Summary of Study ST001520

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	ST001520
Study Title	Stool unknowns profiled using hybrid nontargeted methods (part-II)
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:	SU001594
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
SA127952	9024-2-SE	Modulen	Female	Baseline
SA127976	9016-2-SE	Modulen	Female	Baseline
SA127986	9034-2-SE	Modulen	Female	Baseline
SA128006	9038-2-SE	Modulen	Female	Baseline
SA127953	9024-2-SB	Modulen	Female	Day 12
SA127977	9016-2-SB	Modulen	Female	Day 12
SA127987	9034-2-SB	Modulen	Female	Day 12
SA128007	9038-2-SB	Modulen	Female	Day 12
SA127954	9024-2-SA	Modulen	Female	Day 15
SA127978	9016-2-SA	Modulen	Female	Day 15
SA127988	9034-2-SA	Modulen	Female	Day 15
SA128008	9038-2-SA	Modulen	Female	Day 15
SA127955	9024-2-SD	Modulen	Female	Day 5
SA127979	9016-2-SD	Modulen	Female	Day 5
SA127989	9034-2-SD	Modulen	Female	Day 5
SA128009	9038-2-SD	Modulen	Female	Day 5
SA127980	9016-2-SC	Modulen	Female	Day 9
SA127990	9034-2-SC	Modulen	Female	Day 9
SA128010	9038-2-SC	Modulen	Female	Day 9
SA127869	9003-2-SE	Modulen	Male	Baseline
SA127897	9009-2-SE	Modulen	Male	Baseline
SA127947	9029-2-SE	Modulen	Male	Baseline
SA127956	9025-2-SE	Modulen	Male	Baseline
SA127981	9013-2-SE	Modulen	Male	Baseline
SA127996	9032-2-SE	Modulen	Male	Baseline
SA127870	9003-2-SB	Modulen	Male	Day 12
SA127898	9009-2-SB	Modulen	Male	Day 12
SA127948	9029-2-SB	Modulen	Male	Day 12
SA127957	9025-2-SB	Modulen	Male	Day 12
SA127982	9013-2-SB	Modulen	Male	Day 12
SA127997	9032-2-SB	Modulen	Male	Day 12
SA127871	9003-2-SA	Modulen	Male	Day 15
SA127899	9009-2-SA	Modulen	Male	Day 15
SA127949	9029-2-SA	Modulen	Male	Day 15
SA127958	9025-2-SA	Modulen	Male	Day 15
SA127983	9013-2-SA	Modulen	Male	Day 15
SA127998	9032-2-SA	Modulen	Male	Day 15
SA127900	9009-2-SD	Modulen	Male	Day 5
SA127950	9029-2-SD	Modulen	Male	Day 5
SA127959	9025-2-SD	Modulen	Male	Day 5
SA127984	9013-2-SD	Modulen	Male	Day 5
SA127999	9032-2-SD	Modulen	Male	Day 5
SA127872	9003-2-SC	Modulen	Male	Day 9
SA127901	9009-2-SC	Modulen	Male	Day 9
SA127951	9029-2-SC	Modulen	Male	Day 9
SA127960	9025-2-SC	Modulen	Male	Day 9
SA127985	9013-2-SC	Modulen	Male	Day 9
SA128000	9032-2-SC	Modulen	Male	Day 9
SA128016	QCPS15	NA	NA	NA
SA128017	QCPS13	NA	NA	NA
SA128018	QCPS16	NA	NA	NA
SA128019	QCPS14	NA	NA	NA
SA128020	QCPS19	NA	NA	NA
SA128021	QCPS20	NA	NA	NA
SA128022	QCPS12	NA	NA	NA
SA128023	QCPS18	NA	NA	NA
SA128024	QCPS17	NA	NA	NA
SA128025	QCPS09	NA	NA	NA
SA128026	QCPS04	NA	NA	NA
SA128027	QCPS03	NA	NA	NA
SA128028	QCPS02	NA	NA	NA
SA128029	QCPS05	NA	NA	NA
SA128030	QCPS06	NA	NA	NA
SA128031	QCPS10	NA	NA	NA
SA128032	QCPS08	NA	NA	NA
SA128033	QCPS07	NA	NA	NA
SA128034	QCPS11	NA	NA	NA
SA127878	9027-3-SE	Vegan	Female	Baseline
SA127887	9014-3-SE	Vegan	Female	Baseline
SA127912	9026-3-SE	Vegan	Female	Baseline
SA127966	9031-3-SE	Vegan	Female	Baseline
SA127879	9027-3-SB	Vegan	Female	Day 12
SA127888	9014-3-SB	Vegan	Female	Day 12
SA127913	9026-3-SB	Vegan	Female	Day 12
SA127967	9031-3-SB	Vegan	Female	Day 12
SA127889	9014-3-SA	Vegan	Female	Day 15
SA127914	9026-3-SA	Vegan	Female	Day 15
SA127968	9031-3-SA	Vegan	Female	Day 15
SA127880	9027-3-SD	Vegan	Female	Day 5
SA127890	9014-3-SD	Vegan	Female	Day 5
SA127915	9026-3-SD	Vegan	Female	Day 5
SA127969	9031-3-SD	Vegan	Female	Day 5
SA127881	9027-3-SC	Vegan	Female	Day 9
SA127891	9014-3-SC	Vegan	Female	Day 9
SA127916	9026-3-SC	Vegan	Female	Day 9
SA127970	9031-3-SC	Vegan	Female	Day 9
SA127873	9002-3-SE	Vegan	Male	Baseline
SA127902	9008-3-SE	Vegan	Male	Baseline
SA127922	9004-3-SE	Vegan	Male	Baseline
SA127932	9017-3-SE	Vegan	Male	Baseline
SA127933	9035-3-SE	Vegan	Male	Baseline
SA127934	9022-3-SE	Vegan	Male	Baseline
SA127874	9002-3-SB	Vegan	Male	Day 12
SA127903	9008-3-SB	Vegan	Male	Day 12
SA127923	9004-3-SB	Vegan	Male	Day 12
SA127935	9022-3-SB	Vegan	Male	Day 12
SA127936	9035-3-SB	Vegan	Male	Day 12
SA127937	9017-3-SB	Vegan	Male	Day 12
SA127875	9002-3-SA	Vegan	Male	Day 15
SA127904	9008-3-SA	Vegan	Male	Day 15

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Collection:

Collection ID:	CO001589
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:	TR001609
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:

TR001609

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:	SP001602
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:

SP001602

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	AN002529	AN002530	AN002531	AN002532
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	peak area	peak area	peak area

Chromatography:

Chromatography ID:	CH001849
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:	CH001850
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:	CH001851
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:	CH001852
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:	MS002347
Analysis ID:	AN002529
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:	MS002348
Analysis ID:	AN002530
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:	MS002349
Analysis ID:	AN002531
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:	MS002350
Analysis ID:	AN002532
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