#METABOLOMICS WORKBENCH Codreags00_20210603_143951 DATATRACK_ID:2675 STUDY_ID:ST001848 ANALYSIS_ID:AN002992 PROJECT_ID:PR001165 VERSION 1 CREATED_ON June 28, 2021, 1:50 pm #PROJECT PR:PROJECT_TITLE Untargeted mass spectrometry reveals impact of high fat diet on peripheral amino PR:PROJECT_TITLE acid regulation in a mouse model of Alzheimer’s Disease PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY Recent research regarding amino acid metabolism has shown that there may be a PR:PROJECT_SUMMARY link between obesity and Alzheimer’s Disease (AD). This work reports a PR:PROJECT_SUMMARY metabolomics study using untargeted mass spectrometry-based metabolomic PR:PROJECT_SUMMARY strategies to investigate the metabolic changes that occur in AD and obesity. PR:PROJECT_SUMMARY APPSwe/PS1ΔE9 (APP/PSEN1) transgenic mice (to represent familial or early-onset PR:PROJECT_SUMMARY AD) and wild-type litter mater controls were fed either a high-fat diet (HFD, PR:PROJECT_SUMMARY 60% kcal from lard), low-fat diet (LFD, 10% kcal from lard) from 2 months of PR:PROJECT_SUMMARY age, or reversal diet (REV, high-fat followed by low-fat from 9.5 months). PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME Codreanu PR:FIRST_NAME Simona PR:ADDRESS 3009, Liberty Hills Drive PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 6153477458 #STUDY ST:STUDY_TITLE Untargeted mass spectrometry reveals impact of high fat diet on peripheral amino ST:STUDY_TITLE acid regulation in a mouse model of Alzheimer’s Disease ST:STUDY_SUMMARY APPSwe/PS1ΔE9 (APP/PSEN1) transgenic mice (to represent familial or early-onset ST:STUDY_SUMMARY AD) and wild-type litter mater controls were fed either a high-fat diet (HFD, ST:STUDY_SUMMARY 60% kcal from lard), low-fat diet (LFD, 10% kcal from lard) from 2 months of ST:STUDY_SUMMARY age, or reversal diet (REV, high-fat followed by low-fat from 9.5 months). ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME Codreanu ST:FIRST_NAME Simona ST:ADDRESS 3009, Liberty Hills Drive ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:NUM_GROUPS 6 ST:TOTAL_SUBJECTS 36 ST:NUM_MALES 18 ST:NUM_FEMALES 18 ST:STUDY_TYPE untargeted metabolomics analysis ST:PHONE 6153477458 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN APPSwe/PS1ΔE9 (APP/PSEN1) SU:AGE_OR_AGE_RANGE 12 month SU:GENDER Male and female #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 958_S01 S01 gender:M | genotype:WT | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_LFD_WT_958_S01 SUBJECT_SAMPLE_FACTORS 960_S02 S02 gender:M | genotype:WT | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_LFD_WT_960_S02 SUBJECT_SAMPLE_FACTORS 907_S03 S03 gender:M | genotype:WT | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_LFD_WT_907_S03 SUBJECT_SAMPLE_FACTORS 959_S04 S04 gender:M | genotype:APP | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_LFD_APP_959_S04 SUBJECT_SAMPLE_FACTORS 904_S05 S05 gender:M | genotype:APP | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_LFD_APP_904_S05 SUBJECT_SAMPLE_FACTORS 903_S06 S06 gender:M | genotype:APP | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_LFD_APP_903_S06 SUBJECT_SAMPLE_FACTORS 1092_S07 S07 gender:M | genotype:WT | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_HFD_WT_1092_S07 SUBJECT_SAMPLE_FACTORS 909_S08 S08 gender:M | genotype:WT | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_HFD_WT_909_S08 SUBJECT_SAMPLE_FACTORS 908_S09 S09 gender:M | genotype:WT | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_HFD_WT_908_S09 SUBJECT_SAMPLE_FACTORS 1091_S10 S10 gender:M | genotype:APP | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_HFD_APP_1091_S10 SUBJECT_SAMPLE_FACTORS 1090_S11 S11 gender:M | genotype:APP | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_HFD_APP_1090_S11 SUBJECT_SAMPLE_FACTORS 1162_S12 S12 gender:M | genotype:APP | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_HFD_APP_1162_S12 SUBJECT_SAMPLE_FACTORS 695_S13 S13 gender:M | genotype:WT | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_REV_WT_695_S13 SUBJECT_SAMPLE_FACTORS 696_S14 S14 gender:M | genotype:WT | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_REV_WT_696_S14 SUBJECT_SAMPLE_FACTORS 983_S15 S15 gender:M | genotype:WT | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_REV_WT_983_S15 SUBJECT_SAMPLE_FACTORS 694_S16 S16 gender:M | genotype:APP | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_REV_APP_694_S16 SUBJECT_SAMPLE_FACTORS 697_S17 S17 gender:M | genotype:APP | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_REV_APP_697_S17 SUBJECT_SAMPLE_FACTORS 981_S18 S18 gender:M | genotype:APP | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_M_REV_APP_981_S18 SUBJECT_SAMPLE_FACTORS 961_S19 S19 gender:F | genotype:WT | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_LFD_WT_961_S19 SUBJECT_SAMPLE_FACTORS 962_S20 S20 gender:F | genotype:WT | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_LFD_WT_962_S20 SUBJECT_SAMPLE_FACTORS 964_S21 S21 gender:F | genotype:WT | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_LFD_WT_964_S21 SUBJECT_SAMPLE_FACTORS 963_S22 S22 gender:F | genotype:APP | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_LFD_APP_963_S22 SUBJECT_SAMPLE_FACTORS 965_S23 S23 gender:F | genotype:APP | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_LFD_APP_965_S23 SUBJECT_SAMPLE_FACTORS 984_S24 S24 gender:F | genotype:APP | treatment:LFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_LFD_APP_984_S24 SUBJECT_SAMPLE_FACTORS 749_S25 S25 gender:F | genotype:WT | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_HFD_WT_749_S25 SUBJECT_SAMPLE_FACTORS 753_S26 S26 gender:F | genotype:WT | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_HFD_WT_753_S26 SUBJECT_SAMPLE_FACTORS 1015_S27 S27 gender:F | genotype:WT | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_HFD_WT_1015_S27 SUBJECT_SAMPLE_FACTORS 750_S28 S28 gender:F | genotype:APP | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_HFD_APP_750_S28 SUBJECT_SAMPLE_FACTORS 751_S29 S29 gender:F | genotype:APP | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_HFD_APP_751_S29 SUBJECT_SAMPLE_FACTORS 752_S30 S30 gender:F | genotype:APP | treatment:HFD RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_HFD_APP_752_S30 SUBJECT_SAMPLE_FACTORS 691_S31 S31 gender:F | genotype:WT | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_REV_WT_691_S31 SUBJECT_SAMPLE_FACTORS 692_S32 S32 gender:F | genotype:WT | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_REV_WT_692_S32 SUBJECT_SAMPLE_FACTORS 693_S33 S33 gender:F | genotype:WT | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_REV_WT_693_S33 SUBJECT_SAMPLE_FACTORS 954_S34 S34 gender:F | genotype:APP | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_REV_APP_954_S34 SUBJECT_SAMPLE_FACTORS 956_S35 S35 gender:F | genotype:APP | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_REV_APP_956_S35 SUBJECT_SAMPLE_FACTORS 877_S36 S36 gender:F | genotype:APP | treatment:REV RAW_FILE_NAME=SC_20190826_RPLCp_FMS_Liv_F_REV_APP_877_S36 #COLLECTION CO:COLLECTION_SUMMARY Liver samples from male and female APPSwe/PS1ΔE9 (APP/PSEN1) transgenic mice CO:COLLECTION_SUMMARY and wild-type (WT) littermates were collected after sacrifice at 12 months. CO:COLLECTION_SUMMARY These mice are a subset of mice for which behavioral and biochemical data have CO:COLLECTION_SUMMARY already been published, including weight change and diabetic state as assessed CO:COLLECTION_SUMMARY by glucose tolerance tests.After sacrifice, liver tissue samples were taken and CO:COLLECTION_SUMMARY put immediately on dry ice and stored at −80°C until used in the analytical CO:COLLECTION_SUMMARY workflow described herein. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Briefly, at 2-months of age, standard lab chow (Purina 5001, 4% kcal/fat) was TR:TREATMENT_SUMMARY replaced with either HFD in which 60% kcal are derived from lard, 20% from TR:TREATMENT_SUMMARY carbohydrate and 20% from protein, or low-fat diet (LFD) control in which 10% TR:TREATMENT_SUMMARY kcal are derived from lard and additional calories are derived from corn starch, TR:TREATMENT_SUMMARY with 70% from carbohydrates and 20% from protein.33 For the reversal group TR:TREATMENT_SUMMARY (REV), a subset of mice on the HFD was provided with a LFD, at 9.5 months of TR:TREATMENT_SUMMARY age, for the study duration.33 All other mice were maintained on their original TR:TREATMENT_SUMMARY experimental diets until the end of the study, which was an additional 10 weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For liver samples, frozen tissues were first individually pulverized using a SP:SAMPLEPREP_SUMMARY mortar and pestle followed by lysing in 1 mL ice-cold lysis buffer ((1:1:2, SP:SAMPLEPREP_SUMMARY ACN:MeOH:ammonium bicarbonate (0.1 M, pH 8.0) (LC-MS grade)). Individual samples SP:SAMPLEPREP_SUMMARY were sonicated using a probe tip sonicator, 10 pulses, at 30% power, and cooled SP:SAMPLEPREP_SUMMARY on ice between samples. A bicinchoninic acid (BCA) protein assay was used to SP:SAMPLEPREP_SUMMARY determine the protein concentration for each individual sample and adjusted to a SP:SAMPLEPREP_SUMMARY total amount of protein of 200 µg. For untargeted MS studies, SIL-ISs biotin-D2 SP:SAMPLEPREP_SUMMARY and phenylalanine-D8 were added to each sample to assess sample processing steps SP:SAMPLEPREP_SUMMARY (metabolite extraction and reconstitution). Following lysis and addition of SP:SAMPLEPREP_SUMMARY SIL-ISs, protein precipitation was performed by adding 800 µL of ice-cold SP:SAMPLEPREP_SUMMARY methanol (4x by volume). Samples were incubated at -80°C overnight. Following SP:SAMPLEPREP_SUMMARY incubation, samples were centrifuged at 10,000 rpm for 10 min. The supernatant SP:SAMPLEPREP_SUMMARY containing metabolites were dried in vacuo and metabolite extracts were stored SP:SAMPLEPREP_SUMMARY frozen at -80 °C until ready to use. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Following lysis and standard addition, protein precipitation was performed by SP:EXTRACTION_METHOD adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at SP:EXTRACTION_METHOD -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm SP:EXTRACTION_METHOD for 10 min to eliminate proteins. The supernatants containing metabolites were SP:EXTRACTION_METHOD dried via speed-vacuum. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Hypersil Gold Hypersil Gold, 1.9 mm, 2.1 mm x 100 mm CH:FLOW_GRADIENT 0.250 CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 100% water, 0.1% Formic Acid CH:SOLVENT_B 100% acetonitrile, 0.1% Formic Acid CH:INJECTION_TEMPERATURE 8 CH:SAMPLE_INJECTION 5 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS XCalibur software for data acquisition Progenesis QI 2.0 for data processing MS:MS_RESULTS_FILE ST001848_AN002992_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END