Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

The overall goal of this procedure is to extract lipoproteins from bacterial cells for direct applications and to further prepare N-terminal lipopeptides for structural analysis by MALDI-TOF mass spectrometry. This method can help answer key questions in the immunology field, as the lipoproteins'N-terminal structure influences Toll-like receptor recognition and signaling, impacting the host's immune response. The unique advantage of this technique is the optional but intentional formation of sodium adducts, which promotes fragmentation towards the diagnostic dehydroalanyl ion, aiding in structural assignment of the lipoprotein's acylation state.

Grow bacteria in 15 milliliters of tryptic soy broth or similar rich media to late exponential phase. Harvest the cells by centrifugation before washing once with Tris-buffered saline EDTA or TBSE. resuspend the cells in 800 microliters of TBSE with one millimolar PMSF and 0.5 milligram per milliliter lysozyme.

Transfer the solution to a 2.0-milliliter threaded microcentrifuge tube with screw cap and O-ring prior to incubating for 20 minutes at 37 degrees Celsius. Add approximately 800 microliters of 0.1 millimeter zirconia silica beads to the tube, then disrupt the cells by shaking at maximum speed on a homogenizer for five cycles of 30 seconds each with a two-minute rest on ice between each cycle. Centrifuge the sample at 3, 000 times g for five minutes at four degrees Celsius to pellet beads and unbroken cells.

Transfer the supernatant to a new 2.0-milliliter microcentrifuge tube, then keep on ice. Add 200 microliters of TBSE to the remaining pellet and return to the homogenizer for an additional cycle. Centrifuge as before and combine the supernatant with the previous supernatant.

Supplement the supernatant with TX-114 surfactant to a final concentration of 2%by adding an equal volume of 4%surfactant in ice-cold TBSE. Incubate the supplemented supernatant on ice for one hour, mixing by inversion every 15 minutes. Transfer the tube to a 37-degrees Celsius water bath, and incubate for 10 minutes to induce phase separation.

Centrifuge the sample at 10, 000 times g for 10 minutes at room temperature to maintain biphasic separation. Gently pipette off the upper aqueous phase and discard. Add ice-cold TBSE to the lower surfactant phase to refill the tube to its original volume and invert to mix.

Incubate on ice for 10 minutes. Following incubation, transfer the tube to a 37-degrees Celsius water bath and incubate for 10 minutes to induce phase separation, then centrifuge at 10, 000 time g for 10 minutes at room temperature. After repeating these steps once more for a total of three separations, remove the upper aqueous phase and discard.

Remove the pellet of precipitated proteins that formed during the course of the extractions by adding one volume of ice-cold TBSE to the surfactant phase. Centrifuge at four degrees Celsius and 16, 000 times g for two minutes to pellet the insoluble protein. Immediately transfer the supernatant to a fresh 2.0-milliliter microcentrifuge tube containing 1, 250 microliters of 100%acetone.

Mix the sample by inversion and incubate overnight at minus 20 degrees Celsius to precipitate the protein. The next day, centrifuge the sample at 16, 000 times g for 20 minutes at room temperature to pellet the lipoproteins with attention to the orientation of the tube. Wash the pellet twice with 100%acetone before decanting the acetone and allowing the sample to air dry.

Then, add 20 to 40 microliters of 10-millimolar Tris-HCL, pH 8.0, and thoroughly resuspend the pellet by pipetting up and down against the wall with the precipitated lipoproteins. Separate the lipoproteins by SDS-PAGE using standard methods. Transfer the lipoproteins to a nitrocellulose transfer membrane using a standard electro blotting procedure.

Transfer the nitrocellulose membrane to a container and cover with Ponceau S solution. Rock gently for five minutes or until red-pink bands are visible. Pour off Ponceau S solution and carefully rinse the nitrocellulose membrane with distilled water to remove excess stain.

With a clean razor blade, excise the desired band and transfer to a microcentrifuge tube. Wash the membrane three times with one milliliter of distilled water to completely de-stain the band. After transferring the section to a clean surface, use a clean razor blade to dice the nitrocellulose strip into small pieces of approximately one millimeter by one millimeter.

Collect the pieces into a 0.5-milliliter low protein binding microcentrifuge tube. Finally, wash the pieces twice with 500 microliters of freshly-prepared 50-millimolar ammonium bicarbonate, pH 7.8, in a HPLC-grade water. Resuspend the nitrocellulose pieces in 20 microliters of a 20-microgram per milliliter solution of Trypsin in 50-millimolar ammonium bicarbonate.

Vortex the resuspended nitrocellulose pieces to mix. Then spin the tube briefly to ensure that all pieces are fully covered by the Trypsin solution. Cover the tube lid using paraffin film to prevent evaporation and incubate the digest overnight at 37 degrees Celsius.

Spin the sample at 16, 000 times g for 30 seconds and remove the liquid by pipetting. Then, add 50 microliters of 0.5%trifluoroacetic acid in HPLC-grade water. After vortexing to mix, spin the sample briefly to ensure all pieces are covered by the solution and incubate the sample for 10 minutes at room temperature.

Following removal of the liquid by pipetting, repeat this step with 50 microliters of 10%acetonitrile, and then repeat again with 50 microliters of 20%acetonitrile. To elute the tightly-bound lipopeptides, add 15 microliters of freshly-made 10 milligrams per milliliter CHCA matrix dissolved in chloroform-methanol. Incubate for 10 minutes at room temperature with intermittent vortexing.

To promote sodium adduct formation, supplement the solution of CHCA in chloroform-methanol with aqueous sodium bicarbonate to a final concentration of one millimolar. After spinning the sample briefly, use a pipette to carefully transfer the liquid to a new low protein binding microcentrifuge tube. This solution will contain the bulk of the N-terminal lipopeptides.

Deposit one microliter of the eluted lipopeptides with CHCA onto a polished steel MALDI target. Immediately proceed to mass spectrometry. Example mass spectra of the stepwise nitrocellulose wash solutions of the enterococcus faecalis lipoprotein PnrA are shown here.

With each subsequent wash, changes in signal intensity and a decrease in individual peaks are observed, culminating in the highly-enriched N-terminal lipopeptide ion in the final elution fraction. MS/MS spectrum of the parent lipopeptide ion reveals that it is the lysoform, with fragment ions corresponding to the N-terminal PnrA tryptic peptide featuring an alpha-amino linked acyl chain and a monoacylglycerol structure. Addition of sodium bicarbonate to the eluted lipopeptide fraction of the Escherichia coli lipoprotein Lpp results in a 22-Dalton increase from the calculated N-terminal mass.

MS/MS analysis of the parent ion versus its sodium adduct demonstrates preferential fragmentation towards the dehydroalanyl ion of the sodiated parent through neutral elimination of the diaco thioglyceryl moiety. This aids in structural determination of the N-terminal acylation state. While carrying out this procedure, it is worthwhile to remember that each stepwise nitrocellulose wash fraction can be analyzed by mass spectrometry, allowing for both protein identification and N-terminal characterization of the lipopeptide in a single experiment.

Following lipoprotein extraction from bacteria, other experiments such as cell-based assays measuring Toll-like receptor signaling can be performed to answer additional questions like how the lipoprotein acylation state influences the host's immune response.