Summary of Study ST001522

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.

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Study ID	ST001522
Study Title	Plasma unknowns profiled using hybrid nontargeted methods (part-IV)
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	plasma 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	2021-08-10
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:	SU001596
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
Species Group:	Mammals

Factors:

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

mb_sample_id	local_sample_id	Study_Diet	Sex	Time
SA128198	9024-2-PE	Modulen	Female	Baseline
SA128213	9016-2-PE	Modulen	Female	Baseline
SA128215	9034-2-PE	Modulen	Female	Baseline
SA128220	9038-2-PE	Modulen	Female	Baseline
SA128197	9024-2-PB	Modulen	Female	Day 12
SA128212	9016-2-PB	Modulen	Female	Day 12
SA128219	9038-2-PB	Modulen	Female	Day 12
SA128223	9034-2-PB	Modulen	Female	Day 12
SA128202	9024-2-PA	Modulen	Female	Day 15
SA128209	9016-2-PA	Modulen	Female	Day 15
SA128218	9038-2-PA	Modulen	Female	Day 15
SA128224	9034-2-PA	Modulen	Female	Day 15
SA128195	9024-2-PD	Modulen	Female	Day 5
SA128204	9016-2-PD	Modulen	Female	Day 5
SA128216	9034-2-PD	Modulen	Female	Day 5
SA128221	9038-2-PD	Modulen	Female	Day 5
SA128196	9024-2-PC	Modulen	Female	Day 9
SA128211	9016-2-PC	Modulen	Female	Day 9
SA128217	9034-2-PC	Modulen	Female	Day 9
SA128222	9038-2-PC	Modulen	Female	Day 9
SA128200	9025-2-PE	Modulen	Male	Baseline
SA128208	9013-2-PE	Modulen	Male	Baseline
SA128225	9029-2-PE	Modulen	Male	Baseline
SA128230	9032-2-PE	Modulen	Male	Baseline
SA128243	9009-2-PE	Modulen	Male	Baseline
SA128244	9003-2-PE	Modulen	Male	Baseline
SA128203	9025-2-PB	Modulen	Male	Day 12
SA128207	9013-2-PB	Modulen	Male	Day 12
SA128229	9032-2-PB	Modulen	Male	Day 12
SA128233	9029-2-PB	Modulen	Male	Day 12
SA128236	9009-2-PB	Modulen	Male	Day 12
SA128241	9003-2-PB	Modulen	Male	Day 12
SA128210	9029-2-PA	Modulen	Male	Day 15
SA128227	9013-2-PA	Modulen	Male	Day 15
SA128228	9032-2-PA	Modulen	Male	Day 15
SA128234	9025-2-PA	Modulen	Male	Day 15
SA128235	9009-2-PA	Modulen	Male	Day 15
SA128240	9003-2-PA	Modulen	Male	Day 15
SA128201	9025-2-PD	Modulen	Male	Day 5
SA128205	9013-2-PD	Modulen	Male	Day 5
SA128226	9029-2-PD	Modulen	Male	Day 5
SA128231	9032-2-PD	Modulen	Male	Day 5
SA128239	9003-2-PD	Modulen	Male	Day 5
SA128242	9009-2-PD	Modulen	Male	Day 5
SA128199	9025-2-PC	Modulen	Male	Day 9
SA128206	9013-2-PC	Modulen	Male	Day 9
SA128214	9029-2-PC	Modulen	Male	Day 9
SA128232	9032-2-PC	Modulen	Male	Day 9
SA128237	9009-2-PC	Modulen	Male	Day 9
SA128238	9003-2-PC	Modulen	Male	Day 9
SA128245	QPP10	NA	NA	NA
SA128246	QPP09	NA	NA	NA
SA128247	QPP02	NA	NA	NA
SA128248	QPP08	NA	NA	NA
SA128249	QPP03	NA	NA	NA
SA128250	QPP07	NA	NA	NA
SA128251	QPP06	NA	NA	NA
SA128252	QPP05	NA	NA	NA
SA128253	QPP04	NA	NA	NA
SA128254	QPP01	NA	NA	NA
SA128261	9026-3-PE	Vegan	Female	Baseline
SA128265	9027-3-PE	Vegan	Female	Baseline
SA128269	9031-3-PE	Vegan	Female	Baseline
SA128285	9014-3-PE	Vegan	Female	Baseline
SA128255	9026-3-PB	Vegan	Female	Day 12
SA128258	9027-3-PB	Vegan	Female	Day 12
SA128272	9031-3-PB	Vegan	Female	Day 12
SA128289	9014-3-PB	Vegan	Female	Day 12
SA128259	9027-3-PA	Vegan	Female	Day 15
SA128273	9031-3-PA	Vegan	Female	Day 15
SA128276	9026-3-PA	Vegan	Female	Day 15
SA128288	9014-3-PA	Vegan	Female	Day 15
SA128262	9026-3-PD	Vegan	Female	Day 5
SA128263	9027-3-PD	Vegan	Female	Day 5
SA128267	9031-3-PD	Vegan	Female	Day 5
SA128286	9014-3-PD	Vegan	Female	Day 5
SA128256	9026-3-PC	Vegan	Female	Day 9
SA128264	9027-3-PC	Vegan	Female	Day 9
SA128268	9031-3-PC	Vegan	Female	Day 9
SA128290	9014-3-PC	Vegan	Female	Day 9
SA128260	9002-3-PE	Vegan	Male	Baseline
SA128271	9004-3-PE	Vegan	Male	Baseline
SA128277	9035-3-PE	Vegan	Male	Baseline
SA128279	9008-3-PE	Vegan	Male	Baseline
SA128284	9017-3-PE	Vegan	Male	Baseline
SA128304	9022-3-PE	Vegan	Male	Baseline
SA128274	9004-3-PB	Vegan	Male	Day 12
SA128280	9017-3-PB	Vegan	Male	Day 12
SA128287	9002-3-PB	Vegan	Male	Day 12
SA128293	9008-3-PB	Vegan	Male	Day 12
SA128297	9035-3-PB	Vegan	Male	Day 12
SA128301	9022-3-PB	Vegan	Male	Day 12
SA128278	9004-3-PA	Vegan	Male	Day 15
SA128281	9017-3-PA	Vegan	Male	Day 15
SA128292	9035-3-PA	Vegan	Male	Day 15
SA128295	9002-3-PA	Vegan	Male	Day 15
SA128296	9008-3-PA	Vegan	Male	Day 15
SA128302	9022-3-PA	Vegan	Male	Day 15
SA128257	9002-3-PD	Vegan	Male	Day 5
SA128266	9035-3-PD	Vegan	Male	Day 5

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Collection:

Collection ID:	CO001591
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:	Blood (plasma)
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001611
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:

TR001611

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:	SP001604
Sampleprep Summary:	Plasma samples were thawed on ice and 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:

SP001604

Sampleprep Summary:

Plasma samples were thawed on ice and 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	AN002537	AN002538	AN002539	AN002540
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	peak area	unitless peak areas	unitless peak areas	unitless peak areas

Chromatography:

Chromatography ID:	CH001857
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:	CH001858
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:	CH001859
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:	CH001860
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:	MS002355
Analysis ID:	AN002537
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:	MS002356
Analysis ID:	AN002538
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:	MS002357
Analysis ID:	AN002539
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:	MS002358
Analysis ID:	AN002540
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