Summary of Study ST002758

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 PR001719. The data can be accessed directly via it's Project DOI: 10.21228/M8HF01 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	ST002758
Study Title	Metabolic responses of normal rat kidneys to a high salt intake (Plasma)
Study Type	Time-course metabolomics experiment
Study Summary	In this study, novel methods were developed which allowed continuous (24/7) measurement of arterial blood pressure and renal blood flow in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2 and metabolites. Specifically, the study determined the effects of a high salt (HS; 4.0% NaCl) diet upon whole kidney O2 consumption and arterial and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats. A separate group of rats was studied to determine changes in the cortex and outer medulla tissue metabolomic profiles before and following the switch from a 0.4% to 4.0% NaCl diet.
Institute	Medical College of Wisconsin
Department	Physiology
Laboratory	Dr. Allen W. Cowley
Last Name	Cowley
First Name	Allen
Address	8701 W. Watertown Plank Rd, Milwaukee, WI 53226
Email	cowley@mcw.edu
Phone	4149558277
Submit Date	2023-06-26
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2023-07-02
Release Version	1

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Project:

Project ID:	PR001719
Project DOI:	doi: 10.21228/M8HF01
Project Title:	SD Rat Metabolomics in Response to Salt
Project Type:	Untargeted Four-Mode Metabolomics
Project Summary:	This study analyzed the effects of a high salt (HS; 4.0% NaCl) diet upon the kidney, arterial plasma, and renal venous plasma metabolomic profiles of normal Sprague-Dawley rats.
Institute:	Medical College of Wisconsin
Department:	Physiology
Laboratory:	Dr. Allen W. Cowley
Last Name:	Cowley
First Name:	Allen
Address:	8701 W. Watertown Plank Rd, Milwaukee, WI 53226
Email:	cowley@mcw.edu
Phone:	414-955-8277
Funding Source:	NHLBI
Contributors:	Satoshi Shimada, Brian R. Hoffmann, Chun Yang, Theresa Kurth, Andrew S. Greene, Mingyu Liang, Ranjan K. Dash, Allen W. Cowley Jr

Subject:

Subject ID:	SU002865
Subject Type:	Mammal
Subject Species:	Rattus norvegicus
Taxonomy ID:	10116

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment	Source
SA290237	20211007_Cowley3_Plasma_22_73-434-HS14-A_C18pos	HS14	Artery Plasma
SA290238	20211013_Cowley3_Plasma_28_61-429-HS14-A_C18neg_rep	HS14	Artery Plasma
SA290239	20211020_Cowley3_Plasma_2_49-421-HS14-A_HILICpos_rep	HS14	Artery Plasma
SA290240	20211020_Cowley3_Plasma_9_85-463-HS14-A_HILICpos_rep	HS14	Artery Plasma
SA290241	20211020_Cowley3_Plasma_23_73-434-HS14-A_HILICpos	HS14	Artery Plasma
SA290242	20211013_Cowley3_Plasma_28_61-429-HS14-A_C18neg	HS14	Artery Plasma
SA290243	20211020_Cowley3_Plasma_9_85-463-HS14-A_HILICpos	HS14	Artery Plasma
SA290244	20211013_Cowley3_Plasma_27_37-417-HS14-A_C18neg	HS14	Artery Plasma
SA290245	20211013_Cowley3_Plasma_18_49-421-HS14-A_C18neg	HS14	Artery Plasma
SA290246	20211013_Cowley3_Plasma_16_85-463-HS14-A_C18neg_rep	HS14	Artery Plasma
SA290247	20211013_Cowley3_Plasma_18_49-421-HS14-A_C18neg_rep	HS14	Artery Plasma
SA290248	20211013_Cowley3_Plasma_21_73-434-HS14-A_C18neg	HS14	Artery Plasma
SA290249	20211020_Cowley3_Plasma_23_73-434-HS14-A_HILICpos_rep	HS14	Artery Plasma
SA290250	20211013_Cowley3_Plasma_21_73-434-HS14-A_C18neg_rep	HS14	Artery Plasma
SA290251	20211013_Cowley3_Plasma_27_37-417-HS14-A_C18neg_rep	HS14	Artery Plasma
SA290252	20211020_Cowley3_Plasma_27_37-417-HS14-A_HILICpos	HS14	Artery Plasma
SA290253	20211029_Cowley3_Plasma_32_37-417-HS14-A_HILICneg_rep	HS14	Artery Plasma
SA290254	20211029_Cowley3_Plasma_32_37-417-HS14-A_HILICneg	HS14	Artery Plasma
SA290255	20211029_Cowley3_Plasma_34_73-434-HS14-A_HILICneg	HS14	Artery Plasma
SA290256	20211029_Cowley3_Plasma_34_73-434-HS14-A_HILICneg_rep	HS14	Artery Plasma
SA290257	20211029_Cowley3_Plasma_39_49-421-HS14-A_HILICneg_rep	HS14	Artery Plasma
SA290258	20211029_Cowley3_Plasma_39_49-421-HS14-A_HILICneg	HS14	Artery Plasma
SA290259	20211029_Cowley3_Plasma_28_61-429-HS14-A_HILICneg_rep	HS14	Artery Plasma
SA290260	20211029_Cowley3_Plasma_28_61-429-HS14-A_HILICneg	HS14	Artery Plasma
SA290261	20211020_Cowley3_Plasma_39_61-429-HS14-A_HILICpos	HS14	Artery Plasma
SA290262	20211020_Cowley3_Plasma_27_37-417-HS14-A_HILICpos_rep	HS14	Artery Plasma
SA290263	20211020_Cowley3_Plasma_39_61-429-HS14-A_HILICpos_rep	HS14	Artery Plasma
SA290264	20211029_Cowley3_Plasma_10_85-463-HS14-A_HILICneg	HS14	Artery Plasma
SA290265	20211029_Cowley3_Plasma_10_85-463-HS14-A_HILICneg_rep	HS14	Artery Plasma
SA290266	20211013_Cowley3_Plasma_16_85-463-HS14-A_C18neg	HS14	Artery Plasma
SA290267	20211020_Cowley3_Plasma_2_49-421-HS14-A_HILICpos	HS14	Artery Plasma
SA290268	20211007_Cowley3_Plasma_29_37-417-HS14-A_C18pos	HS14	Artery Plasma
SA290269	20211007_Cowley3_Plasma_29_37-417_HS14-A_C18pos_rep	HS14	Artery Plasma
SA290270	20211007_Cowley3_Plasma_31_85-463-HS14-A_C18pos	HS14	Artery Plasma
SA290271	20211007_Cowley3_Plasma_31_85-463-HS14-A_C18pos_rep	HS14	Artery Plasma
SA290272	20211007_Cowley3_Plasma_37_49-421-HS14-A_C18pos_rep	HS14	Artery Plasma
SA290273	20211007_Cowley3_Plasma_37_49-421-HS14-A_C18pos	HS14	Artery Plasma
SA290274	20211007_Cowley3_Plasma_28_61-429-HS14-A_C18pos	HS14	Artery Plasma
SA290275	20211007_Cowley3_Plasma_28_61-429-HS14-A_C18pos_rep	HS14	Artery Plasma
SA290276	20211007_Cowley3_Plasma_22_73-434-HS14-A_C18pos_rep	HS14	Artery Plasma
SA290277	20211007_Cowley3_Plasma_20_86-463-HS14-RV_C18pos	HS14	Vein Plasma
SA290278	20211007_Cowley3_Plasma_20_86-463-HS14-RV_C18pos_rep	HS14	Vein Plasma
SA290279	20211007_Cowley3_Plasma_24_50-421-HS14-RV_C18pos	HS14	Vein Plasma
SA290280	20211007_Cowley3_Plasma_11_62-429-HS14-RV_C18pos_rep	HS14	Vein Plasma
SA290281	20211007_Cowley3_Plasma_11_62-429-HS14-RV_C18pos	HS14	Vein Plasma
SA290282	20211020_Cowley3_Plasma_7_74-434-HS14-RV_HILICpos	HS14	Vein Plasma
SA290283	20211007_Cowley3_Plasma_7_38-417-HS14-RV_C18pos	HS14	Vein Plasma
SA290284	20211007_Cowley3_Plasma_7_38-417-HS14-RV_C18pos_rep	HS14	Vein Plasma
SA290285	20211013_Cowley3_Plasma_10_86-463-HS14-RV_C18neg_rep	HS14	Vein Plasma
SA290286	20211013_Cowley3_Plasma_11_38-417-HS14-RV_C18neg	HS14	Vein Plasma
SA290287	20211007_Cowley3_Plasma_24_50-421-HS14-RV_C18pos_rep	HS14	Vein Plasma
SA290288	20211013_Cowley3_Plasma_34_74-434-HS14-RV_C18neg_rep	HS14	Vein Plasma
SA290289	20211013_Cowley3_Plasma_34_74-434-HS14-RV_C18neg	HS14	Vein Plasma
SA290290	20211013_Cowley3_Plasma_31_50-421-HS14-RV_C18neg	HS14	Vein Plasma
SA290291	20211020_Cowley3_Plasma_7_74-434-HS14-RV_HILICpos_rep	HS14	Vein Plasma
SA290292	20211013_Cowley3_Plasma_11_38-417-HS14-RV_C18neg_rep	HS14	Vein Plasma
SA290293	20211013_Cowley3_Plasma_13_62-429-HS14-RV_C18neg	HS14	Vein Plasma
SA290294	20211013_Cowley3_Plasma_13_62-429-HS14-RV_C18neg_rep	HS14	Vein Plasma
SA290295	20211013_Cowley3_Plasma_31_50-421-HS14-RV_C18neg_rep	HS14	Vein Plasma
SA290296	20211020_Cowley3_Plasma_33_38-417-HS14-RV_HILICpos	HS14	Vein Plasma
SA290297	20211029_Cowley3_Plasma_11_62-429-HS14-RV_HILICneg	HS14	Vein Plasma
SA290298	20211029_Cowley3_Plasma_8_50-421-HS14-RV_HILICneg_rep	HS14	Vein Plasma
SA290299	20211029_Cowley3_Plasma_8_50-421-HS14-RV_HILICneg	HS14	Vein Plasma
SA290300	20211029_Cowley3_Plasma_11_62-429-HS14-RV_HILICneg_rep	HS14	Vein Plasma
SA290301	20211029_Cowley3_Plasma_16_86-463-HS14-RV_HILICneg	HS14	Vein Plasma
SA290302	20211029_Cowley3_Plasma_31_38-417-HS14-RV_HILICneg_rep	HS14	Vein Plasma
SA290303	20211029_Cowley3_Plasma_31_38-417-HS14-RV_HILICneg	HS14	Vein Plasma
SA290304	20211029_Cowley3_Plasma_16_86-463-HS14-RV_HILICneg_rep	HS14	Vein Plasma
SA290305	20211029_Cowley3_Plasma_5_74-434-HS14-RV_HILICneg_rep	HS14	Vein Plasma
SA290306	20211029_Cowley3_Plasma_5_74-434-HS14-RV_HILICneg	HS14	Vein Plasma
SA290307	20211020_Cowley3_Plasma_25_50-421-HS14-RV_HILICpos_rep	HS14	Vein Plasma
SA290308	20211020_Cowley3_Plasma_25_50-421-HS14-RV_HILICpos	HS14	Vein Plasma
SA290309	20211020_Cowley3_Plasma_22_62-429-HS14-RV_HILICpos_rep	HS14	Vein Plasma
SA290310	20211020_Cowley3_Plasma_30_86-463-HS14-RV_HILICpos_rep	HS14	Vein Plasma
SA290311	20211013_Cowley3_Plasma_10_86-463-HS14-RV_C18neg	HS14	Vein Plasma
SA290312	20211007_Cowley3_Plasma_1_74-434-HS14-RV_C18pos	HS14	Vein Plasma
SA290313	20211007_Cowley3_Plasma_1_74-434-HS14-RV_C18pos_rep	HS14	Vein Plasma
SA290314	20211020_Cowley3_Plasma_33_38-417-HS14-RV_HILICpos_rep	HS14	Vein Plasma
SA290315	20211020_Cowley3_Plasma_22_62-429-HS14-RV_HILICpos	HS14	Vein Plasma
SA290316	20211020_Cowley3_Plasma_30_86-463-HS14-RV_HILICpos	HS14	Vein Plasma
SA290317	20211029_Cowley3_Plasma_24_40-417-HS21-A_HILICneg_rep	HS21	Artery Plasma
SA290318	20211029_Cowley3_Plasma_25_52-421-HS21-A_HILICneg	HS21	Artery Plasma
SA290319	20211029_Cowley3_Plasma_24_40-417-HS21-A_HILICneg	HS21	Artery Plasma
SA290320	20211029_Cowley3_Plasma_23_76-434-HS21-A_HILICneg_rep	HS21	Artery Plasma
SA290321	20211029_Cowley3_Plasma_23_76-434-HS21-A_HILICneg	HS21	Artery Plasma
SA290322	20211029_Cowley3_Plasma_25_52-421-HS21-A_HILICneg_rep	HS21	Artery Plasma
SA290323	20211029_Cowley3_Plasma_26_88-463-HS21-A_HILICneg	HS21	Artery Plasma
SA290324	20211007_Cowley3_Plasma_30_52-421-HS21-A_C18pos	HS21	Artery Plasma
SA290325	20211007_Cowley3_Plasma_30_52-421-HS21-A_C18pos_rep	HS21	Artery Plasma
SA290326	20211007_Cowley3_Plasma_12_40-417-HS21-A_C18pos	HS21	Artery Plasma
SA290327	20211007_Cowley3_Plasma_10_88-463-HS21-A_C18pos_rep	HS21	Artery Plasma
SA290328	20211007_Cowley3_Plasma_10_88-463-HS21-A_C18pos	HS21	Artery Plasma
SA290329	20211029_Cowley3_Plasma_12_64-429-HS21-A_HILICneg_rep	HS21	Artery Plasma
SA290330	20211029_Cowley3_Plasma_12_64-429-HS21-A_HILICneg	HS21	Artery Plasma
SA290331	20211013_Cowley3_Plasma_37_52-421-HS21-A_C18neg_rep	HS21	Artery Plasma
SA290332	20211013_Cowley3_Plasma_37_52-421-HS21-A_C18neg	HS21	Artery Plasma
SA290333	20211013_Cowley3_Plasma_39_76-434-HS21-A_C18neg	HS21	Artery Plasma
SA290334	20211013_Cowley3_Plasma_39_76-434-HS21-A_C18neg_rep	HS21	Artery Plasma
SA290335	20211007_Cowley3_Plasma_5_76-434-HS21-A_C18pos_rep	HS21	Artery Plasma
SA290336	20211013_Cowley3_Plasma_32_64-429-HS21-A_C18neg_rep	HS21	Artery Plasma

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Collection:

Collection ID:	CO002858
Collection Summary:	Plasma was collected through an arterial and renal venous catheter throughout the study (200 µL of arterial and renal venous blood were sampled at the day 7, 14, and 21). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.
Sample Type:	Arterial plasma; Venous plasma
Storage Conditions:	-80?

Treatment:

Treatment ID:	TR002874
Treatment Summary:	Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Treatment ID:

TR002874

Treatment Summary:

Rats (n=7, 10-11 weeks of age) were performed renal blood flow (RBF) probe implantation and femoral arterial catheterization5. Briefly, rats were anesthetized with isoflurane and arterial catheter was inserted. Following an abdominal incision, RBF probe was implanted on left renal artery and the cable was exposed at nape of the neck via the subcutaneous route. In addition to the RBF probe implantation, renal venous catheter was inserted through the femoral vein and placed in the left renal vein and secured to the luminal wall with 10-0 nylon. RBF and BP via arterial line were measured by conscious freely moving rats and recorded on average of every minute for 24 h/day. After 7-10 days of recovery period, 200 µL of arterial and renal venous blood were sampled and that blood was replaced from donor rats before and following 7, 14 and 21 days after the switch in diet from 0.4% (LS) to 4.0% (HS) salt diet (Dyets Inc, Bethlehem, PA). Overnight urine (18 hours) from the day before the blood draw was collected on ice. The kidneys were collected either at 14 days of HS (HS14) or 21 days of HS (HS21). The kidneys of only LS fed SD rats were also collected for comparison. The collected kidneys (n=5 for each group for metabolomics and mRNAseq analysis) were dissected to cortex and outer medulla and snap frozen with liquid nitrogen. Plasma, urine and tissue were stored in -80°C until further analysis.

Sample Preparation:

Sampleprep ID:	SP002871
Sampleprep Summary:	Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Sampleprep ID:

SP002871

Sampleprep Summary:

Plasma/Urine Metabolite Extraction. Metabolites were extracted from 20 µL of plasma and 20 µL of urine from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34. Metabolites were extracted using 500 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer; the sample was part of the water fraction. Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards. Each sample was then vortexed for 30 seconds on the highest setting, subject to one minute of mixing with the Tissue Lyser II in pre-chilled cassettes, and then sonicated at 30 Hz for 5 minutes of 30 seconds on 30 seconds off in an ice water bath. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction. Following the extraction, samples were centrifuged at 21,000 x g at 4°C and supernatant from each metabolite extract was equally divided into five 2 mL microcentrifuge tubes. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use. Tissue Metabolite Extraction. Metabolites were extracted from 20 mg of kidney cortex and medulla from each SD rat in the study according to standard operating procedures in the Mass Spectrometry and Protein Chemistry Service at The Jackson Laboratory34 as described for the plasma and urine samples with slight modification. Metabolites were extracted using 1000 µL of an ice cold 2:2:1 methanol:acetonitrile:water (MeOH:ACN:H2O) buffer containing internal standards as above per 20 mg of sample to ensure the extraction equivalents were normalized. Each sample had a 5 mm stainless steel bead added, then were pulverized in extraction buffer for two minutes usingTissue Lyser II. Samples were then placed in the -20°C freezer overnight (16 hours) for extraction and the supernatant was collected as with the urine/plasma samples. Each sample supernatant was divided into five equal volume aliquots, one for each of the four modes and the rest to create equal representation pools of all samples, one for each mode. Each aliquot was then dried down using a vacuum centrifuge for storage at -80°C until further use.

Combined analysis:

Analysis ID	AN004475	AN004476	AN004477	AN004478
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish	Thermo Vanquish
Column	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
MS Type	ESI	ESI	ESI	ESI
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE	POSITIVE	NEGATIVE
Units	Area	Area	Area	Area

Chromatography:

Chromatography ID:	CH003360
Chromatography Summary:	This chromatography method was utilized for all C18 positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 EC-C18 (2.1 x 50 mm; 2.7-Micron)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 98% A1/2% B1, 1-13 minutes from 98% A1/2% B1 to 10% A1/90% B1, 13-15 minutes at 10% A1/90% B1, 15-16 minutes from 10% A1/90% B1 to 98% A1/2% B1, and was re-equilibrated from 16-25 minutes at 98% A1/2% B1
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003361
Chromatography Summary:	This chromatography method was utilized for all C18 negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	100% water, 0.2% acetic acid
Solvent B:	100% acetonitrile, 0.2% acetic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH003362
Chromatography Summary:	This chromatography method was utilized for all HILIC positive polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium formate in H2O with 0.1% formic acid (Solvent A2)
Solvent B:	90% ACN with 10 mM ammonium formate in H2O with 0.1% formic acid (Solvent B2)
Chromatography Type:	HILIC

Chromatography ID:	CH003363
Chromatography Summary:	This chromatography method was utilized for all HILIC negative polarity runs in this study.
Instrument Name:	Thermo Vanquish
Column Name:	Agilent InfiinityLab Poroshell 120 HILIC-Z (2.1 x 50 mm; 2.7 micron; #689775-924)
Column Temperature:	25C
Flow Gradient:	0-1 minutes at 2% A/98% B, 1-11 minutes from 2% A/98% B to 30% A/70% B, 11-12 minutes from 30% A/70% B to 40% A/60% B, 12-16 minutes from 40% A/60% B to 95% A/5% B, was held at 95% A/5% B from 16-18 minutes, 18-20 minutes from 95% A/5% B to 2% A/98% B, and was re-equilibrated from 20-25 minutes at 2% A/98% B
Flow Rate:	0.1 mL/minute
Internal Standard:	Caffeine, 1-napthylamine, and 9-anthracene carboxylic acid were all added at 0.5 ng/ µL in the extraction buffer as internal standards
Solvent A:	10 mM ammonium acetate in H2O, pH 9.0 with 0.1% AffinityLab Deactivator Inhibitor (Agilent, #5191-3940; Solvent A3)
Solvent B:	85% ACN with 10 mM ammonium acetate in H2O with 0.1% AffinityLab Deactivator Inhibitor (Solvent B3)
Chromatography Type:	HILIC

MS:

MS ID:	MS004222
Analysis ID:	AN004475
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 positive plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004223
Analysis ID:	AN004476
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	C18 negative plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE

MS ID:	MS004224
Analysis ID:	AN004477
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC positive plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	POSITIVE

MS ID:	MS004225
Analysis ID:	AN004478
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HILIC negative plasma data: The tandem mass spectrometry RAW data files (consisting of MS1 and MS2 spectra collected) were analyzed using Thermo Compound Discoverer (v3.2.0.421). The MS1 and MS2 data was searched against the Thermo mzCloud database, ChemSpider database, Metabolika Pathways, and mzLogic predicted composition in the Compound Discoverer workflow.
Ion Mode:	NEGATIVE