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Lipidomic Analysis of Caenorhabditis elegans Embryos
秀丽隐杆线虫胚胎的脂质组学分析   

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Abstract

Metabolomic is an emerging field of system biology. Lipidomic, a branch of metabolomic, aims to characterize lipophilic metabolites in biological systems. Caenorhabditis elegans (C. elegans) is a genetically tractable and versatile animal model for novel discovery of lipid metabolism. In addition, C. elegans embryo is simple and homogeneous. Here, we demonstrate detailed procedures of C. elegans culture, embryo isolation, lipid extraction and metabolomic data analysis.

Keywords: C. elegans(秀丽隐杆线虫), Embryo(胚胎), Lipid(脂质), LC-MS(LC-MS), Untargeted metabolomic(非靶向代谢组学)

Background

The metazoan model Caenorhabditis elegans (C. elegans) offer a unique platform to discover novel functions and biological roles of metabolic enzymes. Current studies of genomic, transcriptomic and proteomic have advanced our understanding to appreciate the complexity of metabolic networks in C. elegans (Watson et al., 2015). Metabolomic is an emerging tool of system biology aiming to determine small molecule metabolites within biological systems. A number of C. elegans studies have used different metabolomic approaches, including nuclear magnetic resonance (NMR) spectroscopy, gas/liquid chromatography coupled mass spectrometry (GC/LC-MS), to dissect the metabolic networks in whole worm (Atherton et al., 2008; Hughes et al., 2009; Castro et al., 2012; Patti et al., 2014; Morgan et al., 2015; Wang et al., 2015; Wan et al., 2017). Currently, there is no metabolomic-based approach to characterize the lipid metabolites in C. elegans embryos. In this study, LC-MS-based untargeted lipidomic method is chosen for several reasons. First, LC-MS is sufficiently sensitive to analyze small quantity of C. elegans embryos. Second, untargeted metabolomic provides an unbiased view of detectable metabolites, which is crucial to generate a large amount of information. Subsequently, a hypothesis can be formulated based on the global metabolomic analysis. Last but not least, C. elegans embryo is considered simple and homogeneous compared to the whole worm. Taken together, these technical advantages provide opportunities for in-depth analysis of lipid metabolism in developing C. elegans embryos.

Materials and Reagents

  1. C. elegans culture
    1. Aluminum foil
    2. Autoclave tape (Fisher Scientific, catalog number: 15904 )
    3. 90 x 15 mm disposable plastic Petri dishes (China biotech corporation)
    4. 14 ml polypropylene round-bottom tubes (Corning, Falcon®, catalog number: 352059 )
    5. 15 ml centrifuge tubes (Corning, catalog number: 430791 )
    6. 50 ml centrifuge tubes (Corning, catalog number: 430829 )
    7. 250 ml NalgeneTM PPCO centrifuge bottles (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3120-0250 )
    8. Microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
    9. Glass slide
    10. 0.3 mm diameter platinum/iridium wire (Shineteh Instruments)
    11. 0.22 μm pore size syringe filter unit (EMD Millipore, catalog number: SLGP033RB )
    12. C. elegans N2 (wild type strain)
    13. C. elegans control-RNAi (Mock) and G6PD-RNAi (Gi) adults and embryos
    14. E. coli OP50 (University of Minnesota, C. elegans Genetics Center)
    15. E. coli HT115(DE3) harboring control-RNAi (Mock) and G6PD-RNAi (Gi) plasmids
      Note: The details of these plasmids, including design and construction, are described in a previous report (Yang et al., 2013).
    16. Ultrawater
    17. Bleach (NaOCl) (Clorox)
    18. Sodium hydroxide (NaOH) (Merck, catalog number: 1064980500 )
    19. M9 buffer
    20. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-5KG-R )
    21. Agar (BioShop, catalog number: AGR001.1 )
    22. Peptone (Oxoid, catalog number: LP0037 )
    23. Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 1051041000 )
    24. Potassium phosphate monobasic (KH2PO4) (Avantor Performance Materials, J.T. Baker, catalog number: 3246-05 )
    25. Cholesterol (Sigma-Aldrich, catalog number: C8667-5G )
    26. Ethanol (Sigma-Aldrich, catalog number: 32221-2.5L )
      Note: This product has been discontinued.
    27. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M2643-500G )
    28. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3881-500G )
    29. Tetracyclin-hydrochloride (Boehringer Mannheim)
    30. Carbenicillin disodium salt (Sigma-Aldrich, catalog number: C1389-5G )
    31. Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518-25G )
    32. Isopropyl-b-D-thiogalactopyranoside (IPTG) (BioShop, catalog number: IPT001.50 )
    33. LB broth (BD, DifcoTM, catalog number: 244610 )
    34. Nematode growth medium (NGM) (see Recipes)
    35. 1 M KPI buffer pH 6.0 (see Recipes)
    36. 5 mg/ml cholesterol (see Recipes)
    37. 1 M MgSO4 (see Recipes)
    38. 1 M CaCl2 (see Recipes)
    39. 10 mg/ml tetracycline-hydrochloride (see Recipes)
    40. 25 mg/ml carbenicillin (see Recipes)
    41. 200 mg/ml ampicillin (see Recipes)
    42. 1 M IPTG (see Recipes)

  2. C. elegans sample preparation
    1. Falcon cell-strainer cap (12 x 75 mm) (Corning, Falcon®, catalog number: 352235 )
    2. Pyrex glass tube (20 x 125 mm) (Corning, PYREX®, catalog number: 9826-20 )
    3. Glass Pasteur pipettes (Kimble Chase Life Science, catalog number: 63A54 )
    4. Glass organic solvent-resistant pipette tips (HBG, catalog number: 1010-19 )
    5. Organic solvent-resistant polypropylene tip (Gilson, catalog number: F161110 )
    6. Organic solvent-resistant polypropylene microcentrifuge tube (STARLAB INTERNATIONAL, catalog number: S1615-5500 )
    7. HPLC vial (WATERS, catalog number: 186000272C )
    8. Chloroform (LC grade) (Merck, catalog number: 1024444000 )
    9. Methanol (HPLC grade) (Avantor Performance Materials, J.T. Baker, catalog number: 9093 )
    10. Chromasolv grade water (H2O) (Sigma-Aldrich, Fluka ,catalog number: 39253-1L-R )
    11. Chromasolv grade acetonitrile (ACN) (Avantor Performance Materials, J.T. Baker, catalog number: 9829-03 )
    12. Chromasolv grade isopropanol (Sigma-Aldrich, Fluka, catalog number: 34965-2.5L )
    13. Ammonium formate (Fluka, catalog number: 70221 )
    14. Formic acid (Sigma-Aldrich, Fluka, catalog number: 56302 )

Equipment

  1. Pyrex narrow mouth Erlenmeyer flasks (Corning, PYREX®, catalog number: 4980-1L )
  2. Lamina flow hood (Chin-Chih H&W ENTERPRISE, catalog number: BSC-4 )
  3. 20 °C incubator (Firstek, catalog number: RI-560 )
  4. 37 °C incubator with shaker (Yihder, catalog number: LM-570RD )
  5. Centrifuge (Beckman Coulter, model: Avanti® J-26XP )
  6. Rotor (Beckman Coulter, model: JA-14 )
  7. Stereomicroscope (Nikon Instruments, model: SMZ745 )
  8. Stainless steel surgical blade (No. 10) and holder (Feather Safety Razor, Japan)
  9. Vorterxer (Scientific Industries, model: Vortex-Genie 2 )
  10. Centrifuge (Eppendorf, model: 5810 R )
  11. High-capacity swing-bucket rotor with four 250 ml buckets (Eppendorf, model: A-4-62 )
  12. -80 °C freezer
  13. 100 ml glass beaker (Corning, PYREX®, catalog number: 1000-100 )
  14. Macropipette (Socorex, catalog number: 835.05 )
  15. Sonicator (Sonics & Materials, model: VCX 400 )
  16. Ultrasonic cleaner (Delta, model: DC200H )
  17. Nitrogen gas spray unit for aluminum block bath evaporation head (TAITEC, model: DTU-2B )
  18. Centrifuge (Eppendorf, model: 5427 R )
  19. Acquity CSH C18 column (particle size of 1.7 μm, 2.1 x 100 mm) (WATERS, catalog number: 186002352 )
  20. Ultra-performance liquid-chromatography (UPLC) system (WATERS, catalog numbers: 176001285 , 700003616 , 700002764 )
  21. SYNAPT G1 HDMS system (WATERS)
  22. Autoclave (Tomin Medical Equipment, catalog number: TM-328 )
  23. 4 °C cooling cabinet (Firstek, catalog number: CC-2 )

Software

  1. MassLynx4.1 (WATERS)
  2. Extended Statistics (EZinfo, WATERS)
  3. MetaboAnalyst (http://www.metaboanalyst.ca)

Procedure

  1. C. elegans media preparation
    1. Prepare NGM media (see Recipes) in a 1 L flask (see Note 1).
    2. Cover and seal the opening of the flask with two layers of aluminum foil, use a short stretch (2 cm) of autoclave tape to mark on top of aluminum foil.
    3. Autoclave NGM media, MgSO4, CaCl2 and KPI solutions (see Recipes) (see Note 2).
    4. Cool media to ~65 °C and add MgSO4, CaCl2, KPI, cholesterol (see Recipes) (see Note 3).
    5. For regular NGM agar plates: pour 20 ml media into each 90 mm Petri dish.
    6. For RNAi NGM agar plates: pour 20 ml media (supplemented with carbenicillin, tetracycline and IPTG [(see Recipes)] into each 90 mm Petri dish.
    7. Allow agar plates to solidify and dry overnight in a lamina flow hood at room temperature (see Note 4).
    8. Inoculate a single bacterial colony of OP50 or HT115(DE3) to 1 ml of LB (for HT115(DE3): 200 μg/ml ampicillin was supplemented) (see Recipes) in a sterilized polypropylene round-bottom tube and grow for 6 h at 37 °C with shaking (200 rpm).
    9. Inoculate 1 ml of bacterial culture to 300 ml of autoclaved LB in a 1 L flask and grow for 16 h at 37 °C with shaking (200 rpm).
    10. Harvest the overnight culture in 250 ml centrifuge bottles (Nalgene) by centrifugation (4,000 x g, 20 min, 4 °C) (Beckman Coulter, Avanti J-26 XP), discard the supernatant.
    11. Resuspend the pellet with fresh LB to a final volume = 7.5 ml (see Note 5).
    12. Seed each regular NGM agar plate with 300 µl concentrated E. coli OP50 culture and spread with a sterilized stainless steel spreader (see Note 6).
    13. Seed each RNAi NGM agar plate with 300 µl concentrated E. coli HT115(DE3) culture supplemented with 200 µg/ml ampicillin and spread with a sterilized stainless steel spreader.
    14. Allow bacteria-seeded plates to dry overnight in a lamina flow hood at room temperature (see Note 7).

  2. C. elegans culture
    1. Use a sterilized surgical blade (passing over the flame of burner) to cut a small chunk of agar (chunking) from OP50 seeded-NGM agar plate containing larvae worms and transfer the chunk of agar to fresh OP50 seeded-NGM agar plate (see Note 8).
    2. Propagate the worms at 20 °C until sufficient gravid adults from offspring are obtained.
    3. Harvest gravid adults by washing the plates with autoclaved ultrawater and collect into a 15 ml centrifuge tube (see Note 9).
    4. Allow the worms settle and then remove the supernatant by aspiration.
    5. Perform hypochlorite bleach by adding 3.5 ml autoclaved ultrawater to resuspend the pellet.
    6. Add 0.5 ml of 5 N NaOH.
    7. Add 1 ml of 5% NaOCl (Bleach), total volume in the tube is 5 ml.
    8. Vortex the tube briefly and leave at room temperature for less than 5 min, vortex 5 sec every few minutes until the majority (80%) of worms are dissolved (observed under dissecting microscope).
    9. Immediately add 8 ml autoclaved ultrawater and invert the tube 5 times for mixing thoroughly.
    10. Centrifuge at 1,258 x g (Eppendorf, 5810 R) for 1 min and discard the supernatant.
    11. Resuspend the pellet with 10 ml autoclaved ultrawater and invert the tube 5 times.
    12. Centrifuge again at 1,258 x g for 1 min (Eppendorf, 5810 R), discard the supernatant and resuspend the pellet (eggs) with 2 ml M9 buffer.
    13. Incubate the egg suspension at 20 °C overnight with shaking (50 rpm) to obtain synchronized L1 worms.
    14. Aliquots L1 worms to RNAi NGM agar plates (see Note 10).
    15. Incubate the plates at 20 °C for 3 days to obtain Mock and G6PD-knockdown (Gi) adult worms (Figure 1A).


      Figure 1. Representative images of sample preparation procedures. A. Worms grown on bacteria-seeded NGM plate at 20 °C for 3 days from synchronized L1. B. Washed eggs harvested in a 15 ml tube (as a pellet) from 5 NGM plates; C. Eggs filtered by Falcon cell-strainer cap; D. Washed eggs transferred to a microcentrifuge tube; E. Egg suspended in ultrawater after Sonication I (see step D2); F. Folch solution added to lysate in a glass tube; G. Initial phase separation formed after vortex and Sonication II (see step D7); H. Enhanced phase separation after centrifugation.

  3. C. elegans sample preparation
    1. Four biological replicates of C. elegans gravid adults (see Note 11) are washed off from NGM plates by ultrawater (see Note 12). Place the 15 ml centrifuge tube on an ice bucket (see Note 13).
    2. Allow the worms to settle and remove the supernatant. Add 10 ml ultrawater to wash worms. Repeat this step to completely remove floating bacteria and eggs in the suspension.
    3. Perform hypochlorite bleach as described in previous section (steps B5-B11) (see Note 14).
    4. Centrifuge the tube at 1,258 x g for 1 min (Eppendorf, 5810 R), discard supernatant (Figure 1B).
    5. Add 1 ml ultrawater to suspend the eggs and keep the tube on ice.
    6. To filter the eggs, add 0.1 ml ultrawater to the top of Falcon cell-strainer cap to equilibrate the membrane.
    7. Once water is eluted from the membrane, transfer the egg suspension to the top of Falcon cell-strainer cap and allow to elute by gravity (Figure 1C).
    8. Add 1 ml ultrawater to wash the membrane.
    9. Centrifuge the tube at 1,258 x g for 5 min at 4 °C (Eppendorf, 5810 R). Discard the supernatant and the cap.
    10. Add 1 ml ultrawater to the tube and transfer the suspension to a microcentrifuge tube.
    11. Add additional 0.1 ml ultrawater to wash the tube and pool all suspension together in a microcentrifuge tube.
    12. Centrifuge the microcentrifuge tube at 1,258 x g for 5 min at 4 °C (Eppendorf, 5810 R).
    13. Aspirate the supernatant carefully (Figure 1D).
    14. Store the pellet in a -80 °C freezer until use (see Note 15).

  4. Lipid extraction
    1. Thaw the pellet on ice and resuspend with 0.5 ml ultrawater.
    2. Perform sonication with samples (tube) being placed in a 100 ml glass beaker filled with ice.
    3. The sonication cycle is set at 2 sec pulse (frequency: 20 kHz; amplitude: 10%) and 5 sec interval, 20 cycles, total 40 sec (Sonicate I, Figure 1E).
    4. Transfer the supernatants to glass tubes with screw cap.
    5. Perform Folch extraction by adding 1 ml ultrawater to each glass tube, followed by adding 6 ml of chloroform/methanol (ratio = 2:1) mixture to each glass tube (see Note 16).
    6. Vortex each glass tube for 30 sec, repeated 4 times (see Note 17) (Figure 1F).
    7. Place the glass tubes on a test tube rack in an ultrasonic cleaner pre-filled with ice and water, sonicate the samples for 15 min (frequency: 40 kHz) (see Note 18) (Sonicate II, Figure 1G)
    8. Centrifuge the tubes at 1,258 x g for 15 min (Eppendorf, 5810 R) to further enhance phase separation (Figure 1H).
    9. Transfer the lower fraction (hydrophobic) to a clean glass tube by a glass Pasteur pipette (see Notes 19 and 20).
    10. Dry the lower fraction under nitrogen flow in a nitrogen evaporator (see Note 21).
    11. Store the dried pellet in a -80 °C freezer until use.

  5. Lipidomic analysis (see Note 22) (steps of lipidomic data analysis are summarized in Figure 2)


    Figure 2. Summary of steps in lipidomic data analysis. A flow chart from sample separation to metabolites by tandem MS is presented.

    1. Add 1,000 μl solvent (isopropanol/acetonitrile/water, 2/1/1, v/v/v) to the lower fraction. Vortex the glass tube for 30 sec, repeat 4 times. Transfer the supernatant to a new microcentrifuge tube (organic solvent-resistant) by using organic solvent-resistant tips and centrifuge at 15,294 x g for 15 min at 4 °C (Eppendorf, 5427 R). Transfer 800 μl supernatant to the HPLC vial for UPLC-qTOF/MS analysis.
    2. Each sample is carried out in three technical replicates for lipidomic profiling.
    3. Prepare mobile phase A (water:acetonitrile [60:40] supplemented with 10 mM ammonium formate and 0.1% formic acid).
    4. Prepare mobile phase B (isopropanol:acetonitrile [90:10] with 10 mM ammonium formate and 0.1% formic acid).
    5. Samples are injected and separated by using Acquity CSH C18 column.
    6. The column temperature is maintained at 55 °C, the flow rate is set at 0.4 ml/min.
    7. The solvent gradient is as follows: 0-2 min, 40-43% solvent B; 2-2.1 min, 43-50% solvent B; 2.1-12 min, 50-54% solvent B; 12-12.1 min, 54-70% solvent B; 12.1-18 min, 70-99% solvent B; 18-20 min, 99-40% solvent B.
    8. An injected volume of 1.5 μl sample is used in electrospray ionization (ESI) positive (+) mode, and 3 μl sample was used in ESI negative (-) mode (see Note 23).
    9. Mass spectrometric analysis is performed in both ESI+ and ESI- modes using the SYNAPT G1 HDMS system. The major parameter settings are as follows: The capillary and cone voltage are set at 3,000 V (2,000 V in ESI- mode) and 30 V, respectively. The desolvation gas flow rate is set at 800 L/h. The desolvation and source temperatures are 400 and 100 °C, respectively.
    10. Chromatograms are acquired over a range of m/z of 20-990 Da at a rate of 10 scans per second (see Note 24) (Figure 3).


      Figure 3. Base peak intensity (BPI) chromatograms of Mock and Gi embryos. A. ESI positive mode; B. ESI negative mode. The y axis represents relative intensity. Blank (solvent control, red line) is shown to indicate the base line.

    11. Raw data are obtained in the centroid mode.
    12. The frequency of LockSpray is set at 0.5 sec per scan and corrected by averaging over 10 scans.
    13. Leucine encephalin (m/z 556.2771 for ESI+ and m/z 554.2615) in water/acetonitrile = 50/50 + 0.1% formic acid is used as LockSpray lockmass for exact mass measurement accuracy.

Data analysis

  1. Use the molecular feature extraction function in MassLynx4.1 (WATERS) to extract information, including time-aligned ion features, mono-isotopic neutral mass, retention time as well as ion signal intensity, from raw data (see Note 25).
  2. The extracted data is further processed by the Extended Statistics (EZinfo, WATERS) to generate unsupervised principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) model (see Note 26) (Figure 4).


    Figure 4. The principal component analysis (PCA) diagram of Mock and Gi embryos in ESI positive mode

  3. From these models, the variable importance in the projection (VIP) score of each variable is obtained to represent its contribution to the grouping (see Note 27).
  4. High VIP score candidates are initially searched against Human Metabolome Database (HMDB) (http://www.hmdb.ca) and METLIN (http://metlin.scripps.edu/index.php) (see Note 28).
  5. Target candidates with high intensity (relative abundance above 200) are selected and validated by tandem mass spectrometry (MS/MS) with SYNAPT G1 HDMS system (WATERS) (see Note 29).
  6. MS/MS spectra are acquired and confirmed by METLIN (http://metlin.scripps.edu/index.php) and LIPID MAPS (http://www.lipidmaps.org/) as well as our in-house database for lipid standards.

Notes

  1. We prepare 500 ml of NGM media in a 1 L flask. If a smaller amount of NGM media is desired, flask size can be changed accordingly. Make sure not to fill media over 2/3 of the flask volume. Overfill media can lead to losing of volume due to spill over during autoclaving and is difficult to thoroughly mix by swirl.
  2. The sterilization cycle lasts 30 min at 121 °C. We usually take out the flask 1 h after sterilization (total time for autoclave is 2 h).
  3. We normally cool the flask (500 ml media) at room temperature for 30 min before adding supplements. Alternatively, the flask can be cooled in ethylene-vinyl acetate (EVA) foam ice bucket (4 L) filled with tap water for 10 min. However, it should be noted that both cooling methods require intermittent swirl and checking temperature through the hand touching by an experienced operator. For smaller volumes, it is recommended to shorten the cooling time accordingly.
  4. The plates are kept in a lamina flow hood (air flow is not required but can speed up the drying) to avoid contamination.
  5. The condensed culture can support sufficient worm growth up to 3 days (from L1 to gravid adult). The concentration factor is 40.
  6. We prefer to spread the bacterial lawn in rectangular shape and avoid touching the wall of plate. Keeping worms on the lawn can prevent losing worms (crawling on walls) upon harvest.
  7. The bacteria-seeded plates are kept in a lamina flow hood to avoid contamination. If the plates are still wet after overnight incubation, plates can be dried with extended time. Alternatively, turn on air flow to accelerate the drying of bacterial lawn in the lamina flow hood.
  8. It is recommended to perform chunking by using plates without contaminated bacteria or mold to avoid cross contamination. Alternatively, after chunking, allow the worms to crawl for a few hours and transfer them again to new plates by picking.
  9. A Petri dish (90 mm diameter) with as many gravid adults as possible yields high level of egg production.
  10. Prior to transferring L1 to RNAi plates, invert the tubes before each aspiration to obtain homogenous L1 suspension. Pipette a drop of suspension (10 μl) on a clean glass slide and count the number of worms in a drop. Make sure to transfer appropriate number of worms on a plate with sufficient food to sustain the growth for 3 days. Overcrowded worms consume bacteria rapidly and lead to starvation. Hence, it is highly recommended to determine the maximum number of worms that can grow for 3 days with enough food on one plate. We grow approximately 500 worms on a 300 μl concentrated bacterial culture-seeded RNAi plate.
  11. We harvest adult worms from 5 plates (90 mm) and pool them in a 15 ml centrifuge tube as one sample. In this case, the worms yield sufficient quantity of embryos to proceed metabolite extraction. 4 separate samples (N = 4) are prepared for each group.
  12. Use ultrawater only, because detergent (like Tween) contaminate the mass spectrometer system.
  13. Low temperature solution enhances the precipitation of worms.
  14. As the number of worms increases, the total bleaching time prolongs accordingly. Nevertheless, instead of increase the bleaching time, which may cause damage to embryos, we vortex the tube more frequently (10 sec every 30 sec) to enhance worm lysis and the release the embryos. It should be noted that worms exhibit embryonic lethality, such as G6PD-knockdown worms, due to the disruption of egg shell integrity (Yang et al., 2013; Chen et al., 2017). Hence the bleach should be done carefully to avoid the potential lysis of embryos. We recommend to examine the integrity of embryos under microscope.
  15. To ensure the consistency of metabolites extraction, samples collected at different date are extracted at the same day with the same batch of extraction solutions.
  16. The mixture of chloroform and methanol is prepared by mixing 2 volume of chloroform with 1 volume of methanol based on the total volume needed for the experiment. The ratio of chloroform, methanol and water is 8:4:3.
  17. An initial phase separation appears after the addition of chloroform and methanol mixture to the suspension.
  18. Sonication enhances the homogenization after vortex. It also removes the debris on the inner side of test tube wall.
  19. The lower fraction (hydrophobic) is lyophilized and stored for lipid-soluble metabolites analysis. The upper fraction (hydrophilic) can be lyophilized and stored for water-soluble metabolites analysis if desired.
  20. When aspirate the lower fraction, be careful not to disturb the middle protein layer, because the protein layer may be unstable and fall into the lower fraction. First, we carefully insert the tip of glass pipette into the lower fraction along the wall of glass tube and minimize touching the protein layer. Second, a minute amount of upper fraction fluid may fill in the front of glass pipette. We simply dispense it in the lower fraction forming ascending bubbles. This action avoids undesired contamination of hydrophilic metabolites.
  21. Upon completion of nitrogen evaporation, immediately cover the glass tube with screw cap to trap nitrogen gas as much as possible in the tube. Inert gas prevents unwanted chemical reactions, like oxidation, damaging the metabolites in the sample.
  22. The lipidomic profiling for UPLC-qTOF/MS analysis is modified from WATERS Application Note (720004107en).
  23. The detection of ESI- mode is less sensitive than that of ESI+ mode, hence, increased amount of sample is required for ESI- mode.
  24. We choose to show base peak intensity (BPI) chromatogram not total ion current (TIC) because BPI looks cleaner than TIC. Therefore, it is easier for us to identify the difference in peaks between sample groups.
  25. We obtain total features of 3,992 metabolites from ESI+ mode and 730 metabolites from ESI- mode.
  26. In addition to the commercial software, we also use MetaboAnalyst for the analysis and visualization of MS data sets in data matrices as well as generating PCA diagram.
  27. A higher VIP score represents a stronger contribution to discrimination between sample groups. We select candidates with VIP > 1.0 and obtain 848 metabolites from ESI+ mode and 198 metabolites from ESI- mode.
  28. When search lipid metabolites against Human Metabolome Database, we select the main adduct type as M+H and M-H for ESI+ and ESI- mode, respectively. The molecular weight tolerance is set as 0.01 Da. For searching the lipid classes, the ESI- mode provides more information on fragment ion property, for example, the composition of the fatty acids.
  29. Depend on the nature of compounds, the collision energy ranges from 6 to 32 V.

Recipes

  1. Nematode growth medium (NGM) media (for 0.5 L)
    1. Dissolve 1.5 g NaCl, 8.5 g agar, and 1.25 g peptone in 488 ml ultrawater (autoclave)
    2. Subsequently add 12.5 ml of 1 M KPI, 0.5 ml of 5 mg/ml cholesterol, 0.5 ml of 1 M MgSO4, 0.5 ml of 1 M CaCl2
    3. For growing E. coli HT115 strain harboring plasmid, 0.5 ml of 10 mg/ml tetracycline-hydrochloride, 1 ml of 25 mg/ml carbenicillin and 0.5 ml of 1 M IPTG are supplemented
  2. 1 M KPI buffer pH 6.0
    1. Dissolve 108.3 g KH2PO4 and 35.6 g K2HPO4 in 950 ml ultrawater
    2. Adjust pH (5.5) to pH 6.0 with 5 N KOH solution
    3. Adjust final volume to 1 L
    4. Autoclave, store at room temperature
  3. 5 mg/ml cholesterol
    Dissolve 0.2 g cholesterol in 40 ml ethanol
    Note: Do not autoclave.
    Store at room temperature
  4. 1 M MgSO4
    Dissolve 12.04 g MgSO4 in 100 ml ultrawater
    Autoclave
    Store at room temperature
  5. 1 M CaCl2
    Dissolve 14.7 g CaCl2 in 100 ml ultrawater
    Autoclave
    Store at room temperature
  6. 10 mg/ml tetracycline-hydrochloride
    Dissolve 150 mg tetracycline-hydrochloride in 15 ml ultrawater
    Note: Do not autoclave.
    Filter with 0.22 μm filter unit (Millipore)
    Aliquot 1 ml to microcentrifuge tubes and store at -80 °C
  7. 25 mg/ml carbenicillin
    Dissolve 375 mg carbenicillin disodium salt in 15 ml ultrawater
    Note: Do not autoclave.
    Filter with 0.22 μm filter unit (Millipore)
    Aliquot 1 ml to microcentrifuge tubes and store at -80 °C
  8. 200 mg/ml ampicillin
    Dissolve 3 g ampicillin in 15 ml ultrawater
    Note: Do not autoclave.
    Filter with 0.22 μm filter unit (Millipore)
    Aliquot 1 ml to microcentrifuge tubes and store at -80 °C
  9. 1 M IPTG
    Dissolve 3.57 g IPTG in 15 ml ultrawater
    Note: Do not autoclave.
    Filter with 0.22 μm filter unit (Millipore)
    Aliquot 1 ml to microcentrifuge tubes and store at -80 °C

Acknowledgments

This work was made possible by grants from the Ministry of Science and Technology of Taiwan (MOST103-2320-B-182-026-MY2 and MOST105-2320-B-182-031-MY2 to DTYC), from the Ministry of Education of Taiwan (EMRPD1E1751, EMRPD1G0181 and EMRPD1G0281 to DTYC), and from Chang Gung Memorial Hospital (BMRP098, CMRPD1F0621 and CMRPD1F0461 to DTYC). This protocol was modified from procedures published in Yang et al. (2013) and Chen et al. (2017).

References

  1. Atherton, H. J., Jones, O. A., Malik, S., Miska, E. A. and Griffin, J. L. (2008). A comparative metabolomic study of NHR-49 in Caenorhabditis elegans and PPAR-α in the mouse. FEBS Lett 582(12): 1661-1666.
  2. Castro, C., Sar, F., Shaw, W. R., Mishima, M., Miska, E. A. and Griffin, J. L. (2012). A metabolomic strategy defines the regulation of lipid content and global metabolism by Δ9 desaturases in Caenorhabditis elegans. BMC Genomics 13: 36.
  3. Chen, T. L., Yang, H. C., Hung, C. Y., Ou, M. H., Pan, Y. Y., Cheng, M. L., Stern, A., Lo, S. J. and Chiu, D. T. (2017). Impaired embryonic development in glucose-6-phosphate dehydrogenase-deficient Caenorhabditis elegans due to abnormal redox homeostasis induced activation of calcium-independent phospholipase and alteration of glycerophospholipid metabolism. Cell Death Dis 8(1): e2545.
  4. Hughes, S. L., Bundy, J. G., Want, E. J., Kille, P. and Sturzenbaum, S. R. (2009). The metabolomic responses of Caenorhabditis elegans to cadmium are largely independent of metallothionein status, but dominated by changes in cystathionine and phytochelatins. J Proteome Res 8(7): 3512-3519.
  5. Morgan, P. G., Higdon, R., Kolker, N., Bauman, A. T., Ilkayeva, O., Newgard, C. B., Kolker, E., Steele, L. M. and Sedensky, M. M. (2015). Comparison of proteomic and metabolomic profiles of mutants of the mitochondrial respiratory chain in Caenorhabditis elegans. Mitochondrion 20: 95-102.
  6. Patti, G. J., Tautenhahn, R., Johannsen, D., Kalisiak, E., Ravussin, E., Bruning, J. C., Dillin, A. and Siuzdak, G. (2014). Meta-analysis of global metabolomic data identifies metabolites associated with life-span extension. Metabolomics 10(4): 737-743.
  7. Wang, W., McReynolds, M. R., Goncalves, J. F., Shu, M., Dhondt, I., Braeckman, B. P., Lange, S. E., Kho, K., Detwiler, A. C., Pacella, M. J. and Hanna-Rose, W. (2015). Comparative metabolomic profiling reveals that dysregulated glycolysis stemming from lack of salvage NAD+ biosynthesis impairs reproductive development in Caenorhabditis elegans. J Biol Chem 290(43): 26163-26179.
  8. Wan, Q. L., Shi, X., Liu, J., Ding, A. J., Pu, Y. Z., Li, Z., Wu, G. S. and Luo, H. R. (2017). Metabolomic signature associated with reproduction-regulated aging in Caenorhabditis elegans. Aging (Albany NY) 9(2): 447-474.
  9. Watson, E., Yilmaz, L. S. and Walhout, A. J. (2015). Understanding metabolic regulation at a systems level: Metabolite sensing, mathematical predictions, and model organisms. Annu Rev Genet 49: 553-575.
  10. Yang, H. C., Chen, T. L., Wu, Y. H., Cheng, K. P., Lin, Y. H., Cheng, M. L., Ho, H. Y., Lo, S. J. and Chiu, D. T. (2013). Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans. Cell Death Dis 4: e616.

简介

在心肌梗死(MI)中,许多心肌细胞凋亡。 这些凋亡心肌细胞被吞噬细胞迅速吞噬。 如果死细胞没有被吞没,其有害物质被释放到外面,导致炎症的诱导。 因此,需要去除这些死细胞。 然而,每个吞噬细胞类型对梗塞心脏中凋亡细胞的去除的贡献仍未解决。 在这里,我们描述了吞噬作用测定的体外方案来比较心脏巨噬细胞和心脏肌成纤维细胞的吞噬能力。
【背景】长期以来一直认为,心脏巨噬细胞消除了在失败的心脏中产生的凋亡细胞。 然而,我们发现负责组织纤维化的心脏肌成纤维细胞也具有在MI后吞噬凋亡细胞的能力(Nakaya等,2017)。 这一发现促使我们比较心脏巨噬细胞和心脏肌成纤维细胞的吞噬能力。 在这里,我们提供了一个详细的体外吞噬试验方案来评估吞噬吞噬的程度。

关键字:秀丽隐杆线虫, 胚胎, 脂质, LC-MS, 非靶向代谢组学

材料和试剂

  1. ℃。线虫文化
    1. 铝箔
    2. 高压灭菌带(Fisher Scientific,目录号:15904)
    3. 90 x 15毫米一次性塑料培养皿(中国生物技术公司)
    4. 14ml聚丙烯圆底管(Corning,Falcon ®,目录号:352059)
    5. 15ml离心管(Corning,目录号:430791)
    6. 50ml离心管(Corning,目录号:430829)
    7. 250ml Nalgene TM PPCO离心机瓶(Thermo Fisher Scientific,Thermo Scientific TM,目录号:3120-0250)
    8. 微量离心管(Corning,Axygen ®,目录号:MCT-150-C)
    9. 玻璃幻灯片
    10. 0.3毫米直径的铂/铱丝(Shineteh Instruments)
    11. 0.22μm孔径注射器过滤器单元(EMD Millipore,目录号:SLGP033RB)
    12. ℃。线虫 N2(野生型菌株)
    13. ℃。线虫 control-RNAi(Mock)和G6PD-RNAi(Gi)成虫和胚胎
    14. 电子。大肠杆菌OP50(明尼苏达大学,秀丽隐杆线虫遗传学中心)
    15. 电子。大肠杆菌携带对照RNAi(Mock)和G6PD-RNAi(Gi)质粒的HT115(DE3)
      注意:这些质粒的细节,包括设计和构造,都在之前的报告(Yang et al。,2013)中有所描述。
    16. Ultrawater
    17. 漂白剂(NaOCl)(Clorox)
    18. 氢氧化钠(NaOH)(Merck,目录号:1064980500)
    19. M9缓冲区
    20. 氯化钠(NaCl)(Sigma-Aldrich,目录号:31434-5KG-R)
    21. 琼脂(BioShop,目录号:AGR001.1)
    22. 蛋白胨(Oxoid,目录号:LP0037)
    23. 磷酸氢二钾(K 2)HPO 4(Merck,目录号:1051041000)
    24. 磷酸二氢钾(KH 2 PO 4)(Avantor Performance Materials,J.T.Baker,目录号:3246-05)
    25. 胆固醇(Sigma-Aldrich,目录号:C8667-5G)
    26. 乙醇(Sigma-Aldrich,目录号:32221-2.5L)
      注意:本产品已停产。
    27. 硫酸镁(MgSO 4)(Sigma-Aldrich,目录号:M2643-500G)
    28. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C3881-500G)
    29. 盐酸四环素(Boehringer Mannheim)
    30. 碳青霉素二钠盐(Sigma-Aldrich,目录号:C1389-5G)
    31. 氨苄青霉素钠盐(Sigma-Aldrich,目录号:A9518-25G)
    32. 异丙基-b-D-硫代吡喃半乳糖苷(IPTG)(BioShop,目录号:IPT001.50)
    33. LB肉汤(BD,Difco TM ,目录号:244610)
    34. 线虫生长培养基(NGM)(见食谱)
    35. 1 M KPI缓冲液pH 6.0(参见食谱)
    36. 5 mg / ml胆固醇(参见食谱)
    37. 1 M MgSO 4(参见食谱)
    38. 1 M CaCl 2 (见配方)
    39. 10mg / ml盐酸四环素(见食谱)
    40. 25mg / ml羧苄青霉素(参见食谱)
    41. 200μg/ ml氨苄青霉素(参见食谱)
    42. 1 M IPTG(见配方)

  2. ℃。线虫样品制备
    1. Falcon细胞过滤器盖(12 x 75 mm)(Corning,Falcon ®,目录号:352235)
    2. 派雷克斯玻璃管(20 x 125 mm)(Corning,PYREX ®,目录号:9826-20)
    3. 玻璃巴斯德移液器(Kimble Chase Life Science,目录号:63A54)
    4. 玻璃有机溶剂移液器吸头(HBG,目录号:1010-19)
    5. 有机耐溶剂聚丙烯头(Gilson,目录号:F161110)
    6. 有机耐溶剂聚丙烯微量离心管(STARLAB INTERNATIONAL,目录号:S1615-5500)
    7. HPLC小瓶(WATERS,目录号:186000272C)
    8. 氯仿(LC级)(默克,目录号:1024444000)
    9. 甲醇(HPLC级)(Avantor Performance Materials,J.T.Baker,目录号:9093)
    10. Chromasolv级水(H 2 O 2)(Sigma-Aldrich,Fluka,目录号:39253-1L-R)
    11. Chromasolv级乙腈(ACN)(Avantor Performance Materials,J.T.Baker,目录号:9829-03)
    12. Chromasolv级异丙醇(Sigma-Aldrich,Fluka,目录号:34965-2.5L)
    13. 甲酸铵(Fluka,目录号:70221)
    14. 甲酸(Sigma-Aldrich,Fluka,目录号:56302)

设备

  1. Pyrex窄口锥形瓶(Corning,PYREX ®,目录号:4980-1L)
  2. Lamina流罩(Chin-Chih H& W ENTERPRISE,目录号:BSC-4)
  3. 20℃培养箱(Firstek,目录号:RI-560)
  4. 37℃培养箱与摇床(Yihder,目录号:LM-570RD)
  5. 离心机(Beckman Coulter,型号:Avanti ® J-26XP)
  6. 转子(Beckman Coulter,型号:JA-14)
  7. 立体显微镜(Nikon Instruments,型号:SMZ745)
  8. 不锈钢手术刀(10号)和保持架(日本羽毛安全剃刀)
  9. Vorterxer(科学工业,型号:Vortex-Genie 2)
  10. 离心机(Eppendorf,型号:5810 R)
  11. 具有四个250毫升桶的大容量摆动转子转子(Eppendorf型号:A-4-62)
  12. -80°C冰柜
  13. 100毫升玻璃烧杯(康宁,PYREX ®,目录号:1000-100)
  14. Macropipette(Socorex,目录号:835.05)
  15. 超声波仪(Sonics& Materials,型号:VCX 400)
  16. 超声波清洗机(Delta,型号:DC200H)
  17. 用于铝块浴蒸发头的氮气喷射单元(TAITEC,型号:DTU-2B)
  18. 离心机(Eppendorf,型号:5427 R)
  19. Acquity CSH C18柱(粒径为1.7μm,2.1×100mm)(WATERS,目录号:186002352)
  20. 超高效液相色谱(UPLC)系统(WATERS,目录号:176001285,700003616,700002764)
  21. SYNAPT G1 HDMS系统(WATERS)
  22. 高压灭菌器(Tomin Medical Equipment,目录号:TM-328)
  23. 4°C冷却柜(Firstek,目录号:CC-2)

软件

  1. MassLynx4.1(WATERS)
  2. 扩展统计(EZinfo,WATERS)
  3. MetaboAnalyst( http://www.metaboanalyst.ca

程序

  1. ℃。线虫媒体准备
    1. 在1升烧瓶中准备NGM培养基(参见食谱)(见注1)
    2. 用两层铝箔覆盖并密封烧瓶的开口,使用短拉伸(2厘米)的高压胶带标记在铝箔的顶部。
    3. 高压灭菌NGM培养基,MgSO 4,CaCl 2和KPI溶液(参见食谱)(见注2)。
    4. 将培养基冷却至〜65℃,加入MgSO 4,CaCl 2,KPI,胆固醇(参见附录3)(见附注3)。
    5. 对于常规的NGM琼脂平板:将20ml培养基倒入每个90mm培养皿中
    6. 对于RNAi NGM琼脂平板:将20ml培养基(补充有羧苄青霉素,四环素和IPTG [(参见食谱)]倒入每个90mm培养皿中。
    7. 允许琼脂板在室温下在层流罩中固化并干燥过夜(见注4)
    8. 将一个单一的OP50或HT115细菌菌落(DE3)接种到1 ml的LB(对于HT115(DE3):200μg/ ml氨苄青霉素补充)(见配方),在灭菌的聚丙烯圆底管中生长6小时37℃振荡(200rpm)
    9. 将1ml细菌培养物接种到1L烧瓶中的300ml高压灭菌的LB中,并在37℃振荡(200rpm)下生长16小时。
    10. 通过离心(4,000 xg,20分钟,4℃)(Beckman Coulter,Avanti J-26 XP)将250ml离心瓶(Nalgene)中的过夜培养物收获,弃去上清液。 >
    11. 用新鲜的LB将沉淀重新悬浮至最终体积= 7.5ml(参见注5)。
    12. 将每个常规的NGM琼脂平板与300μl浓缩E.大肠杆菌 OP50培养,用无菌不锈钢撒布机涂抹(见附注6)
    13. 每个RNAi NGM琼脂平板上加入300μl浓缩的E。大肠杆菌 HT115(DE3)培养物,补充200μg/ ml氨苄青霉素并用灭菌的不锈钢扩张器铺展。
    14. 允许细菌播种板在室温下在层流罩中干燥过夜(见注7)
  2. ℃。线虫文化
    1. 使用灭菌的手术刀片(穿过燃烧器的火焰)从含有幼虫蠕虫的OP50种子-MEMM琼脂平板上切下一小块琼脂(分块),并将大块琼脂转移到新鲜的OP50种子-NGM琼脂平板上8)
    2. 在20°C传播蠕虫,直到获得足够的妊娠后代成虫。
    3. 通过用高压灭菌的超声波清洗板收获妊娠成虫,并收集到15ml离心管中(见注9)。
    4. 让蠕虫沉淀,然后抽出去除上清液。
    5. 通过加入3.5ml高压灭菌的超声波清洗沉淀物,进行次氯酸盐漂白剂
    6. 加入0.5毫升5N NaOH
    7. 加入1ml 5%NaOCl(漂白剂),管中总体积为5ml
    8. 短暂旋转管,在室温下放置不到5分钟,每隔几分钟旋转5秒,直到大部分(80%)的蠕虫溶解(在解剖显微镜下观察)。
    9. 立即加入8毫升高压灭菌的超声波器,并将管子倒置5次,彻底混匀
    10. 离心在1,258 x g(Eppendorf,5810 R)1分钟,弃去上清液。
    11. 用10ml高压灭菌的超声波清洗沉淀物并将管反转5次。
    12. 再次以1,258×g离心1分钟(Eppendorf,5810R),弃去上清液,并用2ml M9缓冲液重新悬浮沉淀(鸡蛋)。
    13. 将蛋悬浮液在20℃下振荡孵育过夜(50rpm)以获得同步的L1蠕虫
    14. 等分试样L1蠕虫转入RNAi NGM琼脂平板(见附注10)
    15. 将板在20℃下孵育3天以获得Mock和G6PD敲低(Gi)成虫(图1A)。


      图1.样品制备程序的代表性图像A.在同步L1下在20℃下在细菌种子NGM板上生长3天的蠕虫。 B.从5个NGM板在15ml管(作为颗粒)中收获的洗涤的蛋; C. Falcon细胞过滤器盖过滤的鸡蛋;将洗涤的卵转移到微量离心管中; E.超声处理后,蛋中悬浮蛋(见步骤D2); F.在玻璃管中加入裂解液中的溶液; G.涡旋和超声处理II后形成初始相分离(见步骤D7); H.离心后增强相分离。

  3. ℃。线虫样品制备
    1. C的四个生物重复。益生菌(见附注11)通过超声波从NGM板洗掉(见附注12)。将15 ml离心管放在冰桶上(见注13)
    2. 让蠕虫沉淀并除去上清液。加入10毫升超级洗涤蠕虫。重复此步骤以完全清除悬浮液中的浮游细菌和蛋。
    3. 执行上一节所述的次氯酸盐漂白剂(步骤B5-B11)(见附注14)。
    4. 将管以1,258×g离心1分钟(Eppendorf,5810R),弃去上清液(图1B)。
    5. 加入1毫升超级蛋鸡悬挂鸡蛋并将管保持在冰上。
    6. 为了过滤蛋,将超过0.1毫升的超细粉末加入Falcon细胞过滤器顶部以平衡膜。
    7. 一旦水从膜上洗脱出来,将蛋悬浮液转移到Falcon细胞过滤器盖的顶部,并允许通过重力洗脱(图1C)。
    8. 加入1毫升超级清洗膜。
    9. 在4℃下离心管(1,258×g)5分钟(Eppendorf,5810R)。丢弃上清液和盖子。
    10. 向管中加入1毫升超滤,并将悬浮液转移到微量离心管中
    11. 再加入0.1毫升的超声波清洗管,并将所有悬浮液一起放入微量离心管中
    12. 在4℃下将离心管以1,258×g离心5分钟(Eppendorf,5810R)。
    13. 仔细吸取上清液(图1D)
    14. 将丸粒储存在-80°C冰箱中直至使用(见附注15)
  4. 脂质提取
    1. 将沉淀物在冰上解冻,并用0.5毫升超滤剂重新悬浮
    2. 将样品(管)放置在装满冰的100ml玻璃烧杯中进行超声处理。
    3. 超声处理周期设置为2秒脉冲(频率:20kHz;幅度:10%)和5秒间隔,20个周期,共40秒(超声处理I,图1E)。
    4. 将上清液转移到带螺帽的玻璃管上
    5. 通过向每个玻璃管中加入1ml超滤器,然后向每个玻璃管中加入6ml氯仿/甲醇(比例= 2:1)混合物进行抽提(参见附注16)。
    6. 涡旋每个玻璃管30秒,重复4次(见附注17)(图1F)
    7. 将玻璃管放置在预先充满冰和水的超声波清洗机中的试管架上,超声波样品15分钟(频率:40 kHz)(见附注18)(Sonicate II,图1G)
    8. 将管以1,258×g离心15分钟(Eppendorf,5810R)以进一步增强相分离(图1H)。
    9. 通过玻璃巴斯德吸管将较低级分(疏水性)转移到干净的玻璃管(见注19和20)。
    10. 在氮气蒸发器中在氮气流下干燥下部馏分(见注21)
    11. 将干燥的颗粒储存在-80°C冰箱中直到使用。

  5. 脂质体分析(见附注22)(脂质体数据分析的步骤总结在图2中)


    图2.脂质体数据分析中的步骤摘要。介绍了通过串联MS从样品分离到代谢物的流程图。

    1. 向低级馏分中加入1,000μl溶剂(异丙醇/乙腈/水,2/1/1,v / v / v)涡旋玻璃管30秒,重复4次。通过使用有机耐溶剂尖端将上清转移到新的微量离心管(有机溶剂耐受性),并在4℃下以15,294×g离心15分钟(Eppendorf,5427R)。将800μl上清液转移到HPLC样品瓶中进行UPLC-qTOF / MS分析。
    2. 每个样品通过三次技术重复进行脂质分析。
    3. 制备补充有10mM甲酸铵和0.1%甲酸的流动相A(水:乙腈[60:40])。
    4. 用10mM甲酸铵和0.1%甲酸制备流动相B(异丙醇:乙腈[90:10])。
    5. 使用Acquity CSH C18色谱柱注射样品并进行分离
    6. 柱温保持在55℃,流速设定为0.4ml / min。
    7. 溶剂梯度如下:0-2分钟,40-43%溶剂B; 2-2.1分钟,43-50%溶剂B; 2.1-12分钟,50-54%溶剂B; 12-12.1分钟,54-70%溶剂B; 12.1-18分钟,70-99%溶剂B; 18-20分钟,99-40%溶剂B.
    8. 注射体积为1.5μl样品用于电喷雾电离(ESI)阳性(+)模式,3μl样品用于ESI阴性( - )模式(见注23)。
    9. 使用SYNAPT G1 HDMS系统在ESI +和ESI-模式下进行质谱分析。主要参数设置如下:毛细管和锥体电压分别设置为3000 V(ESI模式下为2,000 V)和30 V。去溶剂化气体流量设定为800L / h。去溶剂和来源温度分别为400和100°C
    10. 色谱图以20-990 Da的m / z / m范围以每秒10次扫描的速率获取(见注释24)(图3)。


      图3. Mock和Gi胚胎的基本峰强度(BPI)色谱图。 :一种。 ESI阳性模式; B. ESI负模式。 y轴表示相对强度。空白(溶剂控制,红线)显示为基线。

    11. 原始数据以质心模式获得。
    12. LockSpray的频率设置为每次扫描0.5秒,并通过平均超过10次扫描进行校正。
    13. 用于水/乙腈= 50/50 + 0.1%甲酸的亮氨酸脑炎(ESI +和m / z 554.2615的m / z 556.2771)用作LockSpray lockmass,用于精确质量测量精度。

数据分析

  1. 使用MassLynx4.1(WATERS)中的分子特征提取功能从原始数据中提取信息,包括时间对齐离子特征,单同位素中性质量,保留时间以及离子信号强度(见注25)。 />
  2. 提取的数据由扩展统计(EZinfo,WATERS)进一步处理,以产生无监督主成分分析(PCA)和正交偏最小二乘判别分析(OPLS-DA)模型(见附注26)(图4)。 />

    图4. ESI阳性模式中Mock和Gi胚胎的主成分分析(PCA)图

  3. 从这些模型中,获得每个变量的投影(VIP)分数的变量重要性,以表示其对分组的贡献(见注27)。
  4. 最初针对人类代谢组数据库(HMDB)搜索高级VIP成绩候选人( http://www.hmdb .ca )和METLIN( http://metlin.scripps。 edu / index.php )(见注28)。
  5. 通过使用SYNAPT G1 HDMS系统(WATERS)的串联质谱(MS / MS)(见注29)选择和验证具有高强度(相对丰度高于200)的目标候选物。
  6. MS / MS谱由METLIN获得并确认( http://metlin.scripps .edu / index.php )和LIPID MAPS( http:// www。 lipidmaps.org/ )以及我们内部的脂质标准数据库。

笔记

  1. 我们在1升烧瓶中准备500毫升NGM培养基。如果需要更少量的NGM介质,则可以相应地改变烧瓶的尺寸。确保不要将介质填充到烧瓶体积的2/3以上。高压灭菌过程中溢出的介质会导致体积的损失,并且难以通过旋转彻底混合。
  2. 灭菌周期在121°C持续30分钟。我们通常在灭菌后1小时取出烧瓶(高压灭菌器的总时间为2小时)
  3. 我们通常在室温下冷却烧瓶(500ml介质)30分钟,然后加入补充剂。或者,可以将烧瓶在装有自来水的乙烯 - 乙酸乙烯酯(EVA)泡沫冰桶(4L)中冷却10分钟。然而,应该注意的是,这两种冷却方法都需要经过有经验的操作员的手触摸间歇性旋转和检查温度。对于较小的体积,建议相应地缩短冷却时间。
  4. 将板保持在层流罩(不需要空气流,但可加速干燥),以避免污染。
  5. 浓缩培养物可以支持长达3天的足够的蠕虫生长(从L1到妊娠成虫)。浓度因子为40.
  6. 我们更喜欢将细菌草坪铺展成矩形,避免接触板的墙壁。在草坪上保持蠕虫可以防止在收获时丢失蠕虫(爬上墙壁)。
  7. 将细菌种子板保持在层流罩中以避免污染。孵育过夜后,如果平板仍然是湿润的,则可以延长时间对板进行干燥。或者,打开空气流,加速层流罩中细菌草坪的干燥。
  8. 建议使用没有污染细菌或霉菌的平板进行分块,以避免交叉污染。或者,分块之后,允许蠕虫爬行几个小时,然后通过挑选将它们再次转移到新的盘子上。
  9. 具有尽可能多的妊娠成年人的培养皿(直径90mm)产生高水平的产蛋。
  10. 在将L1转移到RNAi板之前,在每次抽吸之前反转管以获得均匀的L1悬浮液。在干净的玻璃片上吸取一滴悬浮液(10μl),并计数滴虫数量。确保在足够的食物的盘子上转运适量的蠕虫,以维持3天的生长。过度拥挤的蠕虫会迅速消耗细菌并导致饥饿。因此,强烈建议确定最大数量的蠕虫可以长达3天,足够的食物在一块板上。我们在300μl浓缩的细菌培养种子RNAi板上生长约500个蠕虫。
  11. 我们从5个板(90毫米)收获成虫,并将它们作为一个样品在15毫升的离心管中储存。在这种情况下,蠕虫产生足够量的胚胎进行代谢物提取。为每个组准备4个独立样本(N = 4)。
  12. 只能使用超高分子,因为洗涤剂(如吐温)污染质谱仪系统。
  13. 低温溶液可增强蠕虫的沉淀
  14. 随着蠕虫数量的增加,总漂白时间相应延长。然而,我们不是增加漂白时间,而是会损害胚胎,我们更频繁地涡旋管(每30秒10秒),以增强蠕虫裂解和释放胚胎。应该指出的是,由于蛋壳完整性的破坏,蠕虫会出现胚胎致死性,例如G6PD敲低蠕虫(Yang等人,2013; Chen等人。 >,2017)。因此,应仔细进行漂白剂以避免胚胎的潜在裂解。我们建议在显微镜下检查胚胎的完整性
  15. 为了确保代谢物提取的一致性,在同一天用相同批次的提取溶液提取不同日期收集的样品。
  16. 基于实验所需的总体积,通过将2体积氯仿与1体积甲醇混合来制备氯仿和甲醇的混合物。氯仿,甲醇和水的比例为8:4:3。
  17. 在向悬浮液中加入氯仿和甲醇混合物后,出现初始相分离
  18. 超声处理增强涡旋后的均质化。也可以去除试管壁内侧的碎屑
  19. 将低级部分(疏水性)冻干并储存用于脂溶性代谢物分析。如果需要,上部分(亲水性)可以冻干并储存用于水溶性代谢物分析。
  20. 当抽吸较低级分时,请注意不要中断蛋白质层,因为蛋白质层可能不稳定并落入较低级分。首先,我们小心地将玻璃移液管的尖端插入玻璃管壁的下部,并最小化接触蛋白质层。第二,微量的上部流体可能会填充在玻璃移液器的前面。我们简单地将其分配到形成上升气泡的较低部分中。此举避免了亲水性代谢物的不良污染
  21. 完成氮气蒸发后,立即用螺帽盖住玻璃管,尽可能多地在管内捕获氮气。惰性气体可防止不必要的化学反应,如氧化,损害样品中的代谢物
  22. UPLC-qTOF / MS分析的脂质组学分析从WATERS应用报告(720004107en)中修改。
  23. ESI-模式的检测比ESI +模式的检测灵敏度低,因此ESI模式需要增加样品量。
  24. 我们选择显示基本峰强度(BPI)色谱图不是总离子电流(TIC),因为BPI看起来比TIC更干净。因此,我们更容易识别样品组之间的峰值差异。
  25. 我们从ESI +模式获得3,992种代谢物的总特征,并从ESI-模式获得730种代谢物
  26. 除了商业软件之外,我们还使用MetaboAnalyst进行数据矩阵中MS数据集的分析和可视化以及生成PCA图。
  27. 较高的VIP分数对于样本群体之间的歧视有较强的贡献。我们选择VIP> 1.0,并从ESI +模式获得848种代谢物,并从ESI-模式获得198种代谢物
  28. 当搜索针对人代谢组数据库的脂质代谢物时,我们分别选择主加合物类型为M + H和M-H用于ESI +和ESI-模式。分子量公差设定为0.01 Da。为了搜索脂类,ESI-模式提供了更多关于碎片离子性质的信息,例如脂肪酸的组成。
  29. 取决于化合物的性质,碰撞能量范围为6至32 V.

食谱

  1. 线虫生长培养基(NGM)培养基(0.5L)
    1. 在488毫升超声波(高压釜)中溶解1.5克NaCl,8.5克琼脂和1.25克蛋白胨
    2. 随后加入12.5ml 1M KPI,0.5ml 5mg / ml胆固醇,0.5ml 1M MgSO 4,0.5ml 1M CaCl 2 / >
    3. 对于含有质粒的生长大肠杆菌HT115菌株,补加0.5ml 10mg / ml四环素盐酸盐,1ml 25mg / ml羧苄青霉素和0.5ml 1M IPTG,补充
  2. 1 M KPI缓冲液pH 6.0
    1. 在950毫升超级电容器中溶解108.3克KH 2 PO 4和35.6克K 2 HPO 4 4 /
    2. 用5N KOH溶液调节pH(5.5)至pH 6.0
    3. 将最终音量调整为1 L
    4. 高压釜,室温下储存
  3. 5 mg / ml胆固醇
    将0.2 g胆固醇溶于40 ml乙醇中 注意:不要高压灭菌。
    在室温下存放
  4. 1 M MgSO 4
    将12.04g MgSO 4溶解在100毫升超级电容器中 高压灭菌器
    在室温下存放
  5. 1 M CaCl 2
    在100毫升的最佳提取物中溶解14.7克的CaCl 2 高压灭菌器
    在室温下存放
  6. 10mg / ml盐酸四环素
    溶解150毫克盐酸四环素在15毫升超大的
    注意:不要高压灭菌。
    用0.22μm过滤器(Millipore)过滤
    将1ml等分至微量离心管并储存在-80°C
  7. 25 mg / ml羧苄西林
    将375毫克羧苄青霉素二钠盐溶解在15毫升超大型的
    中 注意:不要高压灭菌。
    用0.22μm过滤器(Millipore)过滤
    将1ml等分至微量离心管并储存在-80°C
  8. 200μg/ ml氨苄青霉素
    将3 g氨苄青霉素溶解于15 ml最新的
    注意:不要高压灭菌。
    用0.22μm过滤器(Millipore)过滤
    将1ml等分至微量离心管并储存在-80°C
  9. 1 M IPTG
    溶解3.57 g IPTG在15毫升超大的
    注意:不要高压灭菌。
    用0.22μm过滤器(Millipore)过滤
    将1ml等分至微量离心管并储存在-80°C

致谢

这项工作是由台湾科技部(MOST103-2320-B-182-026-MY2和MOST105-2320-B-182-031-MY2至DTYC)提供的,由教育部台湾(EMRPD1E1751,EMRPD1G0181和EMRPD1G0281至DTYC)和长庚纪念医院(BMRP098,CMRPD1F0621和CMRPD1F0461至DTYC)。该方案由Yang等人发表的程序进行了修改。 (2013)和陈等人(2017)。

参考

  1. Atherton,HJ,Jones,OA,Malik,S.,Miska,EA和Griffin,JL(2008)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih。 gov / pubmed / 18435929“target =”_ blank“>小鼠中的秀丽隐杆线虫和PPAR-α中的NHR-49的比较代谢组学研究。 FEBS Lett 582(12):1661-1666。
  2. Castro,C.,Sar,F.,Shaw,WR,Mishima,M.,Miska,EA and Griffin,JL(2012)。< a class =“ke-insertfile”href =“http: ncbi.nlm.nih.gov/pubmed/22264337“target =”_ blank“>代谢组学策略定义了秀丽隐杆线虫中Δ9去饱和酶对脂质含量和全球新陈代谢的调节。 > BMC Genomics 13:36.
  3. Chen,TL,Yang,HC,Hung,CY,Ou,MH,Pan,YY,Cheng,ML,Stern,A.,Lo,SJ和Chiu,DT(2017)。< a class =“ke-insertfile “href =”http://www.ncbi.nlm.nih.gov/pubmed/28079896“target =”_ blank“>葡萄糖-6-磷酸脱氢酶缺陷型秀丽隐杆线虫的胚胎发育受损发生异常氧化还原稳态诱导钙依赖性磷脂酶的活化和甘油磷脂代谢的改变。细胞死亡疾病 8(1):e2545。
  4. Hughes,SL,Bundy,JG,Want,EJ,Kille,P.和Sturzenbaum,SR(2009)。  秀丽隐杆线虫对镉的代谢组学反应在很大程度上独立于金属硫蛋白状态,但由胱硫醚和植物螯合素的变化主导。 J Proteome Res 8(7):3512-3519。
  5. Morgan,P.G.,Higdon,R.,Kolker,N.,Bauman,A.T.,Ilkayeva,O.,Newgard,C.B.,Kolker,E.,Steele,L.M.and Sedensky,M.M。(2015)。 比较线粒体呼吸链突变体的蛋白质组学和代谢组学谱秀丽隐杆线虫。 线粒体 20:95-102。
  6. Patti,GJ,Tautenhahn,R.,Johannsen,D.,Kalisiak,E.,Ravussin,E.,Bruning,JC,Dillin,A.and Siuzdak,G。(2014)。< a class = insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/25530742“target =”_ blank“>全球代谢组学数据的Meta分析可以确定与寿命延长相关的代谢物。 em>代谢组学 10(4):737-743。
  7. Wang,W.,McReynolds,MR,Goncalves,JF,Shu,M.,Dhondt,I.,Braeckman,BP,Lange,SE,Kho,K.,Detwiler,AC,Pacella,MJ和Hanna-Rose, (2015)。比较代谢组学分析表明,糖酵解产生紊乱由于缺乏补救NAD + 生物合成损害了秀丽隐杆线虫的生殖发育。 J Biol Chem 290(43):26163-26179 。
  8. Wan D D D Pu Pu Pu Pu,,,,,,,,,,,,,。。。。。。。。。。ref ref ref =“http://www.ncbi.nlm.nih.gov/pubmed/28177875”target =“_ blank”>与秀丽隐杆线虫中的繁殖调节老化相关的代谢组学签名 < (奥尔巴尼纽约) 9(2):447-474。
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引用:Yang, H., Hung, C., Pan, Y., Lo, S. J. and Chiu, D. T. (2017). Lipidomic Analysis of Caenorhabditis elegans Embryos. Bio-protocol 7(18): e2554. DOI: 10.21769/BioProtoc.2554.
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