Summary of Study ST001519

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 PR001024. The data can be accessed directly via it's Project DOI: 10.21228/M8B984 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.

Study ID	ST001519
Study Title	Stool metabolites of known identity profiled using hybrid nontargeted methods (part-I)
Study Summary	We determined the effect of diet on the composition and metabolic function of human gut microbiome using a controlled feeding experiment with three divergent diets (vegan, omnivore, and an exclusive enteral nutrition diet (EEN) devoid of dietary fiber) in the Food And Resulting Microbial Metabolites (FARMM) study. The study included an antibiotic and polyethylene glycol (Abx/PEG) intervention to dynamically assess the effect of diet on the reconstitution of the gut microbiota and its associated fecal and plasma metabolome. Samples from thirty healthy volunteers between the ages of 18 and 60, 10 mper diet group were analyzed. Self-reported vegans were required to have followed a vegan diet for a minimum of 6 months prior to enrollment. Key exclusion criteria included inflammatory bowel disease, celiac disease, or other chronic intestinal disorders; prior bowel resection surgery other than appendectomy; baseline bowel frequency less than every 2 days or greater than 3 times daily; creatinine concentration greater than the upper limit of normal; diabetes mellitus; currently smoking; body mass index (BMI) <18.5 or >35; and use of antibiotics and probiotics in the prior 6 months. The 10 vegans continued to follow their usual diet as outpatients. All participants completed the Diet History Questionnaire II (DHQ II), a food frequency questionnaire developed by the Risk Factor Monitoring and Methods Branch of the National Cancer Institute. The vegan participants also completed three 24 hour diet recalls with a dietitian in the week prior to starting antibiotics. We randomly assigned the 20 omnivores to receive an omnivore diet or EEN (Modulen® IBD) while residing in an inpatient research unit. The macronutrient composition of Modulen® is protein 36g, fat 47g, and carbohydrate 110g per 1000 Kcal. The two omnivore diets were engineered to have a similar composition to EEN. All the subjects consumed the menu A on day 11 and menu B on days 4 and 14. Diets for omnivores were constructed to provide the expected total calories required per day for the participant to maintain their current weight and were adjusted if there was weight gain or loss of more than 2.5 pounds. On days 6, 7, and 8, inpatients participants received vancomycin 500mg orally every 6 hours and neomycin 1000mg orally every 6 hours. On day 7, participants consumed 4L of polyethylene glycol (PEG) based bowel purgative (GoLytely®). Participants in the omnivore and EEN arms left the inpatient research unit only under the direct supervision of a research staff member. The vegan outpatients reported to the hospital twice on days 6,7 and 8 to receive antibiotics and to consume PEG on day 7. The first stool sample of each day of the inpatient groups was collected, aliquoted and frozen immediately at -80oC. Blood was collected on days 1, 5, 9, 12 and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. The outpatient participants following the vegan diet also had blood collected on days 1, 5, 9, 12, and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. Among these participants, the first stool of the day was collected at home daily and kept on ice packs until it was brought to the research unit where it could be aliquoted and frozen. Samples from vegans were received for aliquoting within 24 hours and on average within 4 hours (Wu et al., 2010). Day 0 stool was not collected from vegans since their diet did not change. The aliquoted amounts for all samples ranged from 500 mg to 1 g. The aliquots were taken from different areas of the sample. Remaining sample was then collected into one residual 50 ml conical tube with a tongue depressor. Any remaining stool was discarded. The University of Pennsylvania Institutional Review Board (IRB) approved the research protocol.
Institute	Broad Institute of MIT and Harvard
Last Name	Clish
First Name	Clary
Address	415 Main Street, Cambridge, MA, 02142, USA
Email	clary@broadinstitute.org
Phone	617-714-7654
Submit Date	2020-11-05
Num Groups	3
Total Subjects	30
Num Males	20
Num Females	10
Study Comments	stool samples collected at baseline and 3-4 timepoints
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2021-04-01
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001024
Project DOI:	doi: 10.21228/M8B984
Project Title:	Role of Diet in the Reconstitution of the Human Gut Microbiome and its Metabolome
Project Type:	Observational study
Project Summary:	We studied the impact of three divergent diets, vegan, omnivore, and a synthetic enteral nutrition (EEN) diet lacking fiber, on the human gut microbiome and its metabolome in a longitudinal analysis that included a microbiota depletion intervention. Hybrid nontargeted LC-MS methods were used to profile stool and plasma metabolites.
Institute:	Broad Institute of MIT and Harvard
Last Name:	Clish
First Name:	Clary
Address:	415 Main Street, Cambridge, MA, 02142, USA
Email:	clary@broadinstitute.org
Phone:	617-714-7654
Funding Source:	Crohn’s & Colitis Foundation, P30 DK 050306, PennCHOP Microbiome Program, and the Penn Center for Nutritional Science and Medicine
Contributors:	Ceylan Tanes, Kyle Bittinger, Yuan Gao, Elliot S. Friedman, Lisa Nessel, Unmesha Roy Paladhi, Lillian Chau, Erika Panfen, Michael A. Fischbach, Jonathan Braun, Ramnik J. Xavier, Clary B. Clish, Hongzhe Li, Frederic D. Bushman, James D. Lewis, Gary D. Wu

Subject:

Subject ID:	SU001593
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	20-60
Weight Or Weight Range:	BMI range: 20-35
Gender:	Male and female
Human Race:	White; American Indian/Alaskan Native; Black/African American
Human Ethnicity:	Hispanic or Latino; Non-Hispanic or Latino
Human Trial Type:	Intervention trial
Human Exclusion Criteria:	Key exclusion criteria included inflammatory bowel disease, celiac disease, or other chronic intestinal disorders; prior bowel resection surgery other than appendectomy; baseline bowel frequency less than every 2 days or greater than 3 times daily; creatinine concentration greater than the upper limit of normal; diabetes mellitus; currently smoking; body mass index (BMI) <18.5 or >35; and use of antibiotics and probiotics in the prior 6 months

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Study_Diet	Sex	Time
SA127786	9024-2-SE	Modulen	Female	Baseline
SA127813	9016-2-SE	Modulen	Female	Baseline
SA127820	9034-2-SE	Modulen	Female	Baseline
SA127840	9038-2-SE	Modulen	Female	Baseline
SA127788	9024-2-SB	Modulen	Female	Day 12
SA127811	9016-2-SB	Modulen	Female	Day 12
SA127824	9034-2-SB	Modulen	Female	Day 12
SA127844	9038-2-SB	Modulen	Female	Day 12
SA127789	9024-2-SA	Modulen	Female	Day 15
SA127814	9016-2-SA	Modulen	Female	Day 15
SA127822	9034-2-SA	Modulen	Female	Day 15
SA127842	9038-2-SA	Modulen	Female	Day 15
SA127787	9024-2-SD	Modulen	Female	Day 5
SA127812	9016-2-SD	Modulen	Female	Day 5
SA127821	9034-2-SD	Modulen	Female	Day 5
SA127841	9038-2-SD	Modulen	Female	Day 5
SA127810	9016-2-SC	Modulen	Female	Day 9
SA127823	9034-2-SC	Modulen	Female	Day 9
SA127843	9038-2-SC	Modulen	Female	Day 9
SA127704	9003-2-SE	Modulen	Male	Baseline
SA127731	9009-2-SE	Modulen	Male	Baseline
SA127782	9029-2-SE	Modulen	Male	Baseline
SA127790	9025-2-SE	Modulen	Male	Baseline
SA127815	9013-2-SE	Modulen	Male	Baseline
SA127831	9032-2-SE	Modulen	Male	Baseline
SA127706	9003-2-SB	Modulen	Male	Day 12
SA127733	9009-2-SB	Modulen	Male	Day 12
SA127781	9029-2-SB	Modulen	Male	Day 12
SA127792	9025-2-SB	Modulen	Male	Day 12
SA127818	9013-2-SB	Modulen	Male	Day 12
SA127834	9032-2-SB	Modulen	Male	Day 12
SA127705	9003-2-SA	Modulen	Male	Day 15
SA127734	9009-2-SA	Modulen	Male	Day 15
SA127784	9029-2-SA	Modulen	Male	Day 15
SA127791	9025-2-SA	Modulen	Male	Day 15
SA127817	9013-2-SA	Modulen	Male	Day 15
SA127830	9032-2-SA	Modulen	Male	Day 15
SA127732	9009-2-SD	Modulen	Male	Day 5
SA127785	9029-2-SD	Modulen	Male	Day 5
SA127793	9025-2-SD	Modulen	Male	Day 5
SA127816	9013-2-SD	Modulen	Male	Day 5
SA127832	9032-2-SD	Modulen	Male	Day 5
SA127703	9003-2-SC	Modulen	Male	Day 9
SA127735	9009-2-SC	Modulen	Male	Day 9
SA127783	9029-2-SC	Modulen	Male	Day 9
SA127794	9025-2-SC	Modulen	Male	Day 9
SA127819	9013-2-SC	Modulen	Male	Day 9
SA127833	9032-2-SC	Modulen	Male	Day 9
SA127850	QCPS15	NA	NA	NA
SA127851	QCPS13	NA	NA	NA
SA127852	QCPS16	NA	NA	NA
SA127853	QCPS14	NA	NA	NA
SA127854	QCPS18	NA	NA	NA
SA127855	QCPS12	NA	NA	NA
SA127856	QCPS20	NA	NA	NA
SA127857	QCPS19	NA	NA	NA
SA127858	QCPS17	NA	NA	NA
SA127859	QCPS02	NA	NA	NA
SA127860	QCPS05	NA	NA	NA
SA127861	QCPS04	NA	NA	NA
SA127862	QCPS03	NA	NA	NA
SA127863	QCPS06	NA	NA	NA
SA127864	QCPS07	NA	NA	NA
SA127865	QCPS10	NA	NA	NA
SA127866	QCPS09	NA	NA	NA
SA127867	QCPS08	NA	NA	NA
SA127868	QCPS11	NA	NA	NA
SA127713	9027-3-SE	Vegan	Female	Baseline
SA127722	9014-3-SE	Vegan	Female	Baseline
SA127748	9026-3-SE	Vegan	Female	Baseline
SA127802	9031-3-SE	Vegan	Female	Baseline
SA127715	9027-3-SB	Vegan	Female	Day 12
SA127724	9014-3-SB	Vegan	Female	Day 12
SA127749	9026-3-SB	Vegan	Female	Day 12
SA127800	9031-3-SB	Vegan	Female	Day 12
SA127721	9014-3-SA	Vegan	Female	Day 15
SA127750	9026-3-SA	Vegan	Female	Day 15
SA127803	9031-3-SA	Vegan	Female	Day 15
SA127712	9027-3-SD	Vegan	Female	Day 5
SA127723	9014-3-SD	Vegan	Female	Day 5
SA127747	9026-3-SD	Vegan	Female	Day 5
SA127804	9031-3-SD	Vegan	Female	Day 5
SA127714	9027-3-SC	Vegan	Female	Day 9
SA127725	9014-3-SC	Vegan	Female	Day 9
SA127746	9026-3-SC	Vegan	Female	Day 9
SA127801	9031-3-SC	Vegan	Female	Day 9
SA127709	9002-3-SE	Vegan	Male	Baseline
SA127740	9008-3-SE	Vegan	Male	Baseline
SA127759	9004-3-SE	Vegan	Male	Baseline
SA127767	9017-3-SE	Vegan	Male	Baseline
SA127773	9022-3-SE	Vegan	Male	Baseline
SA127777	9035-3-SE	Vegan	Male	Baseline
SA127707	9002-3-SB	Vegan	Male	Day 12
SA127737	9008-3-SB	Vegan	Male	Day 12
SA127758	9004-3-SB	Vegan	Male	Day 12
SA127769	9017-3-SB	Vegan	Male	Day 12
SA127778	9022-3-SB	Vegan	Male	Day 12
SA127780	9035-3-SB	Vegan	Male	Day 12
SA127708	9002-3-SA	Vegan	Male	Day 15
SA127738	9008-3-SA	Vegan	Male	Day 15

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Collection:

Collection ID:	CO001588
Collection Summary:	The first stool sample of each day of the inpatient groups was collected, aliquoted and frozen immediately at -80oC. Blood was collected on days 1, 5, 9, 12 and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. The outpatient participants following the vegan diet also had blood collected on days 1, 5, 9, 12, and 15 from which plasma aliquots were immediately isolated and frozen at -80oC. Among these participants, the first stool of the day was collected at home daily and kept on ice packs until it was brought to the research unit where it could be aliquoted and frozen. Samples from vegans were received for aliquoting within 24 hours and on average within 4 hours (Wu et al., 2010). Day 0 stool was not collected from vegans since their diet did not change. The aliquoted amounts for all samples ranged from 500 mg to 1 g. The aliquots were taken from different areas of the sample. Remaining sample was then collected into one residual 50 ml conical tube with a tongue depressor. Any remaining stool was discarded. The University of Pennsylvania Institutional Review Board (IRB) approved the research protocol.
Sample Type:	Feces
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001608
Treatment Summary:	The 10 vegan participants continued to follow their usual diet as outpatients. All participants completed the Diet History Questionnaire II (DHQ II), a food frequency questionnaire developed by the Risk Factor Monitoring and Methods Branch of the National Cancer Institute. The vegan participants also completed three 24 hour diet recalls with a dietitian in the week prior to starting antibiotics. 20 omnivores were randomly assigned to receive an omnivore diet or EEN (Modulen® IBD) while residing in an inpatient research unit. The macronutrient composition of Modulen® is protein 36g, fat 47g, and carbohydrate 110g per 1000 Kcal. The two omnivore diets were engineered to have a similar composition to EEN. All the subjects consumed the menu A on day 11 and menu B on days 4 and 14. Diets for omnivores were constructed to provide the expected total calories required per day for the participant to maintain their current weight and were adjusted if there was weight gain or loss of more than 2.5 pounds. On days 6, 7, and 8, inpatients participants received vancomycin 500mg orally every 6 hours and neomycin 1000mg orally every 6 hours. On day 7, participants consumed 4L of polyethylene glycol (PEG) based bowel purgative (GoLytely®). Participants in the omnivore and EEN arms left the inpatient research unit only under the direct supervision of a research staff member. The vegan outpatients reported to the hospital twice on days 6,7 and 8 to receive antibiotics and to consume PEG on day 7.

Treatment ID:

TR001608

Treatment Summary:

The 10 vegan participants continued to follow their usual diet as outpatients. All participants completed the Diet History Questionnaire II (DHQ II), a food frequency questionnaire developed by the Risk Factor Monitoring and Methods Branch of the National Cancer Institute. The vegan participants also completed three 24 hour diet recalls with a dietitian in the week prior to starting antibiotics. 20 omnivores were randomly assigned to receive an omnivore diet or EEN (Modulen® IBD) while residing in an inpatient research unit. The macronutrient composition of Modulen® is protein 36g, fat 47g, and carbohydrate 110g per 1000 Kcal. The two omnivore diets were engineered to have a similar composition to EEN. All the subjects consumed the menu A on day 11 and menu B on days 4 and 14. Diets for omnivores were constructed to provide the expected total calories required per day for the participant to maintain their current weight and were adjusted if there was weight gain or loss of more than 2.5 pounds. On days 6, 7, and 8, inpatients participants received vancomycin 500mg orally every 6 hours and neomycin 1000mg orally every 6 hours. On day 7, participants consumed 4L of polyethylene glycol (PEG) based bowel purgative (GoLytely®). Participants in the omnivore and EEN arms left the inpatient research unit only under the direct supervision of a research staff member. The vegan outpatients reported to the hospital twice on days 6,7 and 8 to receive antibiotics and to consume PEG on day 7.

Sample Preparation:

Sampleprep ID:	SP001601
Sampleprep Summary:	Stool samples (weight range 42.99-90.35 mg) were homogenized in 4 μL of water per milligram stool sample weight using a bead mill (TissueLyser II; Qiagen) and the aqueous homogenates were aliquoted for metabolite profiling analyses. Plasma samples were thawed and aliquoted for each LC-MS method. LC-MS samples were prepared for four profiling methods as follows: HILIC-pos: Metabolites were extracted by adding 90 μL of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C8-pos: Lipids were extracted by adding 190 μL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, ambient temperature) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. HILIC-neg: Metabolites were extracted by adding 120 μL of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C18-neg: Metabolites were extracted by adding 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis.

Sampleprep ID:

SP001601

Sampleprep Summary:

Stool samples (weight range 42.99-90.35 mg) were homogenized in 4 μL of water per milligram stool sample weight using a bead mill (TissueLyser II; Qiagen) and the aqueous homogenates were aliquoted for metabolite profiling analyses. Plasma samples were thawed and aliquoted for each LC-MS method. LC-MS samples were prepared for four profiling methods as follows: HILIC-pos: Metabolites were extracted by adding 90 μL of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C8-pos: Lipids were extracted by adding 190 μL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL) to a 10 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, ambient temperature) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. HILIC-neg: Metabolites were extracted by adding 120 μL of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis. C18-neg: Metabolites were extracted by adding 90 μL of methanol containing PGE2-d4 as an internal standard (Cayman Chemical Co.; Ann Arbor, MI) to a 30 μL aliquot of stool homogenate or plasma. Samples were vortexed and then centrifuged (10 min, 9,000 x g, 4°C) to pellet protein precipitates. Supernatants were transferred to glass autosampler vials containing inserts for LC-MS analysis.

Combined analysis:

Analysis ID	AN002525	AN002526	AN002527	AN002528
Analysis type	MS	MS	MS	MS
Chromatography type	HILIC	Reversed phase	HILIC	Reversed phase
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters Atlantis HILIC (150 x 2mm,3um)	Waters Acquity BEH C8 (100 x 2.1mm,1.7um)	Phenomenex Luna NH2 (150 x 2.1mm,3um)	Waters Acquity T3 (150 x 2.1 mm,1.7um)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap	Thermo Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE
Units	unitless peak areas	unitless peak areas	unitless peak areas	unitless peak areas

Chromatography:

Chromatography ID:	CH001845
Chromatography Summary:	HILIC-pos: high resolution and accurate mass profiling of polar metabolites using HILIC and positive ion mode full scan MS
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Atlantis HILIC (150 x 2mm,3um)
Column Temperature:	30 ℃
Flow Gradient:	linear
Flow Rate:	250 uL/min
Injection Temperature:	4
Solvent A:	100% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH001846
Chromatography Summary:	C8-pos: high resolution and accurate mass profiling of polar and nonpolar lipids using reversed phase C8 chromatography and positive ion mode full scan MS
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity BEH C8 (100 x 2.1mm,1.7um)
Column Temperature:	40 ℃
Flow Gradient:	linear
Flow Rate:	450 uL/min
Solvent A:	95% water/5% methanol; 0.1% formic acid; 10 mM ammonium acetate
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH001847
Chromatography Summary:	HILIC-neg: high resolution and accurate mass profiling of polar metabolites using HILIC and negative ion mode full scan MS
Instrument Name:	Shimadzu Nexera X2
Column Name:	Phenomenex Luna NH2 (150 x 2.1mm,3um)
Column Temperature:	30 ℃
Flow Gradient:	linear
Flow Rate:	400 uL/min
Injection Temperature:	4
Solvent A:	100% water; 20 mM ammonium acetate, 20 mM ammonium hydroxide
Solvent B:	25% methanol/75% acetonitrile; 10 mM ammonium hydroxide
Chromatography Type:	HILIC

Chromatography ID:	CH001848
Chromatography Summary:	C18-neg: high resolution and accurate mass profiling of metabolites of intermediate polarity using reversed phase C18 chromatography and negative ion mode full scan MS
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity T3 (150 x 2.1 mm,1.7um)
Column Temperature:	45 ℃
Flow Gradient:	linear
Flow Rate:	450 uL/min
Injection Temperature:	4
Solvent A:	100% water; 0.01% formic acid
Solvent B:	100% acetonitrile; 0.01% acetic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002343
Analysis ID:	AN002525
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over m/z 70-800 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, 3.5 kV; capillary temperature, 350°C; probe heater temperature, 300°C; sheath gas, 40; auxiliary gas, 15; and S-lens RF level 40. All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known metabolites was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Compound identities were confirmed using reference standards
Ion Mode:	POSITIVE

MS ID:	MS002344
Analysis ID:	AN002526
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS analyses were carried out using electrospray ionization in the positive ion mode using full scan analysis over m/z 200-1100 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, 3.0 kV; capillary temperature, 300°C; probe heater temperature, 300°C; sheath gas, 50; auxiliary gas, 15; and S-lens RF level 60. All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known lipids was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Lipid identities were confirmed using reference standards representative of different lipid classes and previously characterized reference samples. Lipids were denoted by headgroup and total acyl carbon number and double bond content.
Ion Mode:	POSITIVE

MS ID:	MS002345
Analysis ID:	AN002527
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 60-750 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, -3.0 kV; capillary temperature, 350°C; probe heater temperature, 325°C; sheath gas, 55; auxiliary gas, 10; and S-lens RF level 40. All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known metabolites was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Compound identities were confirmed using reference standards.
Ion Mode:	NEGATIVE

MS ID:	MS002346
Analysis ID:	AN002528
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS analyses were carried out using electrospray ionization in the negative ion mode using full scan analysis over m/z 70-850 at 70,000 resolution and 3 Hz data acquisition rate. Additional MS settings were: ion spray voltage, -3.5 kV; capillary temperature, 320°C; probe heater temperature, 300°C; sheath gas, 45; auxiliary gas, 10; and S-lens RF level 60All raw data were processed using Progenesis QI software (NonLinear Dynamics) for feature alignment, nontargeted signal detection, and signal integration. Targeted processing of a subset of known metabolites was conducted using TraceFinder software (Thermo Fisher Scientific; Waltham, MA). Compound identities were confirmed using reference standards.
Ion Mode:	NEGATIVE