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Jul 2019

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Metabolomic and Lipidomic Analysis of Bone Marrow Derived Macrophages
骨髓衍生巨噬细胞的代谢组学和脂质组学分析   

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Abstract

Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) methods for optimized coverage and quantification. This allows for a comprehensive characterization of cellular metabolism that can be used to understand the impact of a variety of environmental stimuli on macrophage metabolism and function.

Keywords: LC-MS (液质联用), Metabolomics (代谢组学), Lipidomics (脂质组学), Macrophage (巨噬细胞), Metabolism (新陈代谢)

Background

Macrophages, cells of the innate immune system, can adopt a multitude of functional phenotypes in response to cues within the local microenvironment. The activation of macrophages is coupled to, and highly reliant on, specific reprograming of cellular metabolism (Tannahill et al., 2013; Galván-Peña and O’Neill, 2014; Jha et al., 2015; Kelly and O'Neill, 2015; Cordes et al., 2016; Mills and O'Neill 2016; et al.; Mills et al., 2016; Liu et al., 2013; Lampropoulou et al., 2016; Van den Bossche et al., 2017; Williams et al., 2018; Martin et al., 2017). The metabolic reprogramming is wide-spread, involving changes in central metabolism, amino acid metabolism, and lipid remodeling (Galván-Peña and O’Neill, 2014; O’Neill and Pearce, 2016; Van den Bossche et al., 2017). These changes in different pathways are interdependent, allowing cells to produce energy, signaling molecules (e.g., eiconsanoids), and effector molecules (e.g., nitric oxide and reactive oxygen species) for immune functions. Systematic profiling of metabolites and lipids is an important tool to further understand the role these biomolecules play in macrophages.

Development of metabolomic and lipidomic methods has enabled reliable quantification of hundreds of metabolites and lipids simultaneously (Fiehn, 2002; Wenk, 2005). This protocol presents a method for isolation of murine bone marrow derived macrophages (BMDMs), extraction of their lipids and small molecule metabolites and subsequent quantification of their abundance using liquid chromatography–mass spectrometry (LC-MS). BMDMs are a frequently used model for understanding macrophage function and metabolism. Adaptation of the “BUME (butanol and methanol)” extraction method (Löfgren et al., 2012 and 2016), allows for simple and quick extraction of a wide range of both polar metabolites and lipids from a single sample of BMDMs. This reduces variability and gives good recovery. The cell extracts are analyzed by LC-MS, which provides the sensitivity and selectivity required for metabolomic and lipidomic analyses (Theodoridis et al., 2012; Gika et al., 2014). Utilization of parallel LC-MS methods for separate analysis of metabolites and lipids, as outlined in this protocol, provides the broad coverage of the metabolome and lipidome. The LC-MS data are then analyzed using previously developed and publically available analysis tools, MAVEN and LipiDex, giving confident identification and quantification of a wide range metabolite and lipid species (Melamud et al., 2010; Clasquin et al., 2012; Hutchins et al., 2018).

This method can be used to characterize how metabolism in macrophages is altered in response to different stimuli, and how it may be regulated by other microenviromental factors (e.g., nutrient availability). This LC-MS based metabolomics and lipidomics method can be further coupled with isotopic tracing approaches to determine the changes in metabolic flux during immune response, elucidate the mechanisms controlling metabolic rewiring, and facilitate the investigation of the mechanisms connecting altered metabolism to broader macrophage functions.

Materials and Reagents

  1. Isolation and culture of BMDM
    1. 27½ G needle (BD Biosciences, catalog number: 305109 )
    2. 10 ml plastic disposable syringe (Thermo Scientific, catalog number: S7515-10 )
    3. 70 μm cell strainer (Thermo Fisher Scientific, catalog number: 0 87712 )
    4. 0.45 μm PES filter unit (Thermo Fisher Scientific, catalog number: 162-0045 )
    5. 50 ml conical tubes (Thermo Fisher Scientific, catalog number: 339652 )
    6. Countess cell counting chambers (Invitrogen, catalog number: C10283 )
    7. Petri dishes (Fisherbrand, catalog number: FB0875712 )
    8. 6-well culture plates (Eppendorf, catalog number: 00 30720113 )
    9. 175 cm2 flasks (Eppendorf, catalog number: 00 30710029 )
    10. Sterile top filters (Nalgene, catalog number: 596-3320 )
    11. Cell scraper (Corning, catalog number: 3008 )
    12. 6+ weeks old mouse (C57BL/6J, Jackson Laboratory)*
    13.  L929 cells (ATCC®, catalog number: CCL-1TM)
    14. 0.4% Trypan blue stain (Gibco, catalog number: 15250-061 )
    15. 70% Ethanol (Pharmco, catalog number: 1110002000 )
    16. DMEM High Glucose Medium (Sigma-Aldrich, catalog number: D1152 )
    17. Penicillin Streptomycin (Thermo Fisher Scientific, catalog number: 15-140-122 )
    18. Fetal Bovine Serum (FBS, Hyclone, catalog number: 89133-098 )
    19. Dialyzed Fetal Bovine Serum (dFBS, Hyclone, catalog number: 16777-212 )
    20. Recombinant mouse macrophage colony stimulating factor (mCSF, R&D Systems, catalog number: 416ML050 )
    21. RPMI 1640 Medium without Glutamine (Hyclone, catalog number: SH30096.01 )
    22. Glutamine (Thermo Fisher, catalog number: BP379-100 )
    23. HEPES (VWR, catalog number: 16777-032 )
    24. Lipopoylsaccharide from E. coli O111:B4 (LPS, Sigma-Aldrich, catalog number: L3024 )**
    25. Interferon-γ (IFNγ, R&D Systems, catalog number: 485-MI-100 )**
    26. Accutase (STEMCell Technologies, catalog number: 0 7922 )
    27. Trypsin-EDTA (0.05%) (Thermo Fisher Scientific, catalog number: 25300062 )
    28. Phosphate buffered saline (Thermo Fisher Scientific, catalog number: 14190144 )
    29. L929 conditioned media (see Recipes)
    30. BMDM maintenance media (see Recipes)
    31. BMDM differentiation media (see Recipes)
    32. L929 culture media (see Recipes)
    Notes:
    1. *Any mouse model with sufficiently healthy bone marrow can be used.
    2. **Optional based on desired stimulation protocol. LPS and IFN-γ stimulation will induce classical activation.

  2. Metabolite and Lipid Extraction and Sample Preparation
    1. Pipette tips 1,000 μl, 200 μl (VWR, catalog numbers: 89079-470 , 89079-478)
    2. 1.5 ml plastic tube (VWR, catalog number: 89000-028 )
    3. Glass bottles (used for LC-MS grade solvents only, see Notes)
    4. LC-MS polypropylene vials (Thermo Fisher Scientific, catalog number: 03-377-299 )
    5. LC-MS glass vials (Thermo Fisher Scientific, catalog number: 03-FIRVA )
    6. Snap caps (Thermo Fisher Scientific, catalog number: C4011-53 )
    7. Polypropylene microcentrifuge tubes (VWR, catalog number: 89000-028 )
    8. Glass vials (Wheaton, catalog number: 224740 )
    9. Nitrogen gas
    10. 1-Butanol LC-MS grade (Sigma-Aldrich, catalog number: 34867 )
    11. Methanol LC-MS grade (Fisher Chemical, catalog number: A456-4 )
    12. n-Heptane LC-MS grade (Sigma-Aldrich, catalog number: 1036541000 )
    13. Ethyl acetate LC-MS grade (Fisher Scientific, catalog number: E195-4 )
    14. Acetic acid LC-MS grade (Fisher Chemical, catalog number: A11350 )
    15. Water LC-MS grade (Thermo Fisher Scientific, catalog number: W64 )
    16. Tributylamine (Sigma-Aldrich, catalog number: 90780 )
    17. Ammonium Acetate LC-MS grade (Fisher Scientific, catalog number: A114-50 )
    18. Acetonitrile LC-MS grade (Thermo Fisher Scientific, catalog number: A955-4 )
    19. Isopropanol (Thermo Fisher Scientific, catalog number: A461-4 )
    20. SPLASH LipidoMIX Internal Standard (Avanti Polar Lipids, catalog number: 330707 )
    21. Ice

  3. LC-MS Analysis
    1. Acquity UPLC BEH C18 Column, 130Å, 1.7 µm, 2.1 mm x 100 mm (Waters, catalog number: 186002352 )
    2. Acquity UPLC BEH C18 VanGuard Pre-column, 130Å, 1.7 µm, 2.1 mm x 5 mm (Waters, catalog number: 186003975 )
    3. Acquity UPLC CSH C18 Column, 130Å, 1.7 µm, 2.1 mm x 100 mm (Waters, catalog number: 186005297 )
    4. Acquity UPLC CSH C18 VanGuard Pre-column, 130Å, 1.7 µm, 2.1 mm x 5 mm (Waters, catalog number: 186005303 )
    5. Solvent B (100% LC-MS grade methanol)
    6. Solvent A (see Recipes)
    7. Solvent C (see Recipes)
    8. Solvent D (see Recipes)

Equipment

  1. Surgery Scissors (Fisherbrand, catalog number: 08-935 )
  2. Surgery Forceps (BrainTree Scientific, catalog number: FC03-41 )
  3. Thermo Q-Exactive Quadrupole Orbitrap Mass Spectrometer (Thermo Scientific) coupled to a Vanquish Horizon UHPLC (Thermo Scientific)
  4. Nitrogen Stream Sample Concentrator (Techne, catalog number: FSC400D ), equipped with 127 mm needles (Techne, catalog number: F7210 )
  5. Vortexer (VWR, catalog number: 10153-838 )
  6. Micro Centrifuge (Beckman Coulter, model: Microfuge 20 , catalog number: B31599 )
  7. Benchtop Centrifuge (Thermo, model: IEC Centra CL2 )
  8. -20 °C Freezer
  9. 4 °C Fridge
  10. Standard Fume Hood
  11. Pipettes (P1000, P200) 
  12. Sterile Cell Culture Hood
  13. Cell Incubator (37 °C, 5% CO2)
  14. Countess II Cell Counter (AMQAX1000)

Software

  1. Thermo Scientific XCalibur 4.1
  2. MAVEN (v. 682) (http://genomics-pubs.princeton.edu/mzroll/index.php) (Melamud et al., 2010; Clasquin et al., 2012)
  3. MZMine2 (http://mzmine.github.io/) (Pluskal et al., 2010)
  4. MSConvertGUI (ProteoWizard, http://proteowizard.sourceforge.net/download.html) (Kessner et al., 2008)
  5. LipiDex (https://github.com/coongroup/LipiDex) (Hutchins et al., 2018)

Procedure

  1. Isolation of Bone Marrow Cells and Differentiation into Macrophages (Figure 1)
    See additional resource for helpful demonstration of bone marrow isolation (Ying et al., 2013).


    Figure 1. Timing of BMDM Differentiation Protocol. TC treated: tissue culture treated; BMDM: bone marrow derived macrophages.

    1. Euthanize mice by rapid cervical dislocation.
    2. Thoroughly spray mouse with 70% ethanol and bring into sterile hood.
    3. Using aseptic technique, isolate femur and tibia from each leg of mouse.
    4. Using a sharp scissors soaked in ethanol, sever bone proximal to each joint.
    5. Using a 27½ G needle attached to a 10 ml syringe, flush bones with cold, sterile BMDM Differentiation Media into a 10 cm dish, until bone cavity appears white. Use as much media is required to fully flush the bone.
    6. Pipette cell suspension thoroughly to fully resuspend bone marrow cells.
    7. For removal of any muscle or bone fragments, filter cells through 70 µm filter, into a sterile 50 ml conical centrifuge tube.
    8. Centrifuge cells for 3 min at 500 x g, 4 °C.
    9. Discard supernatant and resuspend cell pellet in 100 ml of BMDM Differentiation Media.
    10. Plate 10 ml of cell suspension onto each Petri-dish (non tissue culture treated plates are important here as macrophages will attach too strongly to treated plates)
    11. Place in cell culture incubator for 3 days.
    12. Four days post plating aspirate media from cells and replace with 10 ml fresh BMDM Differentiation Media. Repeat on day 5 and day 6.
    13. Seven days post plating aspirate media from cells and add 4 ml of Accutase per plate. Place back in incubator for approximately 5 min.
    14. Using pipette, gently dislodge cells from plate and combine all cells in a 50 ml conical tube.
    15. Spin down (1,000 x g, 5 min) and resuspend into 20 ml BMDM Maintenance media.
    16. Count cells by staining 1:1 with 0.4% Trypan Blue.
    17. Resuspend cells to 2.5 x 105 cells per ml in BMDM Maintenance Media and plate on TC treated cell culture plates (2 ml per well of 6-well plate). Three replicates (e.g., 3 wells of a 6-well plate) are recommended for each condition. Additional wells should be designated for protein or cell count normalization.
    18. Cell will adhere to plate with in a matter of a couple hours. After adherence, cells are ready for stimulation and/or extraction. To stimulate macrophages, treat with 50 ng/ml of LPS and 10 ng/ml IFN-γ, or other stimuli of interest, for desired duration.

  2. Isolation of Polar Metabolites and Lipids (Figure 2)


    Figure 2. Work flow for extraction of lipids and small polar metabolites. ACN: acetonitrile, IPA: isopropanol.

    1. Before beginning, make the following solutions:
      3:1 1-butanol:methanol (LC-MS grade), with 1% SPLASH LipidoMIX Internal Standard (SPLASH)
      3:1 heptane:ethyl acetate (LC-MS grade)
      1% acetic acid (LC-MS grade)
      65:30:5 Acetonitrile:Isopropanol:Water (LC-MS grade, ACN:IPA:H2O)
    2. Chill 3:1 butanol:methanol (+SPLASH) and 3:1 heptane:ethyl acetate in the -20 °C and 1% acetic acid in the 4 °C for at least an hour prior to beginning.
    3. Aspirate media from cells.
    4. Wash cells twice with room temperature PBS.
    5. Add 300 µl of -20 °C 3:1 butanol:methanol (+SPLASH) to each well, place plates immediately on ice.
    6. While keeping cell plate on ice, scrape cells using cell scraper, and transfer solution from each well to a separate 1.5 ml plastic microcentrifuge tube. Place samples on ice.
      Note: Also perform procedural blanks (N > 2) by performing the same Steps B1-B5 with an empty tube/plate.
    7. Vortex each tube for 1 min, place back on ice.
    8. In the fume hood, add 300 µl -20 °C 3:1 heptane:ethyl acetate to each sample.
    9. Vortex all tubes for 3 min, place back on ice.
    10. In the fume hood, add 300 µl 1% acetic acid to each sample.
    11. Vortex all tubes for 3 min, place back on ice.
    12. Centrifuge all tubes (20,000 x g, 10 min, 4 °C)–two separate layers should form.
    13. Using a pipette, transfer a set volume (~400 µl) of the top organic layer to a glass vial (this is the lipid and fatty acid fraction) (perform in fume hood)
    14. Using a pipette, transfer a set volume (~300 µl) of the bottom aqueous layer to a 1.5 ml plastic tube (this is the polar metabolite fraction) (perform in fume hood)
    15. Completely dry down all samples by nitrogen stream using a N2 sample concentrator in the fume hood. Samples typically dry within 2h at room temperature. Retrieve samples immediately after drying.
    16. Resuspend the metabolite fraction in LC-MS grade water (50 µl) and the lipid fraction in 65:30:5 ACN:IPA:H2O (50 µl).
    17. Transfer metabolite fraction to polypropelyne LC-MS vials and the lipid fraction to glass LC-MS vial, cap and place in LC-MS autosampler for analysis.

  3. Liquid-chromatography Mass spectrometry
    1. LC-MS vials should be placed in sample trays in randomized order. Sample runs should start with injection of an equal volume of at least three appropriate blanks: LC-MS H2O for polar metabolites, 65:30:5 ACN:IPA:H2O for lipids. Blanks should additionally be run throughout, between at least every 10 samples.
    2. Analyses of both the polar metabolite and lipid fractions were performed on a Thermo Q-Exactive mass spectrometer coupled to a Vanquish Horizon UHPLC.
    3. Polar metabolite samples were separated on a 100 x 2.1 mm 1.7uM Acquity UPLC BEH C18 Column (Waters) coupled to a 130Å, 1.7 µm, 2.1 mm x 5 mm Acquity UPLC BEH C18 VanGuard Pre-column (Waters). A gradient of solvent A (97:3 H2O:methanol, 10 mM TBA, 9 mM acetate, pH 8.2) and solvent B (100% methanol) was used at a 0.2ml/min flow rate. The gradient is: 0 min, 5% B; 2.5 min, 5% B; 17 min, 95% B; 21 min, 95% B; 21.5 min, 5% B (Figure 3A). Five µl of each metabolite sample was injected for analysis. Data was collected on a full scan negative mode. Detailed MS parameters are laid out in in Table 1.
    4. Lipid samples were separated on a 100 x 2.1 mm 1.7uM Acquity UPLC CSH C18 Column (Waters) coupled to a 130Å, 1.7 µm, 2.1 mm X 5 mm Acquity UPLC CSH C18 VanGuard Pre-column. A gradient of solvent C (70:30 ACN:H2O, 10 mM NH4Ac) and solvent D (90:10 IPA:ACN, 10 mM NH4Ac) was used at a 0.4 ml/min flow rate. The gradient is: 0 min, 2% D; 2 min, 2% D; 5 min, 30% D; 19 min, 85% D; 20 min 99% D; 27 min, 99% D; 28 min, 2% D; 32 min, 2%D (Figure 3B). Five µl of each lipid sample was injected for analysis. Samples were run twice, once in negative mode and once in positive mode both using data dependent MS2 (ddMS2) analysis. Detailed MS parameters are laid out in in Table 1.

      Table 1. Mass Spectrometry Parameters



      Figure 3. Solvent gradients and elution profiles. A. Solvent gradient and example elution profile for LC-MS polar metabolite method. B. Solvent gradient and example elution profile for LC-MS lipid analysis, profiles from both negative and positive ionization runs are shown.

Data analysis

MS1 data is used for both polar metabolite and free fatty acid quantification using MAVEN software (Melamud et al., 2010; Clasquin et al., 2012). Lipid data is identified and quantified based on full scan–ddMS2 using LipiDex Software (Hutchins et al., 2018). Both methods are described below. Representative data obtained from analysis of metabolite and lipids profiles of unstimulated (M0) and LPS+IFNγ stimulated BMDM (M1) is shown in Figure 4.



Figure 4. Representative metabolite and lipid data. A volcano plot showing fold changes in identified metabolites and lipids in LPS+IFNγ stimulated (M1) BMDM as compared to unstimulated (M0) BMDM. Highlighted are itaconate and succinate, two immunoregulatory metabolites known to be dynamically upregulated in M1 macrophages (Jha et al., 2015; Cordes et al., 2016; Lampropoulou et al., 2016; Mills et al., 2016). Also highlighted is triglyceride 50:2. Triglyceride accumulation has also been shown to occur in M1 macrophages (Köberlin et al., 2014; Lee et al., 2017; Feingold et al., 2012).

Polar Metabolites and Fatty Acids
Polar metabolites and fatty acids are quantified in a targeted fashion by matching the expected exact mass and retention time. The list of retention times and M/z is determined from analysis of metabolite standards using the same LC-MS method for samples as outlined above, and is in the supplementary information (supplemental file “Polar Metabolites RT List .xlsx”). We suggest that particularly with metabolites of interest, users run standards of their own. To do so, dilute standards to a range of concentrations (e.g., 1 μM, 10 μM, 100 μM, 1,000 μM, for inject 1 μl injection volume) in the appropriate sample solvents, and run on LC-MS using the corresponding method. For untargeted metabolomics analysis, see additional resources (Smith et al., 2005 and 2006; Kind et al., 2018; Wang et al., 2019). Additionally, this method will only describe what is necessary for relative quantification of metabolites. For measurement of absolute concentration of metabolites see additional resources (Bennett et al., 2008). We use MAVEN software for integration of peaks (Melamud et al., 2010; Clasquin et al., 2012). This software and more detailed guides on its use are available here.


  1. Convert Thermo generated .raw files to .mzXML files using MSConvertGUI (Holman et al., 2014; Adusumilli et al., 2017).
  2. Open .mzXML files in MAVEN. When properly loaded samples IDs will appear in the samples window to the left.
  3. Make sure “Compounds Widget” is turned on. Load compound list by clicking on compound tab on left and selecting “Load Custom Compound List”. Load the .csv file with known m/z and retention times generated from metabolite standards or as provided in supplemental material. Adjust the ppm window on top right of Maven window to match that of the sensitivity of your instrument.
  4. With compound list loaded, click on name of compound of interest and the peaks corresponding to that metabolite in each sample will appear in the main window. The red line corresponds to the observed retention time according to the imported compound list (Figure 5). To save the data from selected peak double click on the peak–the name of the compound and corresponding parameters will be displayed in a list within the software. Save data only from those peaks whose retention time matches that on the compound list, has a relatively smooth profile, and whose signal is above desired cut off (e.g., 5E4 and at least 2x times the blank). See Figure 5 for example.
    Note: More detailed information about software functions and parameter setting can be found in MAVEN using manual.
  5. When all desired peaks have been saved, export this data from Maven by selecting “Export Peaks to SpreadSheet (.csv) above the list of saved peaks.
  6. Blank the data by subtracting the average signal from the blanks from the signal from each sample. Subsequently normalize data to cell count or protein.
    Note: Only compare relative abundance from samples run within the same sequence, as changes in instrument conditions over time may cause difference in signal response. If absolute quantitation of metabolite concentration is desired, use of isotopic internal standards is recommended.


    Figure 5. Example of a good LC-MS peak. Succinate peaks from unstimulated (M0) and LPS+IFNγ stimulated (M1) BMDM, as displayed by Maven software.


Lipids
Lipid identification and quantification is performed using the open access LipiDex software (Hutchins et al., 2018). Based on full scan-ddMS2 data, LipiDex identifies lipids. The software is coupled to MZmine2 to quantify lipid species. Below is our workflow for this analysis (Pluskal et al., 2010).

  1. Convert Thermo generated .raw files to .mgf files (we use MSConvertGUI software [Holman et al., 2014; Adusumilli et al., 2017]).
  2. Perform Spectrum Search in LipiDex
    1. Open LipiDex and go to Spectrum Searcher.
    2. Upload .mgf files.
    3. Enable the appropriate libraries.
    4. Specify desired parameters for search (e.g., MS1 search tolerance: 0.01, MS2 search tolerance: 0.01).
    5. Click “Spectra Search”–this will produce result .csv files with identifications.
  3. Generate chromatographic feature tables using MZmine2. Do separately for positive and negative ionization runs. Follow step-by-step instructions as provided here.
  4. Perform Peak Finding in LipiDex
    1. Open LipiDex and go to Peak Finder.
    2. Click the MZMine2 option.
    3. Load the positive and negative polarity peak tables generated by MZMine2.
    4. Upload the MS/MS .csv result files, making sure to identify the files with the appropriate polarity (positive or negative).
    5. Set desired filtering parameters, for instance, using the following:
      MS2 filtering parameters: Min. Lipid Spectral Purity (%): 75, Min. MS2 Search Dot Product: 500, Min. MS2 Search Rev. Dot Product: 700.
      Feature Association Parameters: FWHM Window Multiplier: 2.0, Max. Mass Difference (ppm): 15.
      Result Filtering Parameters: Check Adduct/Dimer Filtering and In-source Fragment Filtering. Check Max. RT M.A.D Factor with 3.5. Feature found in n Files: 2.
    6. Click Identify Chromatographic Peaks–this will produce .csv files containing identified lipid species and their quantification.
  5. Blank data using the procedural blanks that were run parallel to samples, and normalize data to cell count or protein content.
  6. Quality Control: The SPLASH LipidoMIX Internal Standard is a mixture of isotopic labeled lipids, representing major lipid classes. SPLASH (1%) was added to the initial extraction buffer (3:1 1-butanol:methanol). The presence of this internal standard in all analyzed lipid samples allows for determination of retention time drift across samples, and potential difference in sample recovery. The retention time drift within a sequence is typically < 0.1min, and the variation of internal standards is < 20%. If the variation is significant, data can be normalized to internal standard of the same lipid class or nearest retention time (if there is no standard of the same class). See supplemental file “SPLASH LipidoMix Stds Compound list .xlsx” for list of compounds in SPLASH LipidoMIX and their respective retention times using the outlined method.

Notes

  1. Keep and measure out all LC-MS grade solvents in clean designated glassware. Do not use this glassware for other purposes and do not wash with tap water or soap. Residual detergent and other material will cause background. When necessary, rinse with HPLC grade solvents only.
  2. Plastic used during making of extraction solvents and the extraction of lipids can cause background, particularly for fatty acids. Always perform a procedural blank to appropriately account for this background signal. Alternative use of glass vials and pipettes in place of plastic microcentrifuge tubes and pipette tips can help reduce background if a considerable amount is observed.

Recipes

  1. BMDM Differentiation Media
    RPMI 1640 without Glutamine
    10% FBS
    20% L929 Conditioned Media
    10 U/ml penicillin/streptomycin
    25 mM HEPES
    2 mM glutamine
  2. BMDM Maintenance Media
    RPMI 1640 without Glutamine
    10% dFBS
    10 U/ml penicillin/streptomycin
    25 mM HEPES
    2 mM glutamine
    20 ng/ml m-CSF
  3. L929 Culture Media
    DMEM High Glucose
    10 U/ml penicillin/streptomycin
    10% FBS
  4. L929 Conditioned Media
    1. Culture L929 cells in L929 culture media until fully confluent
    2. Trypsinize cells, spin down at 1,000 x g for 5 min
    3. Aspirate trypsin, resuspend cell pellet in L929 culture media
    4. Count cells and resuspend to 8 x 103 cells/ml
    5. Add 130 ml of this suspension to a 175 cm2 flask and place in incubator
    6. 7 days later collect all the spent media
    7. Centrifuge at 1,000 x g, 4 °C, for 5 min. Sterile filter using a 0.45 μm filter into a sterile glass bottle
    8. Aliquot to 50 ml plastic centrifuge tubes, and freeze at -20 °C until desired use (use within 6 months)
  5. Solvent A
    97:3 water:methanol (LC-MS grade)
    10 mM tributylamine
    Acetic acid (LC-MS grade), pH to 8.2
    Note: This takes a great deal of vigorous mixing to fully dissolve the tributylamine and reach a stable pH.
  6. Solvent C
    70:30 acetonitrile:water (LC-MS grade)
    10 mM Ammonium Acetate (LC-MS grade)
    250 µl/L Acetic Acid (LC-MS grade) 
  7. Solvent D
    90:10 isopropanol:acetonitrile (LC-MS grade)
    10 mM Ammonium Acetate (LC-MS grade)
    250 µl/L Acetic Acid (LC-MS grade)

Acknowledgments

The methods for bone marrow derived macrophages isolation, metabolites and lipids extraction, LC-MS analysis and data analysis are adapted and optimized based on previous works, each referenced in the respective section.

Competing interests

The authors declare no competing interests.

Ethics

Animal care and experimental procedures were carried out in accordance with a protocol approved by University of Wisconsin-Madison Institutional Animal Care and Use Committee.

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简介

[摘要] 巨噬细胞是高度可塑性的免疫细胞,能够对环境刺激产生广泛的功能表型。巨噬细胞功能的改变通常受到细胞代谢的支持和调节。获取新陈代谢的全面图像对于理解代谢重组在免疫反应中的作用至关重要。本文提出了一种系统定量测定小鼠骨髓源性巨噬细胞(BMDMs)中代谢物和脂质丰度的方法。该方法采用快速两相萃取法同时从BMDMs中提取极性代谢物和脂质。极性代谢物部分和脂质部分随后通过单独的液相色谱-质谱(LC-MS)方法进行分析,以优化覆盖率和定量。这使得对细胞代谢的全面表征可以用来理解各种环境刺激对巨噬细胞代谢和功能的影响。

[背景] 巨噬细胞是先天性免疫系统的细胞,在局部微环境中对信号作出反应时,可采用多种功能表型。巨噬细胞的激活与细胞代谢的特定重编程相结合并高度依赖(Tannahill et al.,2013;Galván-Peña and O'Neill,2014;Jha et al.,2015;Kelly and O'Neill,2015;Cordes et al.,2016;Mills and O'Neill 2016;等人;Mills et al.,2016;Liu et al.,2013;Lampropoulou et al.,2016;Van den Bossche等人,2017年;威廉姆斯等人,2018年;马丁等人,2017年)。代谢重编程广泛存在,包括中枢代谢、氨基酸代谢和脂质重塑的变化(Galván-Peña和O'Neill,2014;O'Neill和Pearce,2016;Van den Bossche等人,2017)。不同途径中的这些变化是相互依赖的,允许细胞产生能量、信号分子(如二十烷醇)和效应分子(如一氧化氮和活性氧物种)来实现免疫功能。代谢产物和脂质的系统分析是进一步了解这些生物分子在巨噬细胞中的作用的重要工具。

代谢组学和脂类学方法的发展使数百种代谢物和脂类同时可靠地定量(Fiehn,2002;Wenk,2005)。本方案提出了一种分离小鼠骨髓源性巨噬细胞(BMDMs)的方法,提取其脂质和小分子代谢物,并随后使用液相色谱-质谱(LC-MS)对其丰度进行量化。BMDMs是了解巨噬细胞功能和代谢的常用模型。采用“BUME(丁醇和甲醇)”提取方法(Löfgren等人,2012年和2016年),可以从单一BMDM样品中简单快速地提取多种极性代谢物和脂质。这减少了可变性,并提供了良好的恢复。细胞提取物通过LC-MS进行分析,这提供了代谢组学和脂类分析所需的灵敏度和选择性(Theodoridis等人,2012年;Gika等人,2014年)。利用平行LC-MS方法分别分析代谢物和脂质,如本方案所述,提供了代谢组和脂质体的广泛覆盖范围。然后使用先前开发和公开的分析工具MAVEN和LipiDex对LC-MS数据进行分析,从而对广泛的代谢物和脂类物种进行可靠的鉴定和量化(Melamud等人,2010;Clasquin等人,2012;Hutchins等人,2018)。

这种方法可以用来描述巨噬细胞的新陈代谢是如何在不同的刺激下发生变化的,以及它是如何被其他微环境因素(如营养素的可用性)调节的。这种基于LC-MS的代谢组学和脂质组学方法可进一步与同位素示踪方法相结合,以确定免疫反应期间代谢通量的变化,阐明控制代谢重组的机制,并有助于研究代谢改变与更广泛的巨噬细胞功能之间的联系机制。

关键字:液质联用, 代谢组学, 脂质组学, 巨噬细胞, 新陈代谢

材料和试剂


 


A、 BMDM的分离培养


1.     271.     27½G针头(BD Biosciences,目录号:305109)


2.     10ml一次性塑料注射器(Thermo Scientific,目录号:S7515-10)


3.     70 μm单元过滤器(赛默飞世尔科学公司,目录号:087712)


4.     0.45 μm PES过滤装置(赛默飞世尔科技公司,目录号:162-0045)


5.     50毫升锥形管(赛默飞世尔科技公司,目录号:339652)


6.     伯爵夫人细胞计数室(Invitrogen,目录号:C10283)


7.     培养皿(Fisherbrand,目录号:FB0875712)


8.     6孔培养板(Eppendorf,目录号:0030720113)


9.     175 cm2烧瓶(Eppendorf,目录号:0030710029)


10.  无菌顶部过滤器(Nalgene,目录号:596-3320)


11.  电池刮板(康宁,目录号:3008)


12.  6周以上大鼠(C57BL/6J,杰克逊实验室)*


13.  L929电池(ATCC®,目录号:CCL-1TM)


14.  0.4%台盼蓝染色(Gibco,目录号:15250-061)


15.  70%乙醇(Pharmco,目录号:1110002000)


16.  DMEM高糖培养基(Sigma-Aldrich,目录号:D1152)


17.  青霉素链霉素(Thermo Fisher Scientific,目录号:15-140-122)


18.  胎牛血清(FBS,Hyclone,目录号:89133-098)


19.  透析胎牛血清(dFBS,Hyclone,目录号:16777-212)


20.  重组小鼠巨噬细胞集落刺激因子(mCSF,研发系统,目录号:416ML050)


21.  不含谷氨酰胺的RPMI 1640培养基(Hyclone,目录号:SH30096.01)


22.  谷氨酰胺(赛默飞世尔,产品目录号:BP379-100)


23.  HEPES(VWR,目录号:16777-032)


24.  大肠杆菌O111:B4中的脂多糖(LPS,Sigma-Aldrich,目录号:L3024)**


25.  干扰素-γ(干扰素γ,研发系统,目录号:485-MI-100)**


26.  Accutase(STEMCell Technologies,目录号:07922)


27.  胰蛋白酶EDTA(0.05%)(Thermo Fisher Scientific,目录号:2530062)


28.  磷酸盐缓冲盐水(赛默飞世尔科技公司,目录号:14190144)


29.  L929条件培养基(见配方)


30.  BMDM维护介质(见配方)


31.  BMDM分化培养基(见配方)


32.  L929培养基(见配方)


笔记:


a。*任何有足够健康骨髓的小鼠模型都可以使用。


b。**根据所需刺激方案选择。LPS和IFN-γ刺激可诱导经典激活。


 


B、 代谢物和脂质提取及样品制备


1.     移液管尖端1000μl,200μl(VWR,目录号:89079-470,89079-478)


2.     1.5 ml塑料管(VWR,目录号:89000-028)


3.     玻璃瓶(仅用于LC-MS级溶剂,见注释)


4.     LC-MS聚丙烯瓶(赛默飞世尔科技公司,目录号:03-377-299)


5.     LC-MS玻璃瓶(Thermo Fisher Scientific,目录号:03-FIRVA)


6.     弹簧帽(赛默飞世尔科技公司,目录号:C4011-53)


7.     聚丙烯微型离心管(VWR,目录号:89000-028)


8.     玻璃瓶(惠顿,目录号:224740)


9.     氮气


10.  富丁醛-347号LC产品目录


11.  LC-MS级甲醇(费希尔化学,目录号:A456-4)


12.  正庚烷LC-MS级(Sigma-Aldrich,目录号:1036541000)


13.  LC-MS级乙酸乙酯(Fisher Scientific,目录号:E195-4)


14.  LC-MS级醋酸(Fisher Chemical,目录号:A11350)


15.  LC-MS级水(赛默飞世尔科技公司,目录号:W64)


16.  三丁胺(Sigma-Aldrich,目录号:90780)


17.  LC-MS级醋酸铵(Fisher Scientific,目录号:A114-50)


18.  乙腈LC-MS级(赛默飞世尔科技公司,目录号:A955-4)


19.  异丙醇(赛默飞世尔科技公司,目录号:A461-4)


20.  SPLASH LipidoMIX内标(Avanti极性脂质,目录号:330707)


21.  冰


 


C、 LC-MS分析


1.     Acquity UPLC BEH C18色谱柱,130汔,1.7µm,2.1 mm x 100 mm(Waters,目录号:186002352)


2.     Acquity UPLC BEH C18 VanGuard Pre-column,130?1.7µm,2.1 mm x 5 mm(Waters,目录号:186003975)


3.     Acquity UPLC CSH C18色谱柱,130峎,1.7µm,2.1 mm x 100 mm(Waters,目录号:186005297)


4.     Acquity UPLC CSH C18 VanGuard Pre-column,130?1.7µm,2.1 mm x 5 mm(Waters,目录号:186005303)


5.     溶剂B(100%LC-MS级甲醇)


6.     溶剂A(见配方)


7.     溶剂C(见配方)


8.     溶剂D(见配方)


 


设备


 


1.     手术剪(Fisherbrand,目录号:08-935)


2.     手术钳(BrainTree Scientific,目录号:FC03-41)


3.     Thermo Q-精密四极轨道质谱仪(Thermo-Scientific)与Vanquish Horizon UHPLC(Thermo Scientific)耦合


4.     氮气流样品浓缩器(Techne,目录号:FSC400D),配备127 mm针(Techne,目录号:F7210)


5.     Vortexer(VWR,目录号:10153-838)


6.     微型离心机(Beckman Coulter,型号:Microfuge 20,目录号:B31599)


7.     台式离心机(Thermo,型号:IEC Centra CL2)


8.     -20 °C冷冻机


9.     4 °C冰箱


10.  标准通风柜


11.  移液管(P1000,P200)


12.  无菌细胞培养罩


13.  细胞培养箱(37°C,5%二氧化碳)


14.  伯爵夫人II手机计数器(AMQAX1000)


 


软件


 


1.     热电科学XCalibur 4.1


2.     马文(682节)(http://genomics-pubs.princeton.edu/mzroll/index.php)(Melamud等人,2010年;Clasquin等人,2012年)


3.     MZMine2型(http://mzmine.github.io/)(Pluskal等人,2010年)


4.     MSConvertGUI(蛋白质向导,http://protowizard.sourceforge.net/download.html)(Kessner等人,2008年)


5.     利必得(https://github.com/coongroup/LipiDex)(Hutchins等人,2018年)


 


程序


 


A、 分离骨髓细胞并分化为巨噬细胞(图1)


有关骨髓分离的有用演示,请参阅其他资源(Ying等人,2013年)。


 






图1。BMDM分化方案的时机选择。TC治疗:组织培养治疗;BMDM:骨髓源性巨噬细胞。


 


1.     用快速颈椎脱位法安乐死小鼠。


2.     用70%乙醇彻底喷洒小鼠,放入无菌罩内。


3.     采用无菌技术,从小鼠每只腿上分离股骨和胫骨。


4.     用浸泡在乙醇中的锋利剪刀,切断每个关节附近的骨头。


5.     用一根27.5克的针头连在10毫升注射器上,用冷的无菌BMDM分化培养基冲洗骨头,放入10厘米的培养皿中,直到骨腔呈白色。使用尽可能多的介质来充分冲洗骨骼。


6.     用移液管彻底吸取细胞悬液,使骨髓细胞完全复苏。


7.     为了去除任何肌肉或骨骼碎片,通过70µm过滤器将细胞过滤到50 ml无菌锥形离心管中。


8.     在500 x g,4°C条件下离心试管3分钟。


9.     弃去上清液,在100 ml BMDM分化培养基中重新悬浮细胞颗粒。


10.  在每个培养皿上放置10毫升的细胞悬液(非组织培养处理过的培养板在这里很重要,因为巨噬细胞会强烈地附着在处理过的培养皿上)


11.  在细胞培养箱中放置3天。


12.  镀后4天从细胞中吸取培养基,用10ml新鲜BMDM分化培养基代替。在第5天和第6天重复。


13.  电镀后7天从细胞中吸取培养基,并在每个培养皿中添加4毫升的Accutase。放回培养箱中约5分钟。


14.  用移液管,轻轻地将细胞从板上移开,并将所有细胞合并在一个50毫升的锥形管中。


15.  降速(1000 x g,5分钟)并重新注入20毫升BMDM维护介质中。


16.  用0.4%台盼蓝1:1染色计数细胞。


17.  在BMDM维持培养基和TC处理过的细胞培养板(6孔板的每孔2 ml)中使细胞再悬浮至2.5 x 105个细胞/ml。对于每种情况,建议三个重复(例如,6孔板的3个孔)。应指定额外的孔用于蛋白质或细胞计数正常化。


18.  细胞会在几个小时内粘在盘子上。贴壁后,细胞准备好进行刺激和/或提取。为了刺激巨噬细胞,用50ng/ml的LPS和10ng/ml的IFN-γ,或其他感兴趣的刺激物,持续一定时间。


 


B、 极性代谢物和脂质的分离(图2)


 






图2。提取脂质和极性小代谢物的工作流程。ACN:乙腈,IPA:异丙醇。


 


1.     在开始之前,请制定以下解决方案:


3: 11-丁醇:甲醇(LC-MS级),含1%SPLASH LipidoMIX内标(SPLASH)


3: 1庚烷:乙酸乙酯(LC-MS等级)


1%乙酸(LC-MS级)


65:30:5秒硝基甲苯:异丙醇:水(LC-MS等级,ACN:IPA公司:水)


2.     冷却3:1丁醇:甲醇(+飞溅)和3:1庚烷:乙酸乙酯在开始之前,在-20°C和1%乙酸中在4°C中放置至少一小时。


3.     从细胞中吸取培养基。


4.     用室温PBS清洗电池两次。


5.     添加300微升-20°C 3:1丁醇:甲醇(+飞溅)对于每个井,立即将板放在冰上。


6.     在保持细胞板在冰上的同时,使用细胞刮板刮取细胞,并将溶液从每个孔转移到一个单独的1.5毫升塑料微型离心管中。将样品放在冰上。


注意:同样,通过对空管/板执行相同的步骤B1-B5来执行程序空白(N>2).


7.     将每个试管旋转1分钟,放回冰上。


8.     在通风柜中,添加300µl-20°C 3:1庚烷:乙酸乙酯每个样品。


9.     旋涡所有管3分钟,放回冰上。


10.  在通风柜中,向每个样品添加300µl 1%乙酸。


11.  旋涡所有管3分钟,放回冰上。


12.  离心分离所有试管(20000 x g,10分钟,4°C)–应形成两个单独的层。


13.  使用移液管,将固定体积(~400µl)的顶部有机层转移到玻璃小瓶中(这是脂质和脂肪酸部分)(在通风橱中进行)


14.  使用移液管,将固定体积(~300µl)底部水层转移到1.5 ml塑料管(这是极性代谢物部分)(在通风橱中进行)


15.  使用通风柜中的N2样品浓缩器通过氮气流完全干燥所有样品。样品通常在室温下干燥2小时。干燥后立即取回样品。


16.  在LC-MS级水(50µl)中再悬浮代谢物部分,在65:30:5中重新悬浮脂质部分ACN:IPA公司:水(50µl)。


17.  将代谢物部分转移到聚丙烯LC-MS小瓶中,并将脂质部分转移到LC-MS玻璃瓶中,加盖并放置在LC-MS自动进样器中进行分析。


 


C、 液相色谱-质谱法


1.     LC-MS小瓶应按随机顺序放置在样本托盘中。样品运行应首先注入至少三个适当空白的等量溶液:极性代谢物的LC-MS H2O,65:30:5ACN:IPA公司:H2O代表脂质。另外,应在至少每10个样品之间进行空白试验。


2.     极性代谢物和脂质组分的分析在热Q-精密质谱仪上进行,该质谱仪与Vanquish Horizon UHPLC耦合。


3.     在100 x 2.1 mm 1.7uM Acquity UPLC BEH C18色谱柱(Waters)上分离极性代谢物样品,并将其连接到130?1.7µm、2.1 mm x 5 mm Acquity UPLC BEH C18 VanGuard预柱(Waters)上。在0.2ml/min流速下使用溶剂A(97:3H2O:甲醇,10 mM TBA,9mM醋酸盐,pH 8.2)和溶剂B(100%甲醇)的梯度。梯度为:0分钟,5%B;2.5分钟,5%B;17分钟,95%B;21分钟,95%B;21.5分钟,5%B(图3A)。每种代谢物样品注入5µl进行分析。在全扫描阴性模式下收集数据。详细的MS参数如表1所示。


4.     在100 x 2.1 mm 1.7 um Acquity UPLC CSH C18色谱柱(Waters)上分离脂质样品,并将其与130?1.7µm、2.1 mm x 5 mm Acquity UPLC CSH C18 VanGuard预柱耦合。溶剂C的梯度(70:30ACN:水,10mM NH4Ac)和溶剂D(90:10IPA:ACN公司,10 mM NH4Ac)在0.4 ml/min流速下使用。梯度为:0分钟,2%D;2分钟,2%D;5分钟,30%D;19分钟,85%D;20分钟99%D;27分钟,99%D;28分钟,2%D;32分钟,2%D(图3B)。每种脂质样品注入5µl进行分析。使用数据相关的MS2(ddMS2)分析,样本运行两次,一次为阴性模式,一次为阳性模式。详细的MS参数如表1所示。


 


表1。质谱参数


 


极性代谢物


脂质/脂肪酸


全扫描


全扫描


ddMS2型


充电


否定的


正/负


分辨率


7万


70万


17.5公里


最大喷射时间


100毫秒


100毫秒


80毫秒


自动增益控制


3E6型


3E6型


5E5型


辅助气体流量


10


12


鞘气流量


35


40


扫气流速


2


1


喷雾电压


3.2千伏


3.5千伏


毛细管温度


320摄氏度


340


加热器温度


300摄氏度


350


循环计数


-


-


5


隔离窗


-


-


1.4米/z


(N) CE/阶梯式


-


-


30, 40


 






图3。溶剂梯度和洗脱曲线。A、 LC-MS极性代谢物法的溶剂梯度和实例洗脱曲线。B、 溶剂梯度和实例洗脱曲线,用于LC-MS脂质分析,显示了正、负电离曲线。


 


数据分析


 


MS1数据用于使用MAVEN软件进行极性代谢物和游离脂肪酸定量(Melamud等人,2010年;Clasquin等人,2012年)。使用LipiDex软件,基于全扫描(ddMS2)识别和量化脂质数据(Hutchins等人,2018年)。两种方法如下所述。图4显示了从非刺激(M0)和LPS+IFNγ刺激的BMDM(M1)的代谢物和脂质谱分析中获得的代表性数据。


 






图4。代表性代谢物和脂质数据。 与未刺激的BM相比,脂类代谢产物(BM 0)在脂类(BM)刺激下(BM 0)的代谢产物(BM 0)中被鉴定为γ-DM。重点介绍衣康酸盐和琥珀酸盐,这两种免疫调节代谢物已知在M1巨噬细胞中动态上调(Jha等人,2015年;Cordes等人,2016年;Lampropoulou等人,2016年;Mills等人,2016年)。同样突出显示的是甘油三酯50:2。甘油三酯积聚也显示在M1巨噬细胞中(Köberlin等人,2014;Lee等人,2017;Feingold等人,2012)。


 


极性代谢物和脂肪酸


极性代谢物和脂肪酸通过匹配预期的准确质量和保留时间以有针对性的方式进行量化。保留时间和M/z的列表是通过对代谢物标准品的分析确定的,使用与上述样品相同的LC-MS方法,并在补充信息中(补充文件“Polar Descellators RT list.xlsx”)。我们建议,特别是对于感兴趣的代谢物,用户应自行制定标准。为此,在适当的样品溶剂中将标准品稀释至一定浓度范围(例如,1μM、10μM、100μM、1000μM,进样体积为1μl),并使用相应的方法在LC-MS上运行。关于非目标代谢组学分析,请参阅其他资源(Smith等人,2005和2006;Kind等人,2018;Wang等人,2019)。此外,该方法仅描述代谢物相对定量所需的内容。有关代谢物绝对浓度的测量,见其他资料(Bennett等人,2008)。我们使用MAVEN软件对峰值进行集成(Melamud等人,2010年;Clasquin等人,2012年)。本软件及其更详细的使用指南可在这里找到。


 


1.     使用MSConvertGUI将热生成的.raw文件转换为.mzXML文件(Holman等人,2014;Adusumilli等人,2017)。


2.     在MAVEN中打开.mzXML文件。正确加载样本后,ID将出现在左侧的“样本”窗口中。


3.     确保“复合小工具”已打开。通过点击左边的复合选项卡并选择“加载自定义复合列表”来加载复合列表。在.csv文件中加载已知的m/z和代谢物标准生成的保留时间或补充材料中提供的保留时间。调整Maven窗口右上角的ppm窗口,以匹配您的仪器的灵敏度。


4.     加载化合物列表后,单击感兴趣化合物的名称,每个样品中对应于该代谢物的峰将出现在主窗口中。红线对应于根据导入化合物列表观察到的保留时间(图5)。要保存所选峰的数据,双击峰-化合物名称和相应参数将显示在软件内的列表中。仅保存那些保留时间与化合物列表上的匹配的峰值的数据,具有相对平滑的轮廓,并且其信号高于期望的截止(例如,5E4和至少2x倍的空白)。如图5所示。


注:有关软件功能和参数设置的详细信息,请参阅MAVEN使用手册。


5.     保存了所有需要的峰值后,从Maven中导出这些数据,方法是在保存的峰值列表上方选择“export peaks to SpreadSheet(.csv)”。


6.     通过从每个样本的信号中减去空白处的平均信号,使数据空白。随后将数据标准化为细胞计数或蛋白质。


注:只比较同一序列内样品的相对丰度,因为仪器条件随时间的变化可能导致信号响应的差异。如果需要绝对定量的代谢物浓度,建议使用同位素内标。


 






图5。良好LC-MS峰的示例。 Maven软件显示未刺激(M0)和LPS+IFNγ刺激(M1)BMDM的琥珀酸峰。




 


脂类


使用开放式LipiDex软件进行脂质识别和量化(Hutchins等人,2018年)。基于全扫描ddMS2数据,LipiDex识别脂质。该软件与MZmine2耦合以量化脂质种类。以下是我们的分析工作流程(Pluskal等人,2010年)。


1.     将热生成的.raw文件转换为.mgf文件(我们使用MSConvertGUI软件[Holman等人,2014;Adusumilli等人,2017])。


2.     在LipiDex中执行频谱搜索


a、 打开LipiDex,进入频谱搜索。


b、 上传.mgf文件。


c、 启用适当的库。


d、 指定所需的搜索参数(例如,MS1搜索容差:0.01,MS2搜索容差:0.01)。


e、 单击“光谱搜索”–这将生成带有标识的结果.csv文件。


3.     使用MZmine2生成色谱特征表。分别进行正、负电离运行。按照此处提供的分步说明进行操作。


4.     在LipiDex中执行峰值查找


a、 打开LipiDex,进入Peak Finder。


b、 单击MZMine2选项。


c、 加载MZMine2生成的正负极性峰值表。


d、 上传MS/MS.csv结果文件,确保文件具有适当的极性(正极或负极)。


e、 例如,使用以下命令设置所需的过滤参数:


MS2过滤参数:最小脂质光谱纯度(%):75,最小MS2搜索点积:500,最小MS2搜索Rev。Dot产品:700。


特征关联参数:半高宽窗乘数:2.0,最大质量差(ppm):15。


结果过滤参数:检查加合物/二聚体过滤和源内片段过滤。检查最大RT M.A.D系数为3.5。在n个文件中找到的功能:2。


f、 点击识别色谱峰-这将产生.csv文件,其中包含已识别的脂质种类及其量化。


5.     空白数据使用与样本平行运行的程序空白,并将数据标准化为细胞计数或蛋白质含量。


6.     质量控制:SPLASH LipidoMIX内标是同位素标记脂质的混合物,代表主要的脂类。在初始提取缓冲液(3:11)中加入飞溅物(1%)-丁醇:甲醇). 在所有分析过的脂质样品中都存在这种内标,可以测定样品的保留时间漂移和样品回收的潜在差异。一个序列内的保留时间漂移通常<0.1min,内标的变化<20%。如果差异显著,可以将数据标准化为同一类脂的内标或最近的保留时间(如果没有同类标准)。见补充文件“SPLASH LipidoMix Stds化合物列表.xlsxSPLASH LipidoMIX中化合物的列表及其各自的保留时间。


 


笔记


 


1.     在干净的指定玻璃器皿中保存并测量所有LC-MS级溶剂。请勿将此玻璃器皿用于其他用途,也不要用自来水或肥皂清洗。残留的清洁剂和其他物质会造成背景。必要时,仅用高效液相色谱级溶剂冲洗。


2.     在提取溶剂和油脂提取过程中使用的塑料会产生背景,尤其是脂肪酸。始终执行一个程序空白,以适当说明此背景信号。如果观察到塑料移液管和微量移液管的使用量,可以减少使用玻璃管和移液管。


 


食谱


 


1.     BMDM分化培养基


不含谷氨酰胺的RPMI 1640


10%FBS


20%L929条件培养基


10 U/ml青霉素/链霉素


25毫米HEPES


2毫米谷氨酰胺


2.     BMDM维护介质


不含谷氨酰胺的RPMI 1640


10%dFBS


10 U/ml青霉素/链霉素


25毫米HEPES


2毫米谷氨酰胺


20 ng/ml m-CSF


3.     L929培养基


DMEM高糖


10 U/ml青霉素/链霉素


10%FBS


4.     L929条件培养基


a、 L929细胞在L929培养基中培养至完全融合


b、 胰蛋白酶化细胞,在1000 x g下旋转5分钟


c、 吸取胰蛋白酶,在L929培养基中重新悬浮细胞颗粒


d、 对细胞计数,并重新消耗至8 x 103个细胞/ml


e、 将130毫升该悬浮液加入175平方厘米的烧瓶中,并置于培养箱中


f、 7天后收集所有用过的介质


g、 在1000 x g,4°C下离心5分钟。使用0.45μm过滤器将无菌过滤器放入无菌玻璃瓶中


h、 等分份放入50毫升塑料离心管中,并在-20°C下冷冻至需要使用(6个月内使用)


5.     溶剂A


97:3分水:甲醇(LC-MS等级)


10毫米三丁胺


乙酸(LC-MS级),pH值为8.2


注:这需要大量的剧烈混合才能完全溶解三丁胺并达到稳定的pH值。


6.     溶剂C


70分30秒二氧化钛:水(LC-MS等级)


10mm醋酸铵(LC-MS级)


250µl/l醋酸(LC-MS级)


7.     溶剂D


90:10是丙丙醇:乙腈(LC-MS等级)


10mm醋酸铵(LC-MS级)


250µl/l醋酸(LC-MS级)


 


致谢


 


骨髓源性巨噬细胞分离、代谢物和脂质提取、LC-MS分析和数据分析的方法在之前的工作基础上进行了调整和优化,每个方法都在相应章节中引用。


 


相互竞争的利益


 


作者声明没有利益冲突。


 


伦理学


 


动物护理和实验程序按照威斯康星大学麦迪逊机构动物护理和使用委员会批准的方案进行。




 


工具书类


 


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8.     Gika,H.G.,Theodoridis,G.A.,Plumb,R.S.和Wilson,I.D.(2014年)。液相色谱-质谱法在代谢组学和代谢组学中的应用现状。药学与生物医学杂志87:12-25。


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17.  Lee,J.W.,Mok,H.J.,Lee,D.Y.,Park,S.C.,Kim,G.S.,Lee,S.E.,Lee,Y.S.,Kim,K.P.和Kim,H.D.(2017年)。基于UPLC-qq/MS的脂质组学方法研究炎性巨噬细胞的脂质改变。蛋白质组学研究16(4):1460-1469。


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引用:Seim, G. L., John, S. V. and Fan, J. (2020). Metabolomic and Lipidomic Analysis of Bone Marrow Derived Macrophages. Bio-protocol 10(14): e3693. DOI: 10.21769/BioProtoc.3693.
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