#METABOLOMICS WORKBENCH kpaglia_20200813_090858 DATATRACK_ID:2118 STUDY_ID:ST001452 ANALYSIS_ID:AN002427 PROJECT_ID:PR000998 VERSION 1 CREATED_ON August 25, 2020, 5:39 pm #PROJECT PR:PROJECT_TITLE Targeted metabolomic analysis on hexosamine biosynthetic pathway in flies on PR:PROJECT_TITLE time restricted feeding PR:PROJECT_TYPE Targeted analysis by HILIC PR:PROJECT_SUMMARY The integration of circadian and metabolic signals is essential for maintaining PR:PROJECT_SUMMARY robust circadian rhythms and ensuring efficient metabolism and energy use. Using PR:PROJECT_SUMMARY Drosophila as an animal model, we showed observed strong correlation between PR:PROJECT_SUMMARY daily daily rhythms of protein O- linked N-acetylglucosaminylation PR:PROJECT_SUMMARY (O-GlcNAcylation) and clock-controlled feeding-fasting cycles, suggesting that PR:PROJECT_SUMMARY O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, PR:PROJECT_SUMMARY daily O-GlcNAcylation rhythms were severely dampened when we subjected flies to PR:PROJECT_SUMMARY time- restricted feeding (TRF) at unnatural feeding time. This suggests the PR:PROJECT_SUMMARY presence of a clock- regulated buffering mechanism that prevents excessive PR:PROJECT_SUMMARY O-GlcNAcylation at non-optimal times of the day-night cycle, which could disrupt PR:PROJECT_SUMMARY circadian health. We performed targeted metabolomic analysis on hexosamine PR:PROJECT_SUMMARY biosynthetic pathway (HBP), which produces UDP-GlcNAc (the substrate for PR:PROJECT_SUMMARY O-GlcNAcylation), to evaluate the daily activity of HBP enzymes under TRF PR:PROJECT_SUMMARY conditions. We found glutamine--fructose-6-phosphate amidotransferase (GFAT) PR:PROJECT_SUMMARY mediates this buffering mechanism. PR:INSTITUTE University of California, Davis PR:DEPARTMENT Department of Entomology and Nematology PR:LABORATORY Chiu Lab PR:LAST_NAME Chiu PR:FIRST_NAME Joanna PR:ADDRESS One Shield Ave, Davis CA 95695 PR:EMAIL jcchiu@ucdavis.edu PR:PHONE 5307521839 PR:FUNDING_SOURCE National Institutes of Health grants R01 GM102225 and R01 DK124068 to JCC #STUDY ST:STUDY_TITLE Targeted metabolomic analysis on hexosamine biosynthetic pathway in flies on ST:STUDY_TITLE time restricted feeding ST:STUDY_TYPE metabolomic identification ST:STUDY_SUMMARY The integration of circadian and metabolic signals is essential for maintaining ST:STUDY_SUMMARY robust circadian rhythms and ensuring efficient metabolism and energy use. Using ST:STUDY_SUMMARY Drosophila as an animal model, we showed observed strong correlation between ST:STUDY_SUMMARY daily daily rhythms of protein O-linked N-acetylglucosaminylation ST:STUDY_SUMMARY (O-GlcNAcylation) and clock-controlled feeding-fasting cycles, suggesting that ST:STUDY_SUMMARY O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, ST:STUDY_SUMMARY daily O-GlcNAcylation rhythms were severely dampened when we subjected flies to ST:STUDY_SUMMARY time-restricted feeding (TRF) at unnatural feeding time. This suggests the ST:STUDY_SUMMARY presence of a clock-regulated buffering mechanism that prevents excessive ST:STUDY_SUMMARY O-GlcNAcylation at non-optimal times of the day-night cycle, which could disrupt ST:STUDY_SUMMARY circadian health. We performed targeted metabolomic analysis on hexosamine ST:STUDY_SUMMARY biosynthetic pathway (HBP), which produces UDP-GlcNAc (the substrate for ST:STUDY_SUMMARY O-GlcNAcylation), to evaluate the daily activity of HBP enzymes under TRF ST:STUDY_SUMMARY conditions. We found glutamine--fructose-6-phosphate amidotransferase (GFAT) ST:STUDY_SUMMARY mediates this buffering mechanism. ST:INSTITUTE University of California, Davis ST:DEPARTMENT Department of Entomology and Nematology ST:LABORATORY Chiu lab ST:LAST_NAME Chiu ST:FIRST_NAME Joanna ST:ADDRESS One Shield Ave, Davis CA 95695 ST:EMAIL jcchiu@ucdavis.edu ST:PHONE (530) 752-1839 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 72 #SUBJECT SU:SUBJECT_TYPE Insect SU:SUBJECT_SPECIES Drosophila melanogaster SU:TAXONOMY_ID 7227 #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 - ZT4 - Rep1_001 Treatment:RF21-3 RAW_FILE_NAME=Liu001_MX451075_negHILIC_ZT24-Rep5-035.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep2_002 Treatment:RF21-3 RAW_FILE_NAME=Liu002_MX451075_negHILIC_ZT4-Rep4-040.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep3_003 Treatment:RF21-3 RAW_FILE_NAME=Liu003_MX451075_negHILIC_ZT8-Rep5-011.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep4_004 Treatment:RF21-3 RAW_FILE_NAME=Liu004_MX451075_negHILIC_ZT20-Rep5-029.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep5_005 Treatment:RF21-3 RAW_FILE_NAME=Liu005_MX451075_negHILIC_ZT12-Rep1-013.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep6_006 Treatment:RF21-3 RAW_FILE_NAME=Liu006_MX451075_negHILIC_ZT8-Rep1-007.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep1_007 Treatment:RF21-3 RAW_FILE_NAME=Liu007_MX451075_negHILIC_ZT16-Rep1-055.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep2_008 Treatment:RF21-3 RAW_FILE_NAME=Liu008_MX451075_negHILIC_ZT12-Rep5-017.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep3_009 Treatment:RF21-3 RAW_FILE_NAME=Liu009_MX451075_negHILIC_ZT4-Rep1-037.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep4_010 Treatment:RF21-3 RAW_FILE_NAME=Liu010_MX451075_negHILIC_ZT4-Rep5-041.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep5_011 Treatment:RF21-3 RAW_FILE_NAME=Liu011_MX451075_negHILIC_ZT16-Rep3-057.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep6_012 Treatment:RF21-3 RAW_FILE_NAME=Liu013_MX451075_negHILIC_ZT4-Rep6-042.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep1_013 Treatment:RF21-3 RAW_FILE_NAME=Liu014_MX451075_negHILIC_ZT16-Rep6-060.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep2_014 Treatment:RF21-3 RAW_FILE_NAME=Liu015_MX451075_negHILIC_ZT16-Rep2-056.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep3_015 Treatment:RF21-3 RAW_FILE_NAME=Liu016_MX451075_negHILIC_ZT8-Rep6-012.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep4_016 Treatment:RF21-3 RAW_FILE_NAME=Liu017_MX451075_negHILIC_ZT20-Rep2-026.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep5_017 Treatment:RF21-3 RAW_FILE_NAME=Liu018_MX451075_negHILIC_ZT20-Rep5-065.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep6_018 Treatment:RF21-3 RAW_FILE_NAME=Liu019_MX451075_negHILIC_ZT12-Rep4-052.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep1_019 Treatment:RF21-3 RAW_FILE_NAME=Liu020_MX451075_negHILIC_ZT4-Rep3-003.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep2_020 Treatment:RF21-3 RAW_FILE_NAME=Liu021_MX451075_negHILIC_ZT20-Rep4-064.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep3_021 Treatment:RF21-3 RAW_FILE_NAME=Liu022_MX451075_negHILIC_ZT12-Rep6-018.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep4_022 Treatment:RF21-3 RAW_FILE_NAME=Liu023_MX451075_negHILIC_ZT12-Rep2-050.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep5_023 Treatment:RF21-3 RAW_FILE_NAME=Liu024_MX451075_negHILIC_ZT4-Rep2-038.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep6_024 Treatment:RF21-3 RAW_FILE_NAME=Liu025_MX451075_negHILIC_ZT8-Rep3-009.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep1_025 Treatment:RF21-3 RAW_FILE_NAME=Liu026_MX451075_negHILIC_ZT16-Rep4-058.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep2_026 Treatment:RF21-3 RAW_FILE_NAME=Liu027_MX451075_negHILIC_ZT24-Rep2-032.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep3_027 Treatment:RF21-3 RAW_FILE_NAME=Liu028_MX451075_negHILIC_ZT16-Rep2-020.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep4_028 Treatment:RF21-3 RAW_FILE_NAME=Liu029_MX451075_negHILIC_ZT16-Rep4-022.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep5_029 Treatment:RF21-3 RAW_FILE_NAME=Liu030_MX451075_negHILIC_ZT16-Rep6-024.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep6_030 Treatment:RF21-3 RAW_FILE_NAME=Liu031_MX451075_negHILIC_ZT24-Rep3-033.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep1_031 Treatment:RF21-3 RAW_FILE_NAME=Liu032_MX451075_negHILIC_ZT24-Rep1-031.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep2_032 Treatment:RF21-3 RAW_FILE_NAME=Liu033_MX451075_negHILIC_ZT20-Rep6-030.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep3_033 Treatment:RF21-3 RAW_FILE_NAME=Liu034_MX451075_negHILIC_ZT4-Rep1-001.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep4_034 Treatment:RF21-3 RAW_FILE_NAME=Liu035_MX451075_negHILIC_ZT4-Rep4-004.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep5_035 Treatment:RF21-3 RAW_FILE_NAME=Liu036_MX451075_negHILIC_ZT4-Rep2-002.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep6_036 Treatment:RF21-3 RAW_FILE_NAME=Liu037_MX451075_negHILIC_ZT24-Rep1-067.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep1_037 Treatment:RF9-15 RAW_FILE_NAME=Liu038_MX451075_negHILIC_ZT16-Rep5-059.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep2_038 Treatment:RF9-15 RAW_FILE_NAME=Liu039_MX451075_negHILIC_ZT8-Rep6-048.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep3_039 Treatment:RF9-15 RAW_FILE_NAME=Liu040_MX451075_negHILIC_ZT16-Rep1-019.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep4_040 Treatment:RF9-15 RAW_FILE_NAME=Liu041_MX451075_negHILIC_ZT20-Rep6-066.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep5_041 Treatment:RF9-15 RAW_FILE_NAME=Liu042_MX451075_negHILIC_ZT12-Rep1-049.wiff SUBJECT_SAMPLE_FACTORS - ZT4 - Rep6_042 Treatment:RF9-15 RAW_FILE_NAME=Liu043_MX451075_negHILIC_ZT24-Rep3-069.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep1_043 Treatment:RF9-15 RAW_FILE_NAME=Liu044_MX451075_negHILIC_ZT20-Rep1-061.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep2_044 Treatment:RF9-15 RAW_FILE_NAME=Liu045_MX451075_negHILIC_ZT12-Rep5-053.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep3_045 Treatment:RF9-15 RAW_FILE_NAME=Liu046_MX451075_negHILIC_ZT8-Rep4-010.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep4_046 Treatment:RF9-15 RAW_FILE_NAME=Liu047_MX451075_negHILIC_ZT8-Rep5-047.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep5_047 Treatment:RF9-15 RAW_FILE_NAME=Liu048_MX451075_negHILIC_ZT12-Rep3-051.wiff SUBJECT_SAMPLE_FACTORS - ZT8 - Rep6_048 Treatment:RF9-15 RAW_FILE_NAME=Liu049_MX451075_negHILIC_ZT16-Rep5-023.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep1_049 Treatment:RF9-15 RAW_FILE_NAME=Liu050_MX451075_negHILIC_ZT24-Rep2-068.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep2_050 Treatment:RF9-15 RAW_FILE_NAME=Liu051_MX451075_negHILIC_ZT12-Rep6-054.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep3_051 Treatment:RF9-15 RAW_FILE_NAME=Liu053_MX451075_negHILIC_ZT24-Rep6-036.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep4_052 Treatment:RF9-15 RAW_FILE_NAME=Liu054_MX451075_negHILIC_ZT16-Rep3-021.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep5_053 Treatment:RF9-15 RAW_FILE_NAME=Liu055_MX451075_negHILIC_ZT24-Rep4-070.wiff SUBJECT_SAMPLE_FACTORS - ZT12 - Rep6_054 Treatment:RF9-15 RAW_FILE_NAME=Liu056_MX451075_negHILIC_ZT12-Rep2-014.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep1_055 Treatment:RF9-15 RAW_FILE_NAME=Liu057_MX451075_negHILIC_ZT8-Rep3-045.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep2_056 Treatment:RF9-15 RAW_FILE_NAME=Liu058_MX451075_negHILIC_ZT20-Rep1-025.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep3_057 Treatment:RF9-15 RAW_FILE_NAME=Liu059_MX451075_negHILIC_ZT4-Rep3-039.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep4_058 Treatment:RF9-15 RAW_FILE_NAME=Liu060_MX451075_negHILIC_ZT24-Rep6-072_2.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep5_059 Treatment:RF9-15 RAW_FILE_NAME=Liu061_MX451075_negHILIC_ZT8-Rep2-044.wiff SUBJECT_SAMPLE_FACTORS - ZT16 - Rep6_060 Treatment:RF9-15 RAW_FILE_NAME=Liu062_MX451075_negHILIC_ZT12-Rep4-016.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep1_061 Treatment:RF9-15 RAW_FILE_NAME=Liu063_MX451075_negHILIC_ZT20-Rep3-027.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep2_062 Treatment:RF9-15 RAW_FILE_NAME=Liu064_MX451075_negHILIC_ZT12-Rep3-015.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep3_063 Treatment:RF9-15 RAW_FILE_NAME=Liu065_MX451075_negHILIC_ZT4-Rep5-005.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep4_064 Treatment:RF9-15 RAW_FILE_NAME=Liu066_MX451075_negHILIC_ZT4-Rep6-006.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep5_065 Treatment:RF9-15 RAW_FILE_NAME=Liu067_MX451075_negHILIC_ZT8-Rep1-043.wiff SUBJECT_SAMPLE_FACTORS - ZT20 - Rep6_066 Treatment:RF9-15 RAW_FILE_NAME=Liu068_MX451075_negHILIC_ZT20-Rep3-063.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep1_067 Treatment:RF9-15 RAW_FILE_NAME=Liu069_MX451075_negHILIC_ZT24-Rep5-071.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep2_068 Treatment:RF9-15 RAW_FILE_NAME=Liu070_MX451075_negHILIC_ZT8-Rep4-046_2.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep3_069 Treatment:RF9-15 RAW_FILE_NAME=Liu071_MX451075_negHILIC_ZT24-Rep4-034.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep4_070 Treatment:RF9-15 RAW_FILE_NAME=Liu072_MX451075_negHILIC_ZT20-Rep2-062.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep5_071 Treatment:RF9-15 RAW_FILE_NAME=Liu073_MX451075_negHILIC_ZT20-Rep4-028.wiff SUBJECT_SAMPLE_FACTORS - ZT24 - Rep6_072 Treatment:RF9-15 RAW_FILE_NAME=Liu074_MX451075_negHILIC_ZT8-Rep2-008-2.wiff #COLLECTION CO:COLLECTION_SUMMARY Flies were flash frozen on dry ice. Bodies were separated from heads and CO:COLLECTION_SUMMARY weighed. CO:SAMPLE_TYPE Fly #TREATMENT TR:TREATMENT_SUMMARY 2 treatments X 6 time points each, 6 replicates Treatment #1: Natural feeding TR:TREATMENT_SUMMARY time (ZT21-3) with 6 time points over the circadian day (ZT 4, 8, 12, 16, 20, TR:TREATMENT_SUMMARY 24) Treatment #2: Unnatural feeding time (ZT9-15) with 6 time points over the TR:TREATMENT_SUMMARY circadian day (ZT 4, 8, 12, 16, 20, 24) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sample Preparation: 1. Mass out 10 mg. 2. Add 1 mL 5:2:2 meOH:CHCl3:H2O SP:SAMPLEPREP_SUMMARY (vol/vol/vol). 3. Genogrind 30 s at 1500 rpm with 2 3 mm stainless steel balls. SP:SAMPLEPREP_SUMMARY 4. Shake 5 min at 4oC. 5. Centrifuge 2 min at 14,000 rcf. 6. Aliquot 2 x 450 uL SP:SAMPLEPREP_SUMMARY supernatant. 7. Pool remaining supernatant. 8. Dry primary aliquots completely SP:SAMPLEPREP_SUMMARY in centrivap. Store backups at -20oC #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Targeted HILIC MS/MS CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME AB Sciex CH:COLUMN_NAME Waters Acquity BEH Amide (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 6500 QTrap MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS HILIC Biogenic Amines analysis by UPLC-QTOF mass spectrometry West Coast MS:MS_COMMENTS Metabolomics Center Genome Center, University of California, Davis, CA, USA MS:MS_COMMENTS Contents Page 1 Instruments Page 1 Page 1 Chemical and Consumables Procedures MS:MS_COMMENTS Page 13 Problems Page 13 Disposal of waste 1. Instruments: ● Agilent 1290 MS:MS_COMMENTS UHPLC-6530-QTOF ● Agilent 1290 UHPLC-6550-QTOF ● Pipettes calibrated MS:MS_COMMENTS following SOP006_2003 ● Ultrasonicator 2. Chemicals and consumables ● Waters MS:MS_COMMENTS Acquity UPLC BEH Amide 2.1x150mm 1.7 μm Column ● Waters Acquity UPLC BEH MS:MS_COMMENTS Amide 1.7 μm Pre-column ● Pipettes calibrated following SOP006_2003 ● MS:MS_COMMENTS Ultrasonicator ● Agilent Tune Mix: G1969-85000 ● Acetonitrile: J.T. Baker MS:MS_COMMENTS LC/MS Grade, 4 L (9829-03) ● Formic Acid: Fluka Mass Spec Grade MS:MS_COMMENTS (94318-250mL-F) ● Ammonium Formate: Fluka, Mass Spec Grade (70221-25G-F) ● MS:MS_COMMENTS Agilent 0.17ID (green) metal tubing: 90 cm 5065-9963 and 20 cm (5065-9931) ● MS:MS_COMMENTS Red Agilent Peek Tubing 5 meters (0.13 ID) (5042-6461) ● Plastic Agilent MS:MS_COMMENTS Connectors (for peek tubing) (0100-1516) ● Stainless Steel Agilent Fitting MS:MS_COMMENTS (5062-2418) 3. Procedure: 3.1 Pre-Run Procedures 3.1.1 Instrument tuning MS:MS_COMMENTS (Instrument in Tune mode) a. Use “Standard Tune” before each run of 300 MS:MS_COMMENTS sample batch. b. Use the “Tuning Solution” (see preparation of solutions MS:MS_COMMENTS below) for the instrument tuning. c. Print the tune report from the standard MS:MS_COMMENTS tune. Rev February 2018 - In ESI(+), check the profile of the calibrant and the MS:MS_COMMENTS intensity of ions m/z 322.0481; m/z 622.0290; and m/z 922.0098, which must be MS:MS_COMMENTS higher than 400k, 500k, and 500k, respectively. d. If the intensity of even one MS:MS_COMMENTS of the selected ions is below this value clean the ion source and repeat the MS:MS_COMMENTS instrument tuning. 3.1.2 Check Reference ions (Instrument in Acquisition mode) MS:MS_COMMENTS a. Use the “Reference Ion Mass Solution” (see preparation of solutions MS:MS_COMMENTS below) for mass correction during the analyses (lock mass). b. Check the MS:MS_COMMENTS following reference ions: - In ESI(+), check the intensity of ions m/z 121.0509 MS:MS_COMMENTS and m/z 980.0164, which should be between 5-20k. Adjust recipe and flow rates to MS:MS_COMMENTS attain this intensity. 3.2 New column installation a. Purge the pumping system MS:MS_COMMENTS of any old buffers and connect the inlet of the column to the injector outlet. MS:MS_COMMENTS Attach the outlet line, but allow flow to go into a beaker instead of to the ion MS:MS_COMMENTS source of mass spectrometer. b. Flush column with 100% Mobile Phase B (see MS:MS_COMMENTS preparation of solution below) with a pump flow rate at 0.4mL/min over 30 MS:MS_COMMENTS minutes. c. Switch to 70% mobile phase A and flush the column for 30 minutes. d. MS:MS_COMMENTS When the mobile phase is flowing freely from the column outlet, stop the flow MS:MS_COMMENTS and attach the column outlet to the ion source of mass spectrometer. e. Switch MS:MS_COMMENTS back to 100% Mobile Phase B and monitor the back pressure until a steady value MS:MS_COMMENTS is achieved. Expected values are between 220-250 bar at the start of the MS:MS_COMMENTS injection. f. Perform 6 "No Injections" and monitor the backpressure on the MS:MS_COMMENTS first and last sample injected. Rev February 2018 NOTE: Use a new column after MS:MS_COMMENTS ~1000 sample injections. The UPLC column must be coupled to a VanGuard MS:MS_COMMENTS pre-column. The VanGuard pre-column is replaced after ~330 sample injections. MS:MS_COMMENTS The number of injections (both solvents and plasma samples) is recorded by an MS:MS_COMMENTS operator in a folder created for each acquisition. 3.3 Preparation of solutions MS:MS_COMMENTS a. Preparation of Tuning Solution ● 88.5 mL acetonitrile ● 1.5 mL H2O ● 10 MS:MS_COMMENTS mL Agilent Low Concentration ESI Tuning Mix ● 5 μL 322 Reference Ion MS:MS_COMMENTS (sonicate before use) ● Degas by sonication for 5 min b. Preparation of MS:MS_COMMENTS Reference Mass Solution ● 95 mL acetonitrile ● 5 mL H2O ● 200 μL 5 mM 921 MS:MS_COMMENTS Reference Ion (sonicate before use) ● 250 μL 10 mM Purine Reference Ion MS:MS_COMMENTS (sonicate before use) ● Degas by sonication for 5 min c. Mobile phase A MS:MS_COMMENTS Positive Mode (100% H2O + 10 mM Ammonium Formate + 0.125% Formic Acid) 1. MS:MS_COMMENTS Pre-rinse three times 1 L glass bottle with pure acetonitrile and water 2. MS:MS_COMMENTS Measure exactly 1000 mL of HPLC/UHPLC MS grade H2O in a pre-rinsed graduated MS:MS_COMMENTS cylinder and add them to the 1 L glass bottle. 3. Add 1.250 mL formic acid 4. MS:MS_COMMENTS Weight 0.630 g of ammonium formate and add them to the glass bottle. Swirl to MS:MS_COMMENTS dissolve ammonium formate. 5. Sonicate for 10 min at room temperature until all MS:MS_COMMENTS the ammonium formate is dissolved. 6. 1 L will last for around 200 samples d. MS:MS_COMMENTS Mobile phase B Positive Mode (95:5 ACN:H2O + 10 mM Ammonium Formate + 0.125% MS:MS_COMMENTS Formic Acid) 1. Pre-rinse three times 1 L glass bottle with pure acetonitrile MS:MS_COMMENTS and water 2. Measure exactly 50 mL of HPLC/UHPLC MS grade H2O in a pre-rinsed MS:MS_COMMENTS graduated cylinder and add them to the 1 L glass bottle. 3. Add 1.250 mL formic MS:MS_COMMENTS acid 4. Weight 0.630 g of ammonium formate and add them to the glass bottle. MS:MS_COMMENTS Swirl to dissolve ammonium formate. 5. Measure exactly 950 mL of HPLC/UHPLC MS MS:MS_COMMENTS grade ACN in a pre-rinsed graduated cylinder and add them to the 1 L glass MS:MS_COMMENTS bottle. 6. Sonicate for 10 min at room temperature until all the ammonium MS:MS_COMMENTS formate is dissolved. This step may require multiple 10 minute sonication steps MS:MS_COMMENTS and swirling until solution is clear. 7. 1 L will last for around 200 samples MS:MS_COMMENTS Rev February 2018 e. Needle wash solution (50:50 ACN/H2O) 1. Pre-rinse three MS:MS_COMMENTS times 1 L glass bottle with pure acetonitrile and water 2. Measure exactly 500 MS:MS_COMMENTS mL of HPLC/UHPLC MS grade H2O in a pre-rinsed graduated cylinder and add them to MS:MS_COMMENTS the 1 L glass bottle. 3. Measure exactly 500 mL of HPLC/UHPLC MS grade ACN in a MS:MS_COMMENTS pre-rinsed graduated cylinder and add them to the 1 L glass bottle. 4. Sonicate MS:MS_COMMENTS for 10 min at room temperature. f. Resuspension solvent preparation for MS:MS_COMMENTS injection into HILIC (See Table 1) 1. Weigh out predetermined amount of internal MS:MS_COMMENTS standards (See supplemental SOP for internal standard resuspension solvent). 2. MS:MS_COMMENTS Add 10 mL of the appropriate solvent for each internal standard, either 80:20 MS:MS_COMMENTS acetonitrile:water, 50:50 acetonitrile:water, 100% water, or 100% methanol. This MS:MS_COMMENTS will depend on compound solubility. 3. Next add 14 mL of water and 63 mL of MS:MS_COMMENTS acetonitrile to a clean 100 mL beaker. 4. Aliquot the predetermined volumes of MS:MS_COMMENTS each internal standard (see supplemental SOP for internal standard resuspension MS:MS_COMMENTS solvent) to the same 100 mL beaker to bring the final volume of the solution to MS:MS_COMMENTS 80 mL. 5. Sonicate for 10 min. 6. Store at 4°C g. Preparation of samples for MS:MS_COMMENTS analysis 1. Resuspend samples in 60 – 200 μL of HILIC-resuspension solvent MS:MS_COMMENTS depending on sample concentration/matrix. 2. We typically use 100 μL for 1⁄2 MS:MS_COMMENTS of 5 mg of tissue and 1⁄2 of 5-10 million cells, but this can vary depending MS:MS_COMMENTS on tissue type and instrument sensitivity. Plasma is typically resuspended in MS:MS_COMMENTS 100 μL. 3. Vortex sample for 10 seconds. 4. Sonicate samples for 5 minutes. 5. MS:MS_COMMENTS Centrifuge for 2 min at 16,100 rcf. 6. Transfer supernatant to LC-MS micro MS:MS_COMMENTS insert in an amber vial. 7. Cap and load into auto sampler 3.4 Pre-run sequence MS:MS_COMMENTS a. Before starting the run inject the following: 1. 3 x "No sample Injection" 2. MS:MS_COMMENTS 2 x Blank sample injection (Resuspension Solvent) 3.5 Run sequence a. After MS:MS_COMMENTS running the pre-run sequence, inject the following: 1. Method Blank 2. Biorec MS:MS_COMMENTS Rev February 2018 3. 10 samples 4. repeat Table 1 Analytes of the MS:MS_COMMENTS Resuspension-mix solution Common Name Formula MS1 m/z RT (min) D9-Caffeine MS:MS_COMMENTS C8D9HN4O2 204.1441 1.26 CUDA C19H36N2O3 341.2799 1.23 D3-Creatinine C4D3H4N3O MS:MS_COMMENTS 117.0850 4.80 D9-Choline C5D9H5NO 113.1635 5.30 D9-TMAO C3D9NO 85.1322 5.71 MS:MS_COMMENTS D3-1-Methylnicotinamide C7H6D3N2O 141.0976 6.26 Val-Tyr-Val C19H29N3O5 380.2180 MS:MS_COMMENTS 6.95 D9-Betaine C5H2D9NO2 127.1427 7.24 D3-AC(2:0) C9D3H14NO4 207.1419 7.28 MS:MS_COMMENTS D3-Histamine, N-methyl- C6H8D3N3 129.1214 7.34 D3-L-Carnitine C7H12D3NO3 MS:MS_COMMENTS 165.1313 7.70 D9-Butyrobetaine C7H6D9NO2 155.1740 7.70 D9-Crotonobetaine MS:MS_COMMENTS C7H4D9NO2 153.1584 7.78 D3-Creatine C4D3H6O2N3 135.0956 8.03 D3-DL-Alanine MS:MS_COMMENTS C3H4D3NO2 93.0738 8.05 D5-L-Glutamine C5H5D5N2O3 152.1078 8.53 D3-DL-Glutamic MS:MS_COMMENTS acid C5H6D3NO4 151.0793 8.72 D3-DL-Aspartic acid C4H4D3NO4 137.0636 9.24 MS:MS_COMMENTS 15N2-L-Arginine [15]N2C6H14N2O2 177.1130 9.45 NOTE: The backpressure should be MS:MS_COMMENTS within the range 220-250 bar at the beginning of each run [elution at 100% of MS:MS_COMMENTS the mobile phase (B)] and should not exceed the range 575-650 bar [elution at MS:MS_COMMENTS 70% of the mobile phase (A)]. NOTE: If the initial backpressure is higher than MS:MS_COMMENTS 220-250 bar, switch LC flow to “Bypass” if pressure decreases change the MS:MS_COMMENTS Needle Seat and Seat Capillary. If pressure does not decrease, Rev February 2018 MS:MS_COMMENTS change the Rotor Seal and/or Sample Needle. If the initial backpressure is still MS:MS_COMMENTS high then replace the VanGuard pre-column. If pressure is still high replace MS:MS_COMMENTS Column. 3.6 HILIC analysis method a. The autosampler, separation and column MS:MS_COMMENTS parameters for the HILIC analysis method are as shown below: Rev February 2018 - MS:MS_COMMENTS Binary Pump Parameters: - Column manager Rev February 2018 The MS conditions are MS:MS_COMMENTS the following: 3.6.1 Positive ion mode MS1 - General parameters - Source MS:MS_COMMENTS parameters Rev February 2018 - Acquisition parameters: - Ref Mass parameters Rev MS:MS_COMMENTS February 2018 - Chromatogram parameters: 3.6.2 Positive ion mode MS/MS MS:MS_COMMENTS -Acquisition Rev February 2018 -Collision Energy -Precursor Selection I Note: 4 MS:MS_COMMENTS methods with different static exclusion ranges in Precursor Selection I tab are MS:MS_COMMENTS used. 1. Range 50-150 m/z (static exclusion start m/z 155, static exclusion end MS:MS_COMMENTS m/z 1700) 2. Range 150-300 m/z (static exclusion start m/z 50, static exclusion MS:MS_COMMENTS end m/z 145, static exclusion start m/z 305, static exclusion end m/z 1700) 3. MS:MS_COMMENTS Range 300-500 m/z (static exclusion start m/z 50, static exclusion end m/z 295, MS:MS_COMMENTS static exclusion start m/z 505, static exclusion end m/z 1700) 4. Range 500-1200 MS:MS_COMMENTS m/z (static exclusion start m/z 50, static exclusion end m/z 495, static MS:MS_COMMENTS exclusion start m/z 1205, static exclusion end m/z 1700) Rev February 2018 MS:MS_COMMENTS -Precursor Selection II -Preferred/Exclude List 3.7 Column Storage Use this MS:MS_COMMENTS procedure to avoid precipitation mobile-phase buffers on the column and in the MS:MS_COMMENTS system. a. Flush column with 50% acetonitrile by setting the pump flow rate to MS:MS_COMMENTS 0.1 mL/min and increase the flow rate to 0.4 mL/min over 5 min; keep the column MS:MS_COMMENTS at this flow rate for 10 min. b. Flush column with 95% acetonitrile 5% H2O by MS:MS_COMMENTS setting the pump flow rate to 0.1 mL/min and increase the flow rate to 0.4 MS:MS_COMMENTS mL/min over 5 min; keep he column at this flow rate for 10 min. c. Remove the MS:MS_COMMENTS column from the system. d. Store the column in the box until the next batch MS:MS_COMMENTS analysis. Add the story usage of the column. Rev February 2018 4. Problems In MS:MS_COMMENTS order to avoid cross-contaminations and artifact formation, disposable MS:MS_COMMENTS consumables are used (Eppendorf plastic tubes, plastic pipette tips) 5. Disposal MS:MS_COMMENTS of waste Chemicals are disposed into appropriate bottles in lab 2.157 under the MS:MS_COMMENTS fume hood before monthly disposal collection. Glass vials and consumables are MS:MS_COMMENTS collected into the plastic bags and stored under the fume hood in lab 2.157 MS:MS_COMMENTS before monthly disposal. Other GC-TOF waste (rubber seals, O-rings etc.) can be MS:MS_COMMENTS disposed into regular waste. Rev February 2018 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS normalized peak height MS_METABOLITE_DATA_START Samples ZT4 - Rep1_001 ZT4 - Rep2_002 ZT4 - Rep3_003 ZT4 - Rep4_004 ZT4 - Rep5_005 ZT4 - Rep6_006 ZT8 - Rep1_007 ZT8 - Rep2_008 ZT8 - Rep3_009 ZT8 - Rep4_010 ZT8 - Rep5_011 ZT8 - Rep6_012 ZT12 - Rep1_013 ZT12 - Rep2_014 ZT12 - Rep3_015 ZT12 - Rep4_016 ZT12 - Rep5_017 ZT12 - Rep6_018 ZT16 - Rep1_019 ZT16 - Rep2_020 ZT16 - Rep3_021 ZT16 - Rep4_022 ZT16 - Rep5_023 ZT16 - Rep6_024 ZT20 - Rep1_025 ZT20 - Rep2_026 ZT20 - Rep3_027 ZT20 - Rep4_028 ZT20 - Rep5_029 ZT20 - Rep6_030 ZT24 - Rep1_031 ZT24 - Rep2_032 ZT24 - Rep3_033 ZT24 - Rep4_034 ZT24 - Rep5_035 ZT24 - Rep6_036 ZT4 - Rep1_037 ZT4 - Rep2_038 ZT4 - Rep3_039 ZT4 - Rep4_040 ZT4 - Rep5_041 ZT4 - Rep6_042 ZT8 - Rep1_043 ZT8 - Rep2_044 ZT8 - Rep3_045 ZT8 - Rep4_046 ZT8 - Rep5_047 ZT8 - Rep6_048 ZT12 - Rep1_049 ZT12 - Rep2_050 ZT12 - Rep3_051 ZT12 - Rep4_052 ZT12 - Rep5_053 ZT12 - Rep6_054 ZT16 - Rep1_055 ZT16 - Rep2_056 ZT16 - Rep3_057 ZT16 - Rep4_058 ZT16 - Rep5_059 ZT16 - Rep6_060 ZT20 - Rep1_061 ZT20 - Rep2_062 ZT20 - Rep3_063 ZT20 - Rep4_064 ZT20 - Rep5_065 ZT20 - Rep6_066 ZT24 - Rep1_067 ZT24 - Rep2_068 ZT24 - Rep3_069 ZT24 - Rep4_070 ZT24 - Rep5_071 ZT24 - Rep6_072 Factors Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF21-3 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 Treatment:RF9-15 N-Acetyl-D-glucosamine 6-phosphate 11.87377778 14.04266667 13.82933333 13.92 13.92977778 13.62488889 13.98044444 14.34222222 13.81511111 14.32622222 14.11822222 13.91111111 13.81422222 13.76977778 14.48444444 14.20088889 14.096 13.84355556 14.34666667 13.82666667 13.85866667 14.19377778 13.71022222 13.92977778 14.65777778 13.76622222 14.04266667 11.84533333 14.14222222 14.18933333 13.40444444 11.85688889 11.86044444 11.83733333 11.856 11.83911111 14.37155556 14.66577778 14.44977778 14.66488889 14.48355556 14.656 14.08444444 13.56888889 14.17422222 15.47377778 14.64 14.60977778 11.86044444 11.87377778 11.84622222 11.85155556 11.85511111 11.83111111 14.33866667 13.87022222 13.79822222 14.24177778 13.91822222 14.60444444 14.07555556 14.29066667 14.36 14.21244444 13.46133333 14.26755556 14.33066667 14.56444444 14.33066667 13.79377778 14.44088889 14.44711111 Uridine diphosphate N-acetylglucosamine 55.92224991 55.40389704 52.15172047 53.20494315 53.38213004 50.25899654 54.34714773 61.02538162 53.78731131 53.04655689 54.64023007 52.62053452 56.43673201 53.4987945 58.30785964 58.14987386 57.16443231 55.34137979 51.97165641 50.01701751 53.23644181 52.76695971 53.01041395 50.78555597 56.56375103 47.52272075 55.32053759 57.77950818 52.9801512 55.9908749 45.36719903 51.56599187 49.62985583 50.50628527 48.7140898 49.38266964 54.82027584 56.62101506 54.40654146 52.11462053 53.04193421 53.51054221 50.76014038 50.49229344 53.70850166 60.56938115 54.89668723 54.40152276 49.75783355 48.66331921 53.07345097 46.79879517 47.72225996 48.70214457 50.78470882 50.36506392 49.83561305 51.51994368 52.29459452 53.35736412 56.67204475 54.6857746 55.27509621 54.21995278 49.23408676 54.6941306 56.95274697 54.4073779 53.3493882 51.18936288 54.24255082 55.82240151 Fructose-6-phosphate 123.6955556 109.1644444 111.5733333 113.1244444 109.9666667 95.60444444 111.2911111 130.62 113.96 125.2666667 135.8866667 134.68 143.3555556 140.2711111 141.6266667 141.3133333 147.2733333 147.3733333 108.6844444 109.9511111 131.2244444 118.3 124.0844444 122.3133333 133.3888889 100.1666667 135.5355556 152.0488889 114.4644444 133.7577778 70.05111111 122.6 85.27555556 107.1022222 104.3466667 116.4222222 142.3888889 143.2022222 135.2044444 126.2955556 138.8911111 142.2511111 126.8555556 108.6977778 119.9844444 143.6911111 122.1066667 120.3777778 138.22 118.9022222 129.4577778 122.0066667 123.8177778 120.9311111 141.4777778 169.4888889 152.0555556 176.7 155.4088889 166.4622222 183.6288889 172.2711111 176.8088889 144.4955556 135.56 160.2933333 161.9444444 178.6733333 166.4755556 133.6288889 181.14 164.2222222 Glutamine 31.66966855 31.17744159 30.88474813 30.85480346 30.89877714 30.74327251 31.31990747 31.75064324 31.03679325 31.19125905 31.75480466 31.04256376 31.01939628 31.10302209 31.33732764 31.35614974 31.65557602 30.97843474 30.81287162 30.58944269 30.9535859 30.93757572 30.90824585 30.85415467 31.00339201 30.59454233 31.00570877 31.21678787 30.75215095 31.33419757 31.11687723 30.53357037 30.57414353 30.60229097 30.57115746 30.54222179 31.141901 31.28971014 30.94331304 30.97166596 30.94977544 31.09122913 30.77303322 30.79086333 31.05032893 31.89750782 30.85158624 30.94658775 30.56201708 30.54134507 30.55481516 30.53451831 30.54821148 30.53460141 30.72120306 30.75076409 30.84483413 30.71728488 30.71550921 30.66391475 31.07400329 30.98040212 31.03363348 31.17029277 30.54566987 30.99884882 30.91896051 30.8943497 30.99994792 30.54063768 31.00114621 31.29192094 Glucosamine-6-phosphate 38.22627967 38.0072 37.79601055 37.94345259 37.84781867 37.72426879 39.30456699 39.21710379 37.67244992 37.78405419 40.10703488 37.56082475 39.34034367 37.32554612 37.78006868 37.41328799 38.56071839 37.52095355 38.09484155 37.80398132 37.36941867 37.67244992 37.58474619 37.77209759 38.03907115 37.49702955 38.10280832 37.88368319 39.77743967 37.73224052 37.04229115 38.12272479 37.07421392 37.22582379 41.819552 37.169972 38.50499167 37.50500432 37.69636688 41.07490155 38.39352255 37.72028288 37.69238079 36.81878395 37.65251839 39.63045248 37.61265332 37.71231099 37.53291519 37.58075935 37.827893 37.66447739 37.68839467 37.01036667 38.81938112 38.01516795 37.42923967 37.88368319 38.04703867 38.21831455 38.08687467 37.79202512 38.41741055 37.4093 37.23380219 37.59271979 38.186453 37.73622635 37.83586335 37.63258619 37.94345259 39.25686139 Uridine 5'-diphosphate 342.352 330.1493333 286.5617778 297.1928889 274.5902222 281.6622222 274.0355556 292.848 257.9964444 265.7155556 285.5448889 255.5928889 279.7671111 266.9173333 295.4364444 267.3333333 269.1822222 253.6053333 283.5573333 252.6346667 284.2968889 281.2462222 248.0586667 282.8177778 321.5982222 228.1831111 309.4417778 285.9608889 306.576 335.8346667 177.9395556 253.6053333 249.2142222 229.8471111 223.9306667 227.952 267.1484444 265.3457778 268.5813333 258.9208889 248.1511111 258.5511111 259.7066667 232.944 269.968 315.1733333 261.6017778 266.8248889 226.8888889 231.6497778 246.7182222 210.6186667 210.5724444 239.0915556 241.68 235.7635556 192.176 267.9804444 283.4648889 233.2213333 267.9804444 251.9413333 249.8613333 222.544 213.392 249.0293333 298.5795556 260.9084444 259.8915556 245.424 275.0524444 235.6248889 Glucose-6-phosphate 418.1066667 384.4377778 391.4533333 377.8955556 362.6044444 297.8155556 359.2533333 451.5755556 399.6777778 465.5377778 454.6111111 451.0711111 477.6422222 489.5955556 485.0333333 451.7977778 516.3511111 470.9755556 325.7377778 357.1444444 435.1555556 369.3311111 408.96 403.3488889 448.7911111 312.1977778 478.18 516.1377778 392.2555556 451.4688889 175.62 401.2022222 255.7044444 342.1577778 335.2044444 377.5688889 499.0022222 484.1822222 464.8622222 401.7688889 481.5177778 466.5266667 388.52 336.8488889 403.2777778 494.5955556 432.9488889 416.4822222 478.0888889 405.9733333 458.3266667 419.4533333 412.9822222 418.4022222 512.7244444 624.0733333 522.0688889 635.5155556 551.74 609.3177778 653.0044444 634.2822222 629.0222222 486.1022222 452.7355556 519.6466667 570.1177778 652.9533333 603.0955556 467.3488889 618.1044444 576.5955556 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index m/z InchiKey N-Acetyl-D-glucosamine 6-phosphate 9.15 300.0499 Uridine diphosphate N-acetylglucosamine 9.64 606.076 LFTYTUAZOPRMMI-MPIASZHXSA-N Fructose-6-phosphate 9.27 259.0233 Glutamine 8.35 145.0623 ZDXPYRJPNDTMRX-VKHMYHEASA-N Glucosamine-6-phosphate 9.8 258.0391 XHMJOUIAFHJHBW-UKFBFLRUSA-N Uridine 5'-diphosphate 10.01 402.9932 Glucose-6-phosphate 9.54 259.0222 NBSCHQHZLSJFNQ-GASJEMHNSA-N METABOLITES_END #END