#METABOLOMICS WORKBENCH jfolz_20191006_104437_mwtab.txt DATATRACK_ID:1830 STUDY_ID:ST001264 ANALYSIS_ID:AN002101 PROJECT_ID:PR000849
VERSION 1
CREATED_ON October 8, 2019, 3:42 pm
#PROJECT
PR:PROJECT_TITLE Antibiotics and Dietary Minerals Lipidomics
PR:PROJECT_TYPE Untargeted Mass Spectrometry
PR:PROJECT_SUMMARY Rats were fed either high sodium/ low potassium diet or low sodium/ high
PR:PROJECT_SUMMARY potassium diet, and either antibiotics, or no antibiotics. Plasma samples were
PR:PROJECT_SUMMARY analyzed using UHPLC MS/MS methodology.
PR:INSTITUTE UC Davis
PR:LABORATORY Oliver Fiehn
PR:LAST_NAME Folz
PR:FIRST_NAME Jacob
PR:ADDRESS 451 Health Science Drive, Davis, CA 95616
PR:EMAIL jfolz@ucdavis.edu
PR:PHONE 7155636311
PR:CONTRIBUTORS Jacob Folz, Young Taek Oh, Ivana Blaženović, Joyce Richey, Oliver Fiehn and
PR:CONTRIBUTORS Jang H. Youn
#STUDY
ST:STUDY_TITLE Antibiotics and Dietary Minerals Lipidomics
ST:STUDY_SUMMARY Plasma samples from Wistar rats fed a control or High-sodium and low-potassium
ST:STUDY_SUMMARY HNaLK diet with or without antibiotic treatment (n = 7 each, a total of 28) were
ST:STUDY_SUMMARY subjected to lipidomics analysis.The HNaLK diet interacts with gut bacteria to
ST:STUDY_SUMMARY alter plasma lipid profiles, which may be related to its health effects.
ST:INSTITUTE University of California, Davis
ST:LAST_NAME Folz
ST:FIRST_NAME Jacob
ST:ADDRESS 451 Health Sciences Dr., Davis, CA, 95616
ST:EMAIL jfolz@ucdavis.edu
ST:PHONE 7155636311
ST:NUM_GROUPS 4
ST:TOTAL_SUBJECTS 28
#SUBJECT
SU:SUBJECT_TYPE Mammal
SU:SUBJECT_SPECIES Rattus norvegicus
SU:TAXONOMY_ID 10116
SU:AGE_OR_AGE_RANGE 10 weeks
SU:GENDER Male
SU:ANIMAL_ANIMAL_SUPPLIER Envigo Laboratories
SU:ANIMAL_LIGHT_CYCLE 12-h light, 6 AM–6 PM; 12-h dark, 6 PM–6 AM
#SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS - Biorec1 Treatment:No Treatment
SUBJECT_SAMPLE_FACTORS - Biorec2 Treatment:No Treatment
SUBJECT_SAMPLE_FACTORS - Biorec3 Treatment:No Treatment
SUBJECT_SAMPLE_FACTORS - Sample1 Treatment:Antibiotics_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample8 Treatment:Antibiotics_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample12 Treatment:Antibiotics_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample18 Treatment:Antibiotics_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample23 Treatment:Antibiotics_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample27 Treatment:Antibiotics_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample13 Treatment:Antibiotics_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample7 Treatment:Antibiotics_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample24 Treatment:Antibiotics_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample11 Treatment:Antibiotics_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample17 Treatment:Antibiotics_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample28 Treatment:Antibiotics_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample2 Treatment:Antibiotics_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample14 Treatment:Antibiotics_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample15 Treatment:None (normal)_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample3 Treatment:None (normal)_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample6 Treatment:None (normal)_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample10 Treatment:None (normal)_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample20 Treatment:None (normal)_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample25 Treatment:None (normal)_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample21 Treatment:None (normal)_High Potasium / Low Sodium
SUBJECT_SAMPLE_FACTORS - Sample4 Treatment:None (normal)_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample5 Treatment:None (normal)_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample9 Treatment:None (normal)_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample16 Treatment:None (normal)_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample19 Treatment:None (normal)_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample22 Treatment:None (normal)_Low Potasium / High Sodium
SUBJECT_SAMPLE_FACTORS - Sample26 Treatment:None (normal)_Low Potasium / High Sodium
#COLLECTION
CO:COLLECTION_SUMMARY After the one-week feeding, animals were anesthetized with isoflurane at ~7 AM,
CO:COLLECTION_SUMMARY and blood samples for lipidomics analysis were collected using a heparinized
CO:COLLECTION_SUMMARY syringe through the abdominal aorta. Blood samples were rapidly spun, and plasma
CO:COLLECTION_SUMMARY was isolated and frozen immediately in liquid N2. The plasma samples were stored
CO:COLLECTION_SUMMARY at -80°C until analysis.
CO:SAMPLE_TYPE Blood (plasma)
#TREATMENT
TR:TREATMENT_SUMMARY Animals were fed for 1 week with diets containing different amounts of Na+ and
TR:TREATMENT_SUMMARY K+ (n = 7 for each diet; a total of 14 rats). The diets were prepared from
TR:TREATMENT_SUMMARY K+-deficient powdered rat diet (TD.88239.P.D Envigo Teklad) with or without
TR:TREATMENT_SUMMARY supplementation with KCl or NaCl.[10] The control ("healthy") diet contained
TR:TREATMENT_SUMMARY 0.29% Na+, which is the level in normal rat diets, and 2% K+, which is higher
TR:TREATMENT_SUMMARY than the normal 1%. In the HNaLK diet, Na+ content was increased from 0.29% to
TR:TREATMENT_SUMMARY 0.79%, a level similar to those in Western diets,[14] and K+ content was
TR:TREATMENT_SUMMARY decreased to 0.1%, which was selected to be low, but not low enough to deplete
TR:TREATMENT_SUMMARY plasma K+. The diets were gelled by heating, dissolving 30 g agarose in 500 mL
TR:TREATMENT_SUMMARY of deionized water, and adding to 500 g of powdered diet supplemented with KCl
TR:TREATMENT_SUMMARY and NaCl.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY Samples (40uL plasma) were extracted using biphasic extraction technique adapted
SP:SAMPLEPREP_SUMMARY from Matyash et al. 2008. In summary 225 µL LC-MS grade methanol was added to
SP:SAMPLEPREP_SUMMARY plasma in 2 mL Eppendorf tube, vortexed for 10 seconds, followed by addition of
SP:SAMPLEPREP_SUMMARY 750 µL methyl tert-butyl ether (MTBE). Each sample was then vortexed for 10
SP:SAMPLEPREP_SUMMARY seconds, shook on orbital shaker at maximum speed for six minutes, followed by
SP:SAMPLEPREP_SUMMARY addition of 188 µL LC-MS grade water. Finally, each sample was vortexed for 10
SP:SAMPLEPREP_SUMMARY seconds and centrifuged for 2 minutes at 14,000 rpm. The top phase of the
SP:SAMPLEPREP_SUMMARY biphasic solution was aliquoted into 1.5 mL microcentrifuge tubes for 350 µL
SP:SAMPLEPREP_SUMMARY aliquots of MTBE phase. Extraction was carried out at 4°C. All extract tubes
SP:SAMPLEPREP_SUMMARY were dried under vacuum and frozen at -80°C until LC-MS/MS analysis. Lipidomics
SP:SAMPLEPREP_SUMMARY analysis followed methods of Cajka et al. 2017 (doi:
SP:SAMPLEPREP_SUMMARY 10.1021/acs.analchem.7b03404). Non-polar lipids were analyzed on Vanquish
SP:SAMPLEPREP_SUMMARY Focused UHPLC coupled to a Q-Exactive HF mass spectrometer (ThermoFisher
SP:SAMPLEPREP_SUMMARY Scientific). Liquid chromatography used a Waters Acquity UPLC CSH C18 column
SP:SAMPLEPREP_SUMMARY (100mm x 2.1mm, 1.7 μm particle size) coupled to an Acquity UPLC CSH C18
SP:SAMPLEPREP_SUMMARY VanGuard precolumn (5 x 2.1 mm; 1.7 μm) (Waters, Milford, MA) with mobile
SP:SAMPLEPREP_SUMMARY phases of 60:40 (v/v) acetonitrile/ water, and 90:10 (v/v)
SP:SAMPLEPREP_SUMMARY isopropanol/acetonitrile, both of which had 10 mM ammonium formate and 0.1%
SP:SAMPLEPREP_SUMMARY formic acid for positive mode ionization analysis, and 10 mM ammonium acetate
SP:SAMPLEPREP_SUMMARY and 0.1% acetic acid for negative mode ionization analysis. Three and five
SP:SAMPLEPREP_SUMMARY microliters of reconstituted sample were injected onto column for positive and
SP:SAMPLEPREP_SUMMARY negative mode ionization analysis respectively. Mobile phase gradient was
SP:SAMPLEPREP_SUMMARY identical to that of Cajka et al. Data were collected in data dependent manner
SP:SAMPLEPREP_SUMMARY with the top four ions from each MS1 scan being selected for MS/MS
SP:SAMPLEPREP_SUMMARY fragmentation. A scan range from 120 to 1200 m/z was used for this analysis.
SP:SAMPLEPREP_SUMMARY Samples were analyzed in a randomized order with a method blank and quality
SP:SAMPLEPREP_SUMMARY control plasma sample analyzed between every ten samples.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY CSH C18 column run in Positive and Negative modes separately.
CH:CHROMATOGRAPHY_TYPE Reversed phase
CH:INSTRUMENT_NAME Thermo Vanquish Focused UHPLC
CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
CH:FLOW_GRADIENT 0 min 15% (B); 0−2 min 30% (B); 2−2.5 min 48% (B); 2.5−11 min 82% (B);
CH:FLOW_GRADIENT 11−11.5 min 99% (B); 11.5−12 min 99% (B); 12−12.1 min 15% (B); and
CH:FLOW_GRADIENT 12.1−15 min 15% (B)
CH:FLOW_RATE 0.6 mL/min
CH:COLUMN_TEMPERATURE 65
CH:SOLVENT_A (A) 60:40 (v/v) acetonitrile:water with ammonium formate (10 mM) and formic acid
CH:SOLVENT_A (0.1%)
CH:SOLVENT_B (B) 90:10 (v/v) isopropanol:acetonitrile with ammonium formate (10 mM) and
CH:SOLVENT_B formic acid (0.1%)
CH:INTERNAL_STANDARD CE(22:1) iSTD Cer(d18:1/17:0) iSTD Cholesterol d7 iSTD CUDA iSTD
CH:INTERNAL_STANDARD DG(12:0/12:0/0:0) iSTD DG(18:1/2:0/0:0) iSTD LPC(17:0) iSTD LPE(17:1) iSTD
CH:INTERNAL_STANDARD MG(17:0/0:0/0:0) iSTD PC(12:0/13:0) iSTD PE(17:0/17:0) iSTD SM(d18:1/17:0) iSTD
CH:INTERNAL_STANDARD Sphingosine(d17:1) iSTD TG d5(17:0/17:1/17:0) iSTD iSTD Ceramide (d18:1/17:0)
CH:INTERNAL_STANDARD iSTD Ceramide (d18:1/17:0) iSTD CUDA iSTD FA d3 (16:0) iSTD LPC (17:0) iSTD LPE
CH:INTERNAL_STANDARD (17:1) iSTD MAG (17:0/0:0/0:0) iSTD PC (12:0/13:0) iSTD PE (17:0/17:0) iSTD PG
CH:INTERNAL_STANDARD (17:0/17:0) iSTD SM (d18:1/17:0)
CH:SAMPLE_INJECTION 3uL (negative) 5uL (positive)
CH:WASHING_BUFFER 100% IPA
CH:TARGET_SAMPLE_TEMPERATURE 4
#ANALYSIS
AN:ANALYSIS_TYPE MS
AN:LABORATORY_NAME Fiehn Lab
AN:OPERATOR_NAME Jacob Folz
AN:DETECTOR_TYPE Orbitrap
AN:ACQUISITION_DATE 02_16_2018
AN:DATA_FORMAT Data Dependent Acquisition
#MS
MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap
MS:INSTRUMENT_TYPE Orbitrap
MS:MS_TYPE ESI
MS:ION_MODE NEGATIVE
MS:MS_COMMENTS Data were processed using open source software MS-DIAL version 2.82.MS-DIAL
MS:MS_COMMENTS performed baseline correction, deconvolution, peak detection, alignment, gap
MS:MS_COMMENTS filling, adduct identification, accurate mass/ retention time (m/z-RT) library
MS:MS_COMMENTS matching, and MS/MS library matching. MS-DIAL parameters were set to 10 for
MS:MS_COMMENTS minimum peak width, 10000 for minimum peak height, MS/MS fragmentation match to
MS:MS_COMMENTS library of at least 80%, MS1 tolerance of 0.005 Da, MS2 tolerance of 0.01 Da,
MS:MS_COMMENTS smoothing level of 3, and retention time window of 0.15 minutes for retention
MS:MS_COMMENTS time matching to an in-house m/z-RT library developed from authentic standards.
MS:MS_COMMENTS Tandem MS spectra were matched to library spectra from the Mass Bank of North
MS:MS_COMMENTS America (MoNA), NIST17, and LipidBlast.[20] Features that appeared in method
MS:MS_COMMENTS blanks were removed from further investigation. Duplicate peaks, isotopes, and
MS:MS_COMMENTS adducts were investigated and removed using Mass Spectral Feature List
MS:MS_COMMENTS Optimizer.[21] Manual inspection of each annotated compound was conducted to
MS:MS_COMMENTS confirm m/z-RT library match, and/or MS/MS library match. Peak height was used
MS:MS_COMMENTS as mass spectral intensity at a specific retention time for each annotated
MS:MS_COMMENTS lipid. After data processing, each sample was normalized to the total summed
MS:MS_COMMENTS intensity of all known features (excluding internal standards).
MS:ACQUISITION_DATE 02_16_2018
MS:COLLISION_ENERGY NCE 20.30.40
MS:IONIZATION Negative
MS:SCAN_RANGE_MOVERZ 120-1200
MS:SCANNING_CYCLE Top 4 ions selected for MS/MS
MS:MS_RESULTS_FILE ST001264_AN002101_Results.txt UNITS:Peak Height (Normalized Intensity) Has m/z:Yes Has RT:Yes RT units:Minutes
#END