The overall goal of the following experiment is to isolate lipopolysaccharide from the outer surface of gram-negative bacteria and to structurally characterize the lipid a portion with the use of tandem MS protocols. This is achieved by chemical extraction of lipopolysaccharide from the bacterial cell surface and subsequent mild acid hydrolysis to liberate lipid A from the polysaccharide portion of lipopolysaccharide and subsequent mild acid hydrolysis to liberate lipid A from the polysaccharide portion of lipopolysaccharide. As a second step, a solution of lipid A is analyzed by electros spray ionization MSMS.
In our procedure, lipid A is analyzed by either collision induced dissociation or ultraviolet photo dissociation based MS MS for structural characterization. The results show differences in fragmentation pattern in mass spectra produced from either collision induced dissociation or ultraviolet photo dissociation based MS MS protocols where UVPD yields a more informative fragmentation pattern. The main advantage of this technique over existing methods is that tandem mass spectrometry allows the elucidation of target analytes based on interpretation of diagnostic fragmentation patterns.
Ultraviolet photos, dissociation in particular is a high energy MSMS method that produces more diverse fragment ions than existing techniques such as collision induced dissociation. Visual demonstration of this method is critical as the UVPD mass spectrometry method used is not yet commercialized. An addition the method uses a laser that has been integrated with a mass spectrometer via custom modification Prior to starting this procedure.
Harvest cells from a 250 mil bacterial culture centrifuge the cells at 10, 000 times G for 10 minutes at four degrees Celsius. Then pour off the media supernatant, wash the cell pellet with 50 mil of one XPBS. After centrifuging to rep pellet the cells pour off the supernatant.
Next resus suspend the cells in 40 mil of one XPBS and divide them between 2 250 mil Teflon centrifuge tubes. Add 25 mil of chloroform and 50 M of methanol to each tube for a single phase bly dire mixture. Mix by inversion and incubate at room temperature for at least 20 minutes.
To ensure complete cell lysis centrifuge the mixture at 2000 times G for 20 minutes at room temperature and discard the supernatant. Wash the LPS pellet with approximately 100 mil of a single phase Bly dire mixture. Centrifuge at 2000 times G for 20 minutes and discard the supernatant.
Add 27 mil of mild acid hydrolysis buffer to the LPS pellet and mix by pipetting up and down until only small particles remain sonicate with a probe tip sonicate to homogenously Resus. Suspend the LPS pellet in solution at a constant duty cycle for 20 seconds at 50%output. Repeat sonication of the sample two times with 20 seconds per burst and approximately five seconds between bursts.
Then boil the samples in a water bath for 30 minutes. Remove the tubes from the water bath and allow the sample to cool to room temperature. Before proceeding to extract lipid A after hydrolysis, convert the SDS solution into a two-phase BLY dire mixture by adding 30 mil of chloroform and 30 mil of methanol for a chloroform methanol, mild acid hydrolysis buffer mixture mixed by inversion and centrifuge.
The S sample for 10 minutes at 2000 times G.Using a glass pipette, extract the lower phase into a clean Teflon centrifuge tube. Following this, perform a second extraction by adding 30 mil of the lower phase from a pre equilibrated two-phase bly dire mixture to the upper phase in the sample tube mixed by inversion and centrifuge 2000 times G for 10 minutes. Extract the lower phase with a glass pipette and pool with the lower phase extracted.Previously.
Wash the pooled lower phases by adding 114 mil of pre equilibrated bly dire upper phase to create a two-phase bly dire mixture mixed by inversion and centrifuge. Jet 2000 times G for 10 minutes with a glass transfer.Pipette. Transfer the lower phase to a clean glass rotary evaporator flask and dry the sample using rotary evaporation.
Next, add five mil of a chloroform methanol mixture to the rotary flask and bath sonicate for at least 30 seconds. To aid in the suspension of lipid A from the sides of the flask. Use a glass transfer pipette to transfer the lipid to a clean glass tube capped with A-P-T-F-E lined phenolic screw cap.
Dry the sample under a stream of nitrogen using a nitrogen dryer. Resuspend the dried lipid in one mil of a chloroform methanol mixture. After addition of solvent vortex the mix to ensure complete resus suspension.
Transfer to a small glass sample vial and dry using a nitrogen dryer. Store the dried sample at minus 20 degrees Celsius until subsequent TLC or MS analysis. The sample may be stored at minus 20 degrees Celsius until subsequent analysis.
Once the sample has been prepared, mix 200 microliters of HPLC grade methanol with 200 microliters of HPLC grade chloroform in an empty small glass sample vial transfer 200 microliters of the chloroform methanol solvent mixture to the vial with lipid A and sonicate the vial contents for five minutes or until all of the material has dissolve. Then set up the mass spectrometer for negative mode electros. Spray ionization fill a 250 microliter syringe with lipid a solution.
Insert the syringe into a syringe pump and directly infuse the diluted lipid. A sample at a flow rate of 2.0 to 3.5 microliters per minute. Optimize and enhance the lipid ion signal by tuning the ion optics.
Collect the mass spectrum of the lipid. A species isolate and activate the target lipid A by selecting CID as the MSM method. Next, increase the CID voltage or normalized collision energy until the precursor lipid.
A species is about 10%relative abundance compared to the highest product ion acquire and average the spectra until sufficient signal to noise is achieved. For the product ions, prepare the sample and mass spectrometer as described in the previous section. Turn on the laser interfaced to the mass spectrometer.
See the text protocol for MS details set up the instrument software so the laser will trigger the excimer laser when the ions enter the HCD cell following this turn on the pulse generator such that the laser is pulsed every two milliseconds. Isolate the lipid a precursor ion by selecting HCD as the MS MS method and adjust the collisional energy to 1%normalized collision energy activate the isolated lipid ion by increasing the laser energy and adjusting the number of laser pulses, which is typically ten six milli joule pulses. Finally acquire and average the spectra until sufficient signal to noise is achieved.
For the UVPD product ions. The negative ion MALDI TOF mass spectrum of lipid A from E Coli K 12 strain W 3 110 yields the single e deprotonated species at mass to charge ratio of 1796.2 as the major observed species. Details of maldi to analysis are discussed within the text protocol.
Alternatively, the same lipid sample subjected to negative mode ESI yields predominantly double deprotonated lipid ions at master charge ratio of 898.1 ly deprotonated lipid ions at master charge ratio of 1796.20 are observable but of lower relative abundance to doubly deprotonated species fragmentation by CID or 193 nanometers. UVPD was performed on the single e deprotonated lipid ion. These techniques can be used to better assign chemical structures, particularly of lipid.
A species containing complex combinations of modifications or previously uncharacterized chemical modifications. Fragmentation profiles are shown with dashed lines representing cleavage sites and are matched with the master charge ratio values below each provided structure. The master charge ratio values and cleavage sites highlighted in red font represent unique product ions associated with UVPD 32 P labeled lipid.
A isolated from two different e coli K 12 strains was analyzed by TLC. The protocol for this procedure is detailed within the text protocol. The K 12 strain W 3 110 contains mostly BIS and tris phosphorylated lipid A or is a more complex TLC pattern is observed with 32 p lipid.
A isolated from e coli strain, WD 1 0 1. WD 1 0 1 produces lipid. A heavily modified with rabino and phosphoethanolamine.
Since both one and four prime phosphates are available for modification, lipid A from WD 1 0 1 can be described as singly modified containing only one el amino arabinose or phosphoethanolamine at either phosphate or doubly modified where both phosphates are modified in a combinatorial manner. In addition to modification at the one and four prime phosphates Palmitate edition is also observed and increases the RF value of lipid a species in this solvent system. This protocol has uniquely paved the way for researchers in the field of microbiology to further explore structure function relationships with respect to how lipid a modifications impact the biology of gram-negative bacteria.
We are also very interested and have demonstrated the power of UVPD based MS to characterize the structures of biologically important molecules, such as glycans proteins, nucleic acids, and other complex lipids. Don't forget that working with organic solvents biologically hazardous material and lasers can be extremely hazardous. It's important to stress that solvents should be handled in ventilated chemical fume hoods.
Appropriate protective equipment such as gloves and goggles should always be worn and the laser used for UVPD should be properly shielded.
