Electrospray Ionization Mass Spectrometry (ESI-MS)

       Electrospray ionization mass spectrometry (ESI-MS) is a highly sensitive method that has been widely used for the selective characterisation and quantification of lipid molecular species from various biological samples. In the traditional analytical approach to lipid analysis using gas chromatography, chemical derivatization is often required prior to analysis to increase the volatility of lipid species. In contrast, ESI is a mild ionization technique that generates intact molecular ions from solution with relatively little fragmentation and no derivatization is required.

       This example describes an ESI-MS method to analyse lipids following an approach of shotgun lipidomics. This uses using electrospray ionization tandem MS for the analysis of lipid molecular species directly from crude biological extracts of tissue or fluids.

     A. Reagents and Equipment

         Reagents and reagent preparation

  • HPLC-grade methanol and chloroform. Prepare the lipid extraction solvent by combining two parts chloroform with one part methanol.
  • MS-grade ammonium acetate.
  • Lipid standards: PC (14:0/14:0, 14:0/16:0, 14:0/18:0,17:0/17:0, 18:0/18:0, 18:1/18:1, 18:2/18:2, 18:3/18:3, 23:0/23:0), LPC (14:0, 15:0, 16:0, 17:0, 18:0, 18:1, 26:0), PE (16:0/16:0, 17:0/17:0, 18:0/18:0), and LPE (14:0, 18:0).
  • Total bovine liver lipid extract.
  • Rat plasma.
  • Dry ice.
  • Liquid nitrogen.
  • Nitrogen gas source.

         Equipment

  • TSQ Vantage triple quadrupole mass spectrotometer.
  • Barnstead Nanopure Infinity water system.
  • Syringe pump
  • Syringes of various volumes.
  • Glass vials and tubes for extraction, agitation, and centrifugation with Teflon-covered liner screw caps or siliconised, low-retention, individually wrapped and sterilised Eppendorf tubes.
  • Centrifuge.
  • 8 mm crimp-top, conical-bottom, 700 μl glass vials with 8-mm natural caps and Teflon septa.
  • 2 ml glass vials with Teflon-lined caps.
  • 8 ml glass vials with Teflon-lined caps.
  • 100 ml volumetric flask.

     B. Assay Procedure

 1. Infusion Solvent Preparation

  1. Dissolve 2.312 g ammonium acetate in a 100 ml volumetric flask and dilute to mark with distilled water to make a final concentration of 300 mM.
  2. Mix chloroform, methanol, and 300 mM Mammonium acetate aqueous solution in a volume ratio of 30/66.5/3.5, respectively. The concentration of ammonium acetate in the final infusion solvent is 10.5 mM.

 2a. Lipid Standard Preparation - Liquid Standards

  1. For lipid standards received in chloroform at 2.5 mg/ml, add 40 μl of each standard to 2 ml glass vials.
  2. Evaporate the chloroform under a constant nitrogen gas stream and then add 1 mL of the infusion solvent to prepare 0.1 mg/ml stock solutions.
  3. Prepare working standard solutions by diluting the stock solutions to 1 nmol/ml with the infusion solvent based on the molecular weights of the individual lipid standards.
  4. Store all stock and working standard solutions in Teflonlined, screw cap glass vials at –80 and –20◦C, respectively, until analysis.

 2b. Lipid Standard Preparation - Powder Standards

  1. For lipid standards received in powder form, prepare 0.1 mg/ml stock solutions by weighing the standards (0.50 ± 0.01 mg) and dissolving in 5 ml of infusion solvent in 8 ml Teflon-lined, screw cap glass vials.
  2. Prepare working standard solutions by diluting the stock solutions to 1 nmol/ml with the infusion solvent based on the molecular weights of the individual lipid standards.
  3. Store all stock and working standard solutions in Teflon-lined, screw cap glass vials at –80 and –20◦C, respectively, until analysis.

 3. Total Bovine Liver Lipid Extract Preparation.

  1. The total bovine liver lipid extract was received at a concentration of 2.5 mg/ml in chloroform.
  2. Add 40 μL of the bovine liver lipid extract to a 2 ml glass vial.
  3. Evaporate the chloroform under a constant nitrogen gas stream and then add 1 ml of infusion solvent to prepare a 0.1 mg/ml stock solution.
  4. Prepare a working bovine liver lipid extract of 0.25 μg/ml by diluting 5 μL of the above stock solution with 1995 μL of the infusion solvent in a 2 ml glass vial.
  5. Store all stock and working standard solutions in Teflon-lined, screw cap glass vials at –80 and –20◦C, respectively, until analysis.

 4a. Lipid Extraction - Plasma

  1. Add 25 μl of rat plasma to a sterile siliconised 0.6 ml Eppendorf tube.
  2. Add 200 μl of cold (–20◦C) chloroform/methanol (2:1).
  3. Vortex the sample for 30 s at room temperature.
  4. Allow the sample to stand for 5 min at room temperature followed by vortexing for 30 s.
  5. Centrifuge the sample at 13,000 × g for 5 min at room temperature to separate precipitated protein.
  6. Pierce the protein disc with a pipette tip, collect the lower organic phase, and transfer it to a new 700 μl glass vial.
  7. Discard the protein interface and upper aqueous phase.
  8. Evaporate the organic phase under vacuum or constant nitrogen gas stream and store at –80◦C.
  9. Immediately prior to analysis, dissolve the evaporated lipid extract in a final volume 20 times that of the original volume of plasma using the infusion solvent.
  10. Add synthetic lipid internal standards 14:0 LPC, 17:0 LPC, 14:0/14:0 PC, and 23:0/23:0 PC to make a final concentration of 1 μM.
  11. Centrifuge the sample at 350 × g for 5 min to remove any particulates prior to MS analysis.

 4b. Lipid Extraction - Tissue

  1. When possible, freeze fresh tissue samples (brain, liver, heart, etc) in liquid nitrogen.
  2. Grind the frozen tissue in a dry ice-chilled mortar and pestle.
  3. Transfer the ground tissue to glass tubes with Teflon-lined screw caps.
  4. Homogenise the tissue with chloroform/methanol (2:1) to a final volume 20 times the weight of the original tissue sample (e.g., 0.5 g in 10 ml of solvent).
  5. Agitate the sample for 30 min in an orbital shaker at room temperature.
  6. Centrifuge the homogenate at 9000 × g for 10 min, recover the supernatant, and transfer it to a new glass tube with glass pipette.
  7. Wash the supernatant with 0.2 volumes (e.g., 2 ml for 10 ml) of water.
  8. Vortex the sample for 30 s and then centrifuge at 1000 × g for 5 min to separate the two phases.
  9. Remove and discard the upper phase and then rinse the interface one or two times with methanol/water (1:1), without mixing the whole preparation.
  10. After centrifugation at 350 × g for 3 min, remove and discard the upper phase.
  11. Evaporate the lower organic phase containing lipids under vacuum or a constant nitrogen gas stream, then store at –80◦C.
  12. Immediately prior to analysis, reconstitute the lipid extracts in infusion solvent to any desired final volume depending on the weight of the original tissue sample and the amount of material that would be infused to the mass spectrometer.
  13. Centrifuge the sample at 350 × g for 5 min to remove any particulates prior to MS analysis.

 5. Mass Spectrometric Analysis

  1. ESI-MS analyses are performed on a tandem quadrupole mass spectrometer equipped with an electrospray ion source and operated in both positive- and negative-ion mode.
  2. Load sample solutions in a gastight 250 μl Hamilton Syringe and deliver them to the mass spectrometer by a syringe pump through a 100 cm capillary transfer line of 150 μm internal diameter and 360 μm outer diameter at a flow rate of 0.3–0.5 μl/min. The construction of the transfer line and the flow rate may differ depending on the ion source and mass spectrometer being used.
  3. Flush the capillary with infusion solvent after each sample infusion to prevent sample to sample cross-contamination.
  4. For full scan MS, set the ion spray voltage to 2.2 kV and the capillary temperature to 200◦C. Collect a 1 min period of signal in profile mode. The ions produced from the full scan mode can be selected for product ion scan.
  5. For tandem MS, set the collision gas pressure to 1.5 mTorr. Use collision energies of 40 eV for LPC, PC, SM and 35 eV for LPE and PE.  Collect 1–6 min of signal in profile mode.
  6. Isobaric species (lipid species with the same m/z value) from different lipid classes can be resolved by PIS and NLS. In contrast, it is impossible to identify isobaric species from full scan MS. For example, the standard lipid species 15:0 LPC and 18:0 LPE have the same chemical formula C23H48NO7P and hence produce the same m/z at 482.32 [M+H]+ and 504.31 [M+Na]+, respectively. PIS of 184.1 only produces the parent ion 15:0 LPC, and NLS 141 produces only the parent ion 18:0 LPE.
  7. Data processing and quantification can be performed using the open source lipid mass spectrum analysis (LIMSA) software. LIMSA is a Microsoft Excel add-on that can perform peak finding, integration, assigning, isotope correction, and quantification with internal standards in an imported MS spectrum. It is available as a free download at http://www.helsinki.fi/science/lipids/software.html.

     C. Notes

  1. Chloroform is a carcinogen and necessary precautions should be taken to limit human exposure. Always work in a fume hood.
  2. All volume measurements for organic solvents should be carried out using glass syringe needles. Do not use plastic pipette tips for organic solvent volume measurements to avoid the danger of introducing polymers and any source of contamination leaching from the plastic pipette tips.
  3. Containers should be resistant to organic solvents. Never use rubber, cork, polyethylene, or Parafilm.
  4. Pierce the protein disc carefully and try to exclude the contamination of the lower organic phase by the upper phase containing salt and other water-soluble materials.
  5. If the 700 μl glass vial does not fit into the centrifuge or the vacuum concentrator, place it in 1.5 ml Eppendorf tube prior to centrifugation or vacuum concentration.
  6. Other triple quadrupole mass spectrometers (such as ABI Sciex API 3000, 4000, 5000, 5500; Waters Quattro Premier XE, ACQUITY TQD, and Agilent 6400 series) capable of performing CID (PIS and constant NLS) and equipped with electrospray ionization can be used.
  7. Optimum infusion flow rates should be optimized depending on the ion source and mass spectrometer being used.
  8. To save time and facilitate the flushing, push the syringe by hand.
  9. The fragmentation of each lipid molecular species depends on the applied collision energy and needs to be optimized depending on each lipid class and type of mass spectrometer used.