Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

All solutions should be prepared with ultrapure water and HPLC grade methanol and chloroform. Prepared solutions that contain organic solvents such as methanol, chloroform, or pyridine and concentrated acids or bases should be prepared and used under a chemical fume hood. All solutions can be stored at RT. Solvents should be measured in a graduated glass cylinder and stored in glass solvent bottles with PTFE lined caps. For long-term storage chloroform-containing solvents should be stored in tinted amber glass bottles to avoid the production of phosgene, a highly reactive acid chloride. PTFE centrifuge tubes and rotary evaporator flasks should be rinsed with methanol and chloroform before use. Follow necessary federal, state and/or institutional waste disposal regulations when disposing of solvents and/or radioactive waste.

1. Large Scale Lipid A Extraction (50 ml to 1.5 L)

1. Prepare a single-phase Bligh-Dyer mixture: chloroform-methanol-1X phosphate buffered saline (PBS), pH 7.4; 1:2:0.8 v/v). Combine 200 ml of chloroform, 400 ml of methanol and 160 ml of PBS in 1 L solvent bottle. Cap bottle and mix by inversion (>30x). Loosen the cap periodically during mixing to vent and store sealed.
2. Prepare a pre-equilibrated two-phase Bligh-Dyer mixture- chloroform:methanol:water (2:2:1.8 v/v). Combine 400 ml of chloroform, 400 ml of methanol and 180 ml of water in 1 L solvent bottle. Cap bottle and mix by inversion, making sure to loosen cap periodically during mixing to vent. Let equilibrate O/N and store sealed.
3. Prepare the mild acid hydrolysis buffer (50 mM sodium acetate, pH 4.5; 1% sodium dodecyl sulfate (SDS)). For 500 ml of mild acid hydrolysis buffer, weigh 2.05 g of sodium acetate and transfer to a 500 ml beaker. Add water to a volume of ~350 ml and stir, then add 50 ml of 10% SDS. Mix and adjust pH to 4.5, transfer to a graduated cylinder, and add water to 500 ml.
4. Prepare chloroform:methanol (4:1, v/v): Measure 100 ml of chloroform and transfer to solvent bottle. Measure 25 ml of methanol and mix with chloroform. Store in amber glass bottle.
5. Inoculate 5 ml of media (Luria broth or other) from a single bacterial colony. Grow O/N at 37 °C, or at required °C for growth. A diagram of the extraction procedure is shown in Figure 2.
6. The next day, measure the OD₆₀₀ and use the 5 ml O/N culture to inoculate 200 ml of culture to a starting OD₆₀₀ of 0.05. Grow cells until an OD₆₀₀ of 0.8-1.0 is reached.
7. Harvest cells via centrifugation at 10,000 x g for 10 min. Longer spins may be required for strains that pellet poorly. Pour off media supernatant.
8. Wash cell pellet with 50 ml of 1x phosphate buffered saline (PBS). Repeat centrifugation to pellet cells. Pour off supernatant and store cell pellet at -20 °C, or proceed to step 1.9.
9. Resuspend cells in 40 ml of 1x PBS and divide between two 250 ml PTFE centrifuge tubes (yielding 20 ml of cell suspension/tube). Add 25 ml of chloroform and 50 ml of methanol to each tube, for a single phase Bligh-Dyer (chloroform: methanol:water; 1:2:0.8 v/v) (Figure 2). Mix by inversion and incubate at RT for >20 min to ensure complete cell lysis.
10. Centrifuge the mixture at 2,000 x g for 20 min. LPS will pellet along with proteins and nucleic acids (Figure 2); however, phospholipids, isoprenyl lipids, and small, hydrophobic peptides will remain in the supernatant. Discard the supernatant.
11. Wash the LPS pellet with ~100 ml single phase Bligh-Dyer mixture. Centrifuge at 2,000 x g for 20 min. Discard supernatant. When isolating lipid A from E. coli or Salmonella, only one wash is required; however, additional wash steps may be needed to reduce phospholipid contamination when isolating lipid A from some organisms.
12. Add 27 ml of mild acid hydrolysis buffer (50 mM sodium acetate, pH 4.5; 1% SDS; see step 1.3) to LPS pellet, and mix by pipetting up and down until only small particles remain. Sonicate to homogeneously resuspend LPS pellet in solution with probe tip sonicator at a constant duty cycle for 20 sec at 50% output. Repeat sonication of sample 2x (20 sec/burst, ~5 sec between bursts).
13. Boil samples in a water bath for 30 min. Caution: make sure the caps are tight, but not fully sealed. Some organisms, like Vibrio cholerae (V. cholerae) require longer incubation times (1 hr) to increase overall yield of lipid A. Remove bottles from water bath and allow sample to cool to RT before proceeding.
14. To extract lipids after hydrolysis, convert the SDS solution into a two-phase Bligh-Dyer (Figure 2) mixture by adding 30 ml of chloroform and 30 ml of methanol, for a chloroform:methanol:water (2:2:1.8, v/v) mixture. Mix by inversion and centrifuge the sample for 10 min at 2,000 x g. Extract the lower phase into a clean PTFE centrifuge tube using a glass pipette.
15. Perform a second extraction by adding 30 ml of lower phase from a pre-equilibrated two-phase Bligh-Dyer mixture (step 1.2), to the upper phase from step 1.14. Mix, then centrifuge at 2,000 x g for 10 min. Extract the lower phase, and pool with the lower phase extracted in step 1.14.
16. Wash the pooled lower phases (60 ml total) by adding 114 ml of pre-equilibrated two phase Bligh-Dyer upper phase (step 1.2) to create a two-phase Bligh-Dyer mixture (chloroform:methanol:water; 2:2:1.8, v/v). Mix. Centrifuge at 2,000 x g for 10 min.
17. Remove the lower phase to a clean glass rotary evaporator flask and dry sample using rotary evaporation (Figure 2).
18. Add 5 ml of chloroform:methanol (4:1, v/v) to the rotary flask, and bath-sonicate (>30 sec) to aid in suspension of lipid from sides of flask. Use a glass transfer pipette to transfer lipid to a clean glass tube (13 x 100 mm or larger) capped with PTFE lined phenolic screw caps. Dry sample under a stream of nitrogen using a nitrogen dryer.
19. Resuspend dried lipid in 1 ml of chloroform:methanol (4:1, v/v). Transfer to small glass sample vial (12 x 32 mm) with a tapered base for more quantitative resuspension using small volumes (see Table of Equipment/Reagents). Dry using a nitrogen dryer.
20. Dried sample can be stored at -20 °C until subsequent TLC (Protocol 2) or MS analysis (Protocol 3, 4, or 5). (Note: Amount of material isolated and used in subsequent protocols is suggested based on what’s sufficient for most organisms. The same lipid sample can be used in Protocols 2-5, taking note of % material removed. See Discussion for more details.).

2. Visualization of Lipid A Species via Thin Layer Chromatography

1. Prepare the TLC mobile phase solvent system (chloroform:pyridine:88% formic acid:water; 50:50:16:5 v/v). Combine 200 ml of chloroform, 200 ml of pyridine, 64 ml of 88% formic acid, and 20 of ml water in a 1 L solvent bottle. Cap bottle, mix by inversion multiple times, and vent.
2. Use a TLC tank that will accommodate 20 x 20 cm plates. Line TLC tank with ~40 cm chromatography paper.
3. To TLC tank add 200 ml of chloroform:pyridine:88% formic acid:water (50:50:16:5, v/v) mixture. Allow tank to pre-equilibrate for >3 hr, often O/N is preferred and more convenient.
4. Remove dried lipid samples from freezer (step 1.20) and let warm to RT.
5. With a razor remove the silica from the top edge of a silica gel 60 TLC plate. Using a dull pencil, draw a line parallel to the bottom of the plate, 2 cm from the bottom. This line is the origin for spotting samples. Mark increments along this reference line to spot samples 1 cm apart.
6. Dissolve dried lipid from step 2.4 in 200 μl of chloroform:methanol (4:1, v/v), vortex and sonicate (3x, ~15 sec each), yields ~100-500 ng/μl lipid A if extracted from E. coli.
7. Using a microcapillary glass pipet, spot one-tenth of the volume (20 μl) onto the TLC plate as marked in step 2.5. Allow samples to air-dry on TLC plate for ~15 min. (Note: Samples in small glass vials can be dried using a nitrogen dryer and stored at -20 °C for subsequent use in protocols 3-5. Take note of % material removed).
8. Place TLC plate containing spotted samples into the pre-equilibrated tank. Once solvent front reaches the top of the TLC plate (~2.5-3 hr), remove the plate and air dry (>60 min). A cold-air gun can be used to ensure complete dryness.
9. While plate is drying, turn on hot plate at 250 °C for charring.
10. In a ventilated chemical fume hood, prepare 10% sulfuric acid-ethanol mixture for charring lipids resolved on the silica TLC plate (concentrated sulfuric acid:100% ethanol; 1:9 v/v). Measure 10 ml of sulfuric acid and add slowly to 90 ml of 100% ethanol. Carefully mix and transfer to a glass chromatographic reagent atomizer.
11. In the fume hood, use a glass chromatographic reagent atomizer to spray the dried TLC plate with 10% sulfuric acid-ethanol mixture. Spray mixture evenly across the plate.
12. Place the TLC plate on the 250 °C hot plate until charred lipid samples appear as black/brown spots (<1 min). Do not overexpose the plate, as this will cause the entire plate to turn brown and make visualization of lipid A species difficult.

3. Structural Characterization of Lipid A via MALDI-TOF Mass Spectrometry

1. From step 1.20, (or step 2.7) remove lipid A sample from freezer and let warm to RT. Resuspend dried lipid A sample in ~20 μl chloroform:methanol (4:1, v/v) and vortex to obtain a ~1-5 μg/μl lipid A solution if extracted from E. coli. (Note: 10% less concentrated if material used from step 2.7).
2. Prepare ATT matrix components. Saturated 6-aza-2-thiothymine in 50% acetonitrile: add 500 μl of water and 500 μl of acetonitrile to a 1.5 ml microcentrifuge tube, and then add >10 mg 6-aza-2-thiothymine so that the 50% acetonitrile is super-saturated. Saturated tribasic ammonium citrate solution: Add >1 mg to 500 μl water, precipitate should be readily apparent. Vortex and centrifuge solutions before use; only saturated supernatant is used.
3. Prepare ATT matrix mixture: saturated 6-aza-2-thiothymine in 50% acetonitrile, saturated tribasic ammonium citrate (20:1, v/v). Mix matrix components together by adding 20 μl of ATT solution to 1 μl saturated tribasic ammonium citrate solution in 500 μl microcentrifuge tube, vortex to mix, and centrifuge before applying to MALDI plate.
4. Prepare MALDI plate by adding 0.5 μl calibrant mixture to the MALDI plate on a spot near where the samples will be deposited, to provide the most accurate mass/charge ratio determination.
5. Deposit 0.5 μl of ATT matrix onto MALDI plate on each spot where lipid A sample will be deposited.
6. Deposit 0.5 μl of sample (2.5% of total sample) onto the spot of ATT matrix, to mix on plate, and acquire spectra by scanning sample for optimal ion signals (Figure 3). Note: Amounts for the most optimal MS signal can vary with organism and need to be determined empirically. To add more material one can spot 0.5 μl multiple times allowing spotted mixture to dry in between addition of more sample. Sample can also be concentrated (dry and resuspend in a smaller volume) or diluted as necessary. More starting material (volume of starting cell culture) can also be used (See Discussion).

4. Electrospray Ionization Mass Spectrometry and Collision Induced Dissociation of Lipid A

1. Prepare a chloroform:methanol solvent mixture (1:1, v/v) by mixing a 200 μl stock of HPLC grade methanol with 200 ml HPLC grade chloroform in a glass solvent bottle.
2. Transfer 200 μl of the chloroform:methanol solvent mixture to the vial with lipid A (e.g. from step 1.20, 2.7, or dried after Protocol 3) and sonicate the lipid a solution for 5 min or until all material is dissolved.
3. Set up the mass spectrometer for negative mode electrospray ionization.
4. Using a 250 μl syringe and a syringe pump, directly infuse the diluted lipid A sample at a flow rate of 2.0-3.5 μl/min.
5. Optimize and enhance the lipid A ion signal by tuning the ion optics. A full mass spectrum can now be collected of the lipid A species (Figure 4).
6. Isolate and activate the target lipid A by selecting CID as the MS/MS method.
7. Increase the CID voltage (or normalized collision energy, NCE) until the precursor lipid A species is about 10% relative abundance compared to the highest product ion.
8. Acquire and average spectra until sufficient signal-to-noise is achieved for the product ions. The number of scans needed is dependent on the signal intensity of the original precursor and can range from 3-300 scans (Figure 5A).

5. MS/MS on Lipid A by Ultraviolet Photodissociation

1. Prepare sample and mass spectrometer as in steps 4.1-4.5.
2. Turn on the laser interfaced to the mass spectrometer. The mass spectrometer was outfitted with a 193 nm excimer laser and modified to allow UV activation in the HCD cell of the instrument. Photodissociation was implemented in a manner similar to that described previously³⁵. We use a modified vacuum manifold with a CaF₂ optical window to transmit photons into the higher-energy C-trap dissociation (HCD) cell. The laser is triggered during MS/MS by a transistor-transistor logic (TTL) signal from mass spectrometer to a pulse/delay generator.
3. Set up the instrument software so that the laser will trigger the excimer laser when the ions enter the HCD cell. This requires a modest modification of the software.
4. Turn on the pulse generator such that the laser is pulsed every 2 msec (500 Hz).
5. Isolate the lipid A precursor ion by selecting HCD as MS/MS method and adjust the collisional energy to 1% NCE. This allows the isolation of precursor ions for ultraviolet dissociation within the HCD cell. Although the HCD cell is used, the software is modified to perform UVPD during this interval.
6. Activate the isolated lipid A ion by increasing the laser energy and adjusting the number of laser pulses. A typical UVPD experiment will be performed using ten 6 ml pulses.
7. As in step 4.8, acquire and average spectra until sufficient signal-to-noise is achieved for the UVPD product ions. The number of scans needed is dependent on the signal intensity of the original precursor and can range from 3-300 scans (Figure 5B).

6. ³²P-Labeling of Lipid A and Subsequent Isolation

1. Inoculate 5 ml of media (Luria broth or other media) from a single colony. Grow O/N at 37 °C or at required temperature.
2. The next day, measure the OD₆₀₀ and use the O/N culture to inoculate 7 ml of culture to a starting OD₆₀₀ of ~0.05, grown in a standard 20 x 150 mm disposable glass culture tube. Add 2.5 μCi/ml of inorganic ³²P. Grow cells until an OD₆₀₀ of 0.8-1.0 is reached. Please follow federal, state, and/or institutional guidelines for the safe and proper handling of radioactive materials.
3. Harvest cells in 16 x 125 mm glass centrifuge tubes with PTFE lined cap using a fixed angle clinical centrifuge at 1,500 x g for 10 min. Remove supernatant into appropriate radioactive waste container. All radioactive waste (e.g. liquid, glass, biohazard) generated in subsequent steps should be disposed of in accordance with federal, state, and/or institutional guidelines.
4. Wash cell pellet with 5 ml of 1x phosphate buffered saline (PBS). Centrifuge for 10 min at 1,500 x g. Discard supernatant.
5. Resuspend cells in 5 ml of single phase Bligh-Dyer mixture (Figure 2) consisting of chloroform:methanol:water (1:2:0.8, v/v). Vortex to mix, and incubate at RT for >20 min to ensure complete cell lysis.
6. Centrifuge in clinical centrifuge at 1,500 x g for 20 min. Gently pour off supernatant, which contains phospholipids and isoprenyl lipids.
7. Resuspend the LPS pellet in 1.8 ml of 50 mM sodium acetate, pH 4.5; 1% SDS buffer by vortexing. Sonicate sample using a bath sonicator until pellet is evenly dispersed (~30 sec).
8. Incubate sample for 30 min in boiling water bath. Make sure caps are tight but not sealed.
9. Remove from water bath and allow sample to cool at RT for 5-10 min.
10. Convert the solution into a two-phase Bligh-Dyer mixture by adding 2 ml of chloroform and 2 ml of methanol, yielding a chloroform:methanol:aqueous (2:2:1.8 v/v) mixture. Vortex to mix, and centrifuge for 10 min in clinical centrifuge at 1,500 x g to separate phases.
11. Extract the lower phase into a clean glass centrifuge tube using a glass pipette (first extraction).
12. Perform a second extraction on the sample by adding 2 ml of pre-equilibrated two-phase Bligh-Dyer lower phase (prepared as in step 1.2) to the remaining upper phase from step 11. Vortex and centrifuge 10 min. Remove lower phase and combine with the lower phase from the first extraction.
13. To the pooled lower phase (~4 ml total volume), add 7.6 ml of pre-equilibrated upper phase (step 1.2), yielding a two-phase Bligh-Dyer solution (chloroform:methanol:water; 2:2:1.8, v/v). Vortex and centrifuge for 10 min.
14. Remove the lower phase to a clean glass tube and dry using a nitrogen dryer. Dried samples can be stored at -20 °C until further use.

7. Visualization of ³²P-labeled Lipid A Species via Thin Layer Chromatography

1. Prepare ³²P-labeled lipid A sample, TLC tank, and TLC plates as described in steps 2.1-2.8.
2. Dissolve ³²P-labeled sample in 500 μl of 4:1 chloroform-methanol (v/v). Vortex and bath sonicate to completely dissolve lipid material. Add 5 μl of sample to scintillation vial containing 5 ml of scintillation cocktail. Count in scintillation counter and calculate total counts/min of sample.
3. When visualizing radiolabeled lipid species, 10 x 20 cm or 20 x 20 cm TLC plates can be used. For 10×20 cm plates, samples should be spotted along the origin marked in step 2.5 so that the plate’s longest dimension is vertical (i.e. samples spotted at origin marked 2 cm above the 10 cm edge).
4. Using a microcapillary pipette, spot 10,000-20,000 cpm/sample on plate and allow spots to dry (>15 min). Samples might need to be concentrated by drying under nitrogen and resuspended in an appropriate volume, to achieve 10,000-20,000 counts/min/spot (2 μl or 2 μl at a time for <10 μl total).
5. Place TLC plate containing radiolabeled samples into the pre-equilibrated tank. Once solvent front reaches the top of the TLC plate (~3 hr), remove the plate and air dry (>60 min). Caution: TLC plates run in the presence of pyridine must be dried thoroughly in a chemical fume hood, as trace amounts of pyridine can damage phosphorimaging screens. A cold-air gun can be used to ensure complete dryness.
6. Wrap the plate in plastic wrap and expose to phosphorImager screen in an autoradiography cassette O/N. The next morning, scan the screen to obtain the image (Figure 6). Using image densitometry analysis software, this method allows for accurate, relative quantification of lipid A species.