Summary of Study ST001452
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 PR000998. The data can be accessed directly via it's Project DOI: 10.21228/M8PQ57 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001452 |
| Study Title | Targeted metabolomic analysis on hexosamine biosynthetic pathway in flies on time restricted feeding |
| Study Type | metabolomic identification |
| Study Summary | The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we showed observed strong correlation between daily daily rhythms of protein O-linked N-acetylglucosaminylation (O-GlcNAcylation) and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms were severely dampened when we subjected flies to time-restricted feeding (TRF) at unnatural feeding time. This suggests the presence of a clock-regulated buffering mechanism that prevents excessive O-GlcNAcylation at non-optimal times of the day-night cycle, which could disrupt circadian health. We performed targeted metabolomic analysis on hexosamine biosynthetic pathway (HBP), which produces UDP-GlcNAc (the substrate for O-GlcNAcylation), to evaluate the daily activity of HBP enzymes under TRF conditions. We found glutamine--fructose-6-phosphate amidotransferase (GFAT) mediates this buffering mechanism. |
| Institute | University of California, Davis |
| Department | Department of Entomology and Nematology |
| Laboratory | Chiu lab |
| Last Name | Chiu |
| First Name | Joanna |
| Address | One Shield Ave, Davis CA 95695 |
| jcchiu@ucdavis.edu | |
| Phone | (530) 752-1839 |
| Submit Date | 2020-08-13 |
| Num Groups | 2 |
| Total Subjects | 72 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | wiff |
| Analysis Type Detail | LC-MS |
| Release Date | 2020-09-10 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000998 |
| Project DOI: | doi: 10.21228/M8PQ57 |
| Project Title: | Targeted metabolomic analysis on hexosamine biosynthetic pathway in flies on time restricted feeding |
| Project Type: | Targeted analysis by HILIC |
| Project Summary: | The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we showed observed strong correlation between daily daily rhythms of protein O- linked N-acetylglucosaminylation (O-GlcNAcylation) and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms were severely dampened when we subjected flies to time- restricted feeding (TRF) at unnatural feeding time. This suggests the presence of a clock- regulated buffering mechanism that prevents excessive O-GlcNAcylation at non-optimal times of the day-night cycle, which could disrupt circadian health. We performed targeted metabolomic analysis on hexosamine biosynthetic pathway (HBP), which produces UDP-GlcNAc (the substrate for O-GlcNAcylation), to evaluate the daily activity of HBP enzymes under TRF conditions. We found glutamine--fructose-6-phosphate amidotransferase (GFAT) mediates this buffering mechanism. |
| Institute: | University of California, Davis |
| Department: | Department of Entomology and Nematology |
| Laboratory: | Chiu Lab |
| Last Name: | Chiu |
| First Name: | Joanna |
| Address: | One Shield Ave, Davis CA 95695 |
| Email: | jcchiu@ucdavis.edu |
| Phone: | 5307521839 |
| Funding Source: | National Institutes of Health grants R01 GM102225 and R01 DK124068 to JCC |
Subject:
| Subject ID: | SU001526 |
| Subject Type: | Insect |
| Subject Species: | Drosophila melanogaster |
| Taxonomy ID: | 7227 |
Factors:
Subject type: Insect; Subject species: Drosophila melanogaster (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA124046 | ZT20 - Rep1_025 | RF21-3 |
| SA124047 | ZT20 - Rep2_026 | RF21-3 |
| SA124048 | ZT20 - Rep4_028 | RF21-3 |
| SA124049 | ZT16 - Rep6_024 | RF21-3 |
| SA124050 | ZT16 - Rep5_023 | RF21-3 |
| SA124051 | ZT16 - Rep3_021 | RF21-3 |
| SA124052 | ZT16 - Rep4_022 | RF21-3 |
| SA124053 | ZT20 - Rep5_029 | RF21-3 |
| SA124054 | ZT24 - Rep1_031 | RF21-3 |
| SA124055 | ZT24 - Rep5_035 | RF21-3 |
| SA124056 | ZT24 - Rep6_036 | RF21-3 |
| SA124057 | ZT4 - Rep1_001 | RF21-3 |
| SA124058 | ZT24 - Rep4_034 | RF21-3 |
| SA124059 | ZT24 - Rep3_033 | RF21-3 |
| SA124060 | ZT16 - Rep2_020 | RF21-3 |
| SA124061 | ZT24 - Rep2_032 | RF21-3 |
| SA124062 | ZT20 - Rep6_030 | RF21-3 |
| SA124063 | ZT20 - Rep3_027 | RF21-3 |
| SA124064 | ZT8 - Rep1_007 | RF21-3 |
| SA124065 | ZT8 - Rep2_008 | RF21-3 |
| SA124066 | ZT8 - Rep3_009 | RF21-3 |
| SA124067 | ZT4 - Rep6_006 | RF21-3 |
| SA124068 | ZT4 - Rep5_005 | RF21-3 |
| SA124069 | ZT4 - Rep2_002 | RF21-3 |
| SA124070 | ZT16 - Rep1_019 | RF21-3 |
| SA124071 | ZT4 - Rep4_004 | RF21-3 |
| SA124072 | ZT8 - Rep4_010 | RF21-3 |
| SA124073 | ZT4 - Rep3_003 | RF21-3 |
| SA124074 | ZT12 - Rep4_016 | RF21-3 |
| SA124075 | ZT12 - Rep6_018 | RF21-3 |
| SA124076 | ZT8 - Rep5_011 | RF21-3 |
| SA124077 | ZT12 - Rep3_015 | RF21-3 |
| SA124078 | ZT12 - Rep5_017 | RF21-3 |
| SA124079 | ZT12 - Rep2_014 | RF21-3 |
| SA124080 | ZT8 - Rep6_012 | RF21-3 |
| SA124081 | ZT12 - Rep1_013 | RF21-3 |
| SA124082 | ZT20 - Rep1_061 | RF9-15 |
| SA124083 | ZT20 - Rep3_063 | RF9-15 |
| SA124084 | ZT20 - Rep2_062 | RF9-15 |
| SA124085 | ZT16 - Rep5_059 | RF9-15 |
| SA124086 | ZT20 - Rep4_064 | RF9-15 |
| SA124087 | ZT16 - Rep3_057 | RF9-15 |
| SA124088 | ZT16 - Rep4_058 | RF9-15 |
| SA124089 | ZT16 - Rep6_060 | RF9-15 |
| SA124090 | ZT24 - Rep3_069 | RF9-15 |
| SA124091 | ZT24 - Rep5_071 | RF9-15 |
| SA124092 | ZT24 - Rep6_072 | RF9-15 |
| SA124093 | ZT16 - Rep2_056 | RF9-15 |
| SA124094 | ZT24 - Rep4_070 | RF9-15 |
| SA124095 | ZT24 - Rep2_068 | RF9-15 |
| SA124096 | ZT20 - Rep6_066 | RF9-15 |
| SA124097 | ZT24 - Rep1_067 | RF9-15 |
| SA124098 | ZT20 - Rep5_065 | RF9-15 |
| SA124099 | ZT4 - Rep1_037 | RF9-15 |
| SA124100 | ZT8 - Rep1_043 | RF9-15 |
| SA124101 | ZT8 - Rep2_044 | RF9-15 |
| SA124102 | ZT8 - Rep3_045 | RF9-15 |
| SA124103 | ZT4 - Rep6_042 | RF9-15 |
| SA124104 | ZT4 - Rep5_041 | RF9-15 |
| SA124105 | ZT4 - Rep2_038 | RF9-15 |
| SA124106 | ZT4 - Rep3_039 | RF9-15 |
| SA124107 | ZT4 - Rep4_040 | RF9-15 |
| SA124108 | ZT8 - Rep4_046 | RF9-15 |
| SA124109 | ZT8 - Rep5_047 | RF9-15 |
| SA124110 | ZT12 - Rep4_052 | RF9-15 |
| SA124111 | ZT12 - Rep5_053 | RF9-15 |
| SA124112 | ZT12 - Rep6_054 | RF9-15 |
| SA124113 | ZT12 - Rep3_051 | RF9-15 |
| SA124114 | ZT12 - Rep2_050 | RF9-15 |
| SA124115 | ZT8 - Rep6_048 | RF9-15 |
| SA124116 | ZT12 - Rep1_049 | RF9-15 |
| SA124117 | ZT16 - Rep1_055 | RF9-15 |
| Showing results 1 to 72 of 72 |
Collection:
| Collection ID: | CO001521 |
| Collection Summary: | Flies were flash frozen on dry ice. Bodies were separated from heads and weighed. |
| Sample Type: | Fly |
Treatment:
| Treatment ID: | TR001541 |
| Treatment Summary: | 2 treatments X 6 time points each, 6 replicates Treatment #1: Natural feeding time (ZT21-3) with 6 time points over the circadian day (ZT 4, 8, 12, 16, 20, 24) Treatment #2: Unnatural feeding time (ZT9-15) with 6 time points over the circadian day (ZT 4, 8, 12, 16, 20, 24) |
Sample Preparation:
| Sampleprep ID: | SP001534 |
| Sampleprep Summary: | Sample Preparation: 1. Mass out 10 mg. 2. Add 1 mL 5:2:2 meOH:CHCl3:H2O (vol/vol/vol). 3. Genogrind 30 s at 1500 rpm with 2 3 mm stainless steel balls. 4. Shake 5 min at 4oC. 5. Centrifuge 2 min at 14,000 rcf. 6. Aliquot 2 x 450 uL supernatant. 7. Pool remaining supernatant. 8. Dry primary aliquots completely in centrivap. Store backups at -20oC |
Combined analysis:
| Analysis ID | AN002427 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | |
| Column | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 6500 QTrap |
| Ion Mode | NEGATIVE |
| Units | normalized peak height |
Chromatography:
| Chromatography ID: | CH001784 |
| Chromatography Summary: | Targeted HILIC MS/MS |
| Column Name: | Waters Acquity BEH Amide (150 x 2.1mm,1.7um) |
| Flow Rate: | 0.4mL/min |
| Solvent A: | 100% water; 0.125% formic acid; 10 mM ammonium formate |
| Solvent B: | 95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS002264 |
| Analysis ID: | AN002427 |
| Instrument Name: | ABI Sciex 6500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | HILIC Biogenic Amines analysis by UPLC-QTOF mass spectrometry West Coast Metabolomics Center Genome Center, University of California, Davis, CA, USA Contents Page 1 Instruments Page 1 Page 1 Chemical and Consumables Procedures Page 13 Problems Page 13 Disposal of waste 1. Instruments: ● Agilent 1290 UHPLC-6530-QTOF ● Agilent 1290 UHPLC-6550-QTOF ● Pipettes calibrated following SOP006_2003 ● Ultrasonicator 2. Chemicals and consumables ● Waters Acquity UPLC BEH Amide 2.1x150mm 1.7 μm Column ● Waters Acquity UPLC BEH Amide 1.7 μm Pre-column ● Pipettes calibrated following SOP006_2003 ● Ultrasonicator ● Agilent Tune Mix: G1969-85000 ● Acetonitrile: J.T. Baker LC/MS Grade, 4 L (9829-03) ● Formic Acid: Fluka Mass Spec Grade (94318-250mL-F) ● Ammonium Formate: Fluka, Mass Spec Grade (70221-25G-F) ● Agilent 0.17ID (green) metal tubing: 90 cm 5065-9963 and 20 cm (5065-9931) ● Red Agilent Peek Tubing 5 meters (0.13 ID) (5042-6461) ● Plastic Agilent Connectors (for peek tubing) (0100-1516) ● Stainless Steel Agilent Fitting (5062-2418) 3. Procedure: 3.1 Pre-Run Procedures 3.1.1 Instrument tuning (Instrument in Tune mode) a. Use “Standard Tune” before each run of 300 sample batch. b. Use the “Tuning Solution” (see preparation of solutions below) for the instrument tuning. c. Print the tune report from the standard tune. Rev February 2018 - In ESI(+), check the profile of the calibrant and the intensity of ions m/z 322.0481; m/z 622.0290; and m/z 922.0098, which must be higher than 400k, 500k, and 500k, respectively. d. If the intensity of even one of the selected ions is below this value clean the ion source and repeat the instrument tuning. 3.1.2 Check Reference ions (Instrument in Acquisition mode) a. Use the “Reference Ion Mass Solution” (see preparation of solutions below) for mass correction during the analyses (lock mass). b. Check the following reference ions: - In ESI(+), check the intensity of ions m/z 121.0509 and m/z 980.0164, which should be between 5-20k. Adjust recipe and flow rates to attain this intensity. 3.2 New column installation a. Purge the pumping system of any old buffers and connect the inlet of the column to the injector outlet. Attach the outlet line, but allow flow to go into a beaker instead of to the ion source of mass spectrometer. b. Flush column with 100% Mobile Phase B (see preparation of solution below) with a pump flow rate at 0.4mL/min over 30 minutes. c. Switch to 70% mobile phase A and flush the column for 30 minutes. d. When the mobile phase is flowing freely from the column outlet, stop the flow and attach the column outlet to the ion source of mass spectrometer. e. Switch back to 100% Mobile Phase B and monitor the back pressure until a steady value is achieved. Expected values are between 220-250 bar at the start of the injection. f. Perform 6 "No Injections" and monitor the backpressure on the first and last sample injected. Rev February 2018 NOTE: Use a new column after ~1000 sample injections. The UPLC column must be coupled to a VanGuard pre-column. The VanGuard pre-column is replaced after ~330 sample injections. The number of injections (both solvents and plasma samples) is recorded by an operator in a folder created for each acquisition. 3.3 Preparation of solutions a. Preparation of Tuning Solution ● 88.5 mL acetonitrile ● 1.5 mL H2O ● 10 mL Agilent Low Concentration ESI Tuning Mix ● 5 μL 322 Reference Ion (sonicate before use) ● Degas by sonication for 5 min b. Preparation of Reference Mass Solution ● 95 mL acetonitrile ● 5 mL H2O ● 200 μL 5 mM 921 Reference Ion (sonicate before use) ● 250 μL 10 mM Purine Reference Ion (sonicate before use) ● Degas by sonication for 5 min c. Mobile phase A Positive Mode (100% H2O + 10 mM Ammonium Formate + 0.125% Formic Acid) 1. Pre-rinse three times 1 L glass bottle with pure acetonitrile and water 2. Measure exactly 1000 mL of HPLC/UHPLC MS grade H2O in a pre-rinsed graduated cylinder and add them to the 1 L glass bottle. 3. Add 1.250 mL formic acid 4. Weight 0.630 g of ammonium formate and add them to the glass bottle. Swirl to dissolve ammonium formate. 5. Sonicate for 10 min at room temperature until all the ammonium formate is dissolved. 6. 1 L will last for around 200 samples d. Mobile phase B Positive Mode (95:5 ACN:H2O + 10 mM Ammonium Formate + 0.125% Formic Acid) 1. Pre-rinse three times 1 L glass bottle with pure acetonitrile and water 2. Measure exactly 50 mL of HPLC/UHPLC MS grade H2O in a pre-rinsed graduated cylinder and add them to the 1 L glass bottle. 3. Add 1.250 mL formic acid 4. Weight 0.630 g of ammonium formate and add them to the glass bottle. Swirl to dissolve ammonium formate. 5. Measure exactly 950 mL of HPLC/UHPLC MS grade ACN in a pre-rinsed graduated cylinder and add them to the 1 L glass bottle. 6. Sonicate for 10 min at room temperature until all the ammonium formate is dissolved. This step may require multiple 10 minute sonication steps and swirling until solution is clear. 7. 1 L will last for around 200 samples Rev February 2018 e. Needle wash solution (50:50 ACN/H2O) 1. Pre-rinse three times 1 L glass bottle with pure acetonitrile and water 2. Measure exactly 500 mL of HPLC/UHPLC MS grade H2O in a pre-rinsed graduated cylinder and add them to the 1 L glass bottle. 3. Measure exactly 500 mL of HPLC/UHPLC MS grade ACN in a pre-rinsed graduated cylinder and add them to the 1 L glass bottle. 4. Sonicate for 10 min at room temperature. f. Resuspension solvent preparation for injection into HILIC (See Table 1) 1. Weigh out predetermined amount of internal standards (See supplemental SOP for internal standard resuspension solvent). 2. Add 10 mL of the appropriate solvent for each internal standard, either 80:20 acetonitrile:water, 50:50 acetonitrile:water, 100% water, or 100% methanol. This will depend on compound solubility. 3. Next add 14 mL of water and 63 mL of acetonitrile to a clean 100 mL beaker. 4. Aliquot the predetermined volumes of each internal standard (see supplemental SOP for internal standard resuspension solvent) to the same 100 mL beaker to bring the final volume of the solution to 80 mL. 5. Sonicate for 10 min. 6. Store at 4°C g. Preparation of samples for analysis 1. Resuspend samples in 60 – 200 μL of HILIC-resuspension solvent depending on sample concentration/matrix. 2. We typically use 100 μL for 1⁄2 of 5 mg of tissue and 1⁄2 of 5-10 million cells, but this can vary depending on tissue type and instrument sensitivity. Plasma is typically resuspended in 100 μL. 3. Vortex sample for 10 seconds. 4. Sonicate samples for 5 minutes. 5. Centrifuge for 2 min at 16,100 rcf. 6. Transfer supernatant to LC-MS micro insert in an amber vial. 7. Cap and load into auto sampler 3.4 Pre-run sequence a. Before starting the run inject the following: 1. 3 x "No sample Injection" 2. 2 x Blank sample injection (Resuspension Solvent) 3.5 Run sequence a. After running the pre-run sequence, inject the following: 1. Method Blank 2. Biorec Rev February 2018 3. 10 samples 4. repeat Table 1 Analytes of the Resuspension-mix solution Common Name Formula MS1 m/z RT (min) D9-Caffeine C8D9HN4O2 204.1441 1.26 CUDA C19H36N2O3 341.2799 1.23 D3-Creatinine C4D3H4N3O 117.0850 4.80 D9-Choline C5D9H5NO 113.1635 5.30 D9-TMAO C3D9NO 85.1322 5.71 D3-1-Methylnicotinamide C7H6D3N2O 141.0976 6.26 Val-Tyr-Val C19H29N3O5 380.2180 6.95 D9-Betaine C5H2D9NO2 127.1427 7.24 D3-AC(2:0) C9D3H14NO4 207.1419 7.28 D3-Histamine, N-methyl- C6H8D3N3 129.1214 7.34 D3-L-Carnitine C7H12D3NO3 165.1313 7.70 D9-Butyrobetaine C7H6D9NO2 155.1740 7.70 D9-Crotonobetaine C7H4D9NO2 153.1584 7.78 D3-Creatine C4D3H6O2N3 135.0956 8.03 D3-DL-Alanine C3H4D3NO2 93.0738 8.05 D5-L-Glutamine C5H5D5N2O3 152.1078 8.53 D3-DL-Glutamic acid C5H6D3NO4 151.0793 8.72 D3-DL-Aspartic acid C4H4D3NO4 137.0636 9.24 15N2-L-Arginine [15]N2C6H14N2O2 177.1130 9.45 NOTE: The backpressure should be within the range 220-250 bar at the beginning of each run [elution at 100% of the mobile phase (B)] and should not exceed the range 575-650 bar [elution at 70% of the mobile phase (A)]. NOTE: If the initial backpressure is higher than 220-250 bar, switch LC flow to “Bypass” if pressure decreases change the Needle Seat and Seat Capillary. If pressure does not decrease, Rev February 2018 change the Rotor Seal and/or Sample Needle. If the initial backpressure is still high then replace the VanGuard pre-column. If pressure is still high replace Column. 3.6 HILIC analysis method a. The autosampler, separation and column parameters for the HILIC analysis method are as shown below: Rev February 2018 - Binary Pump Parameters: - Column manager Rev February 2018 The MS conditions are the following: 3.6.1 Positive ion mode MS1 - General parameters - Source parameters Rev February 2018 - Acquisition parameters: - Ref Mass parameters Rev February 2018 - Chromatogram parameters: 3.6.2 Positive ion mode MS/MS -Acquisition Rev February 2018 -Collision Energy -Precursor Selection I Note: 4 methods with different static exclusion ranges in Precursor Selection I tab are used. 1. Range 50-150 m/z (static exclusion start m/z 155, static exclusion end m/z 1700) 2. Range 150-300 m/z (static exclusion start m/z 50, static exclusion end m/z 145, static exclusion start m/z 305, static exclusion end m/z 1700) 3. Range 300-500 m/z (static exclusion start m/z 50, static exclusion end m/z 295, static exclusion start m/z 505, static exclusion end m/z 1700) 4. Range 500-1200 m/z (static exclusion start m/z 50, static exclusion end m/z 495, static exclusion start m/z 1205, static exclusion end m/z 1700) Rev February 2018 -Precursor Selection II -Preferred/Exclude List 3.7 Column Storage Use this procedure to avoid precipitation mobile-phase buffers on the column and in the system. a. Flush column with 50% acetonitrile by setting the pump flow rate to 0.1 mL/min and increase the flow rate to 0.4 mL/min over 5 min; keep the column at this flow rate for 10 min. b. Flush column with 95% acetonitrile 5% H2O by setting the pump flow rate to 0.1 mL/min and increase the flow rate to 0.4 mL/min over 5 min; keep he column at this flow rate for 10 min. c. Remove the column from the system. d. Store the column in the box until the next batch analysis. Add the story usage of the column. Rev February 2018 4. Problems In order to avoid cross-contaminations and artifact formation, disposable consumables are used (Eppendorf plastic tubes, plastic pipette tips) 5. Disposal of waste Chemicals are disposed into appropriate bottles in lab 2.157 under the fume hood before monthly disposal collection. Glass vials and consumables are collected into the plastic bags and stored under the fume hood in lab 2.157 before monthly disposal. Other GC-TOF waste (rubber seals, O-rings etc.) can be disposed into regular waste. Rev February 2018 |
| Ion Mode: | NEGATIVE |
