chromatography Summary:	For non-targeted GC-MS experiments, metabolites were detected using a Trace 1310 GC coupled to a Thermo ISQ mass spectrometer. Samples (1 µL) were injected at a 10:1 split ratio to a 30 m TG-5MS column (Thermo Scientific, 0.25 mm i.d., 0.25 μm film thickness) with a 1.2 mL/min helium gas flow rate. GC inlet was held at 285°C. The oven program started at 140°C for 1 min, followed by a ramp of 15°C/min to 330°C, and 5 min hold. Masses between 50-650 m/z were scanned at 5 scans/sec under electron impact ionization. Transfer line and ion source were held at 300 and 260°C, respectively. Pooled QC samples were injected after every 6 actual samples.
Instrument Name:	Thermo ISQ
Column Name:	Trace 1310 GC
Chromatography Type:	GC

MS:

MS ID:	MS001845
Analysis ID:	AN001992
Instrument Name:	Thermo ISQ
Instrument Type:	GC-TOF
MS Type:	EI
MS Comments:	Raw data was converted to *.CSV with Waters® Databridge. For idMS/MS (RP-LC-MS runs), a file was converted for low-collision, high-collision, and LockSpray for each sample. Peaks were detected within the XCMS workflow using the Centwave algorithm (Smith et al. 2006).
Ion Mode:	POSITIVE