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

1. Cell Growth and Lysis

1. Grow bacteria in 15 mL tryptic soy broth (TSB) or similar rich media to late exponential phase (OD₆₀₀ of 1.0 – 1.5). Harvest cells by centrifugation, wash once with Tris-buffered saline/EDTA (TBSE) and continue with protocol or freeze until use.
   NOTE: TBSE: 20 mM Tris-hydrochloride (HCl), pH 8.0, 130 mM sodium chloride (NaCl), and 5 mM ethylenediaminetetraacetic acid (EDTA)). Lipoprotein expression and modification can be influenced by growth conditions (e.g. acidity, salinity, growth media) and growth phase¹⁵. Cell growth can be scaled as desired, but 15-mL of cells is recommended for a single preparation of lipoproteins as excess biomass can decrease lipoprotein yield. One 15-mL preparation generally yields enough sample for optimal loading of ~2-4 lanes on a standard SDS-PAGE mini gel.
2. Resuspend cells in 800 μL TBSE with 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 0.5 mg/mL lysozyme. Transfer solution to a 2.0-mL threaded microcentrifuge tube with screw cap and O-ring and incubate for 20 min at 37 °C.
   NOTE: Certain species are not susceptible to lysis by lysozyme. An appropriate lytic enzyme should be substituted to aid in lysis.
3. Add ~800 μL 0.1 mm zirconia/silica beads to the tube and disrupt cells by shaking at maximum speed (7,000 rpm) on a homogenizer for 5 cycles of 30 s each, with 2 min rest on ice between each cycle.
4. Centrifuge sample at 3000 x g for 5 min at 4 °C (to pellet beads and unbroken cells). Transfer the supernatant to a new 2.0-mL microcentrifuge tube and keep on ice.
5. Add 200 μL TBSE to the remaining pellet and return to the homogenizer for an additional cycle. Centrifuge (as above) and combine supernatant with the previous supernatant (should be ~800-1000 μL total volume).

2. Enrichment of Lipoproteins by TX-114 Phase Partitioning

1. Supplement the supernatant with TX-114 surfactant to a final concentration of 2% (vol/vol)by adding an equal volume of 4% (vol/vol) the surfactant in ice-cold TBSE and incubate on ice for 1 h, mixing by inversion every ~15 min.
   NOTE: When chilled, the supernatant and surfactant will be miscible.
2. Transfer the tube to a 37 °C water bath and incubate for 10 min to induce phase separation. Centrifuge the sample at 10,000 x g for 10 min at room temperature to maintain bi-phasic separation.
3. 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 min.
4. Transfer the tube to a 37 °C water bath and incubate for 10 min to induce phase separation, then centrifuge at 10,000 x g for 10 min at room temperature.
5. Repeat steps 2.3-2.4 once more for a total of 3 separations. Remove the upper aqueous phase and discard.
6. Remove pellet of precipitated proteins that formed during the course of extractions (visible at the bottom of the tube), by adding 1 volume of ice-cold TBSE to the surfactant phase. Centrifuge at 4 °C at 16,000 x g for 2 min to pellet insoluble protein.
   NOTE: Sample should remain a single phase. If phase separation occurs, re-chill sample and centrifuge again. The pellet is generally composed of non-lipoprotein contaminants, but can be retained for further analysis.
7. Immediately transfer the supernatant to a fresh 2.0-mL microcentrifuge tube containing 1250 μL of 100% acetone. Pipette up and down thoroughly to wash the sample from the tip as it will be viscous. Mix by inversion and incubate overnight at -20 °C to precipitate protein.
8. Centrifuge the sample at 16,000 x g for 20 min at room temperature to pellet lipoproteins with attention to the orientation of the tube. Lipoproteins will form a thin, white film along the outside wall of the tube.
9. Wash pellet twice with 100% acetone. Decant acetone and allow sample to air dry.
10. Add 20-40 μL of 10 mM Tris-HCl, pH 8.0 or a standard Laemmli SDS-PAGE sample buffer¹⁶ and thoroughly resuspend by pipetting up and down against the wall with the precipitated lipoproteins. Scrape the side of the tube with the pipette tip to dislodge lipoproteins.
    NOTE: Lipoproteins will not dissolve in Tris buffer, but rather form a suspension.
    1. Store lipoproteins at -20 °C until use.

3. SDS-PAGE, Electroblotting, and Staining with Ponceau S

1. Separate lipoproteins by SDS-PAGE using standard methods¹⁶.
   NOTE: The appropriate gel acrylamide percentage will vary depending on its intended use and size of lipoproteins. Here, E. faecalis lipoproteins were separated over a 10% Tris-glycine gel, while a 16.5% Tris-tricine gel was used for the smaller E. coli lipoprotein Lpp¹⁷.
2. Transfer the lipoproteins to a nitrocellulose transfer membrane using a standard electroblotting procedure.
   NOTE: A semi-dry transfer using Bjerrum Schafer-Nielsen buffer plus 0.1% SDS was performed at 25 V, 1.3 mA for 15 min¹⁸. Alternately, polyvinylidene difluoride (PVDF) membrane could be used, however as it is more hydrophobic than nitrocellulose, it may reduce efficient elution of hydrophobic lipopeptides from the membrane at later steps.
3. Transfer the nitrocellulose membrane to a container and cover with Ponceau S solution (0.2% (w/v) Ponceau S in 5% acetic acid). Rock gently for 5 min or until red-pink bands are visible.
4. Pour off Ponceau S solution and carefully rinse the nitrocellulose membrane with dH₂O to remove excess stain.
   NOTE: Ponceau-stained bands will destain rapidly. If bands disappear, repeat staining process.
5. With a clean razor blade, excise the desired band and transfer to a microcentrifuge tube. Wash three times with 1 mL of dH₂O to completely destain the band.
   NOTE: The protocol can be paused here. Store the excised section covered with water at -20 °C until use.
6. Transfer the section to a clean surface and with a clean razor blade, dice the nitrocellulose strip into small pieces of approximately 1 mm x 1 mm. Collect the pieces into a 0.5-mL low protein binding microcentrifuge tube.
7. Wash the pieces twice with 0.5 mL of freshly-prepared 50 mM ammonium bicarbonate (NH₄HCO₃), pH 7.8 in high-performance liquid chromatography (HPLC) grade water.

4. Tryptic Digestion, Lipopeptide Extraction from Nitrocellulose Membrane, Deposition onto MALDI Target, and Data Acquisition

1. Resuspend nitrocellulose pieces in 20 μL of a 20 μg/mL solution of trypsin in 50 mM NH₄HCO₃, pH 7.8 in HPLC grade water. Vortex to mix, then spin 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 digest overnight at 37 °C.
2. Spin the sample at 16,000 x g for 30 s and remove the liquid by pipetting.
   NOTE: This removes trypsinized hydrophilic peptides that can be examined later by MALDI-TOF MS for protein identification.
3. Add 50 μL of 0.5% trifluoroacetic acid (TFA) in HPLC grade water. Vortex to mix, then spin the sample briefly to ensure all pieces are covered by the solution. Incubate for 10 min at room temperature. Remove the liquid by pipetting.
   CAUTION: TFA is harmful if inhaled. Handle with caution and use in the chemical fume hood. Avoid contact with skin.
4. Repeat step 4.3 with 50 μL of 10% acetonitrile in HPLC grade water.
   CAUTION: Acetonitrile is harmful if inhaled. Handle with caution and use in the chemical fume hood. Avoid contact with skin.
5. Repeat step 4.3 with 50 μL of 20% acetonitrile in HPLC grade water.
   NOTE: This removes any moderately hydrophobic peptides loosely bound to the nitrocellulose.
6. To elute the tightly-bound lipopeptides, add 15 μL of freshly-made 10 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) matrix dissolved in chloroform-methanol (2:1, v/v) and incubate for 10 min at room temperature with intermittent vortexing.
   1. Alternately, add 15 μL chloroform-methanol to elute lipopeptides and mix with matrix at a later time. Other matrices, such as 2,5-dihydroxybenzoic acid (DHB), should be tested as alternatives to CHCA if unsatisfactory results are obtained for a particular lipopeptide composition.
      CAUTION: Both chloroform and methanol are harmful if inhaled. Handle with caution and use in the chemical fume hood. Avoid contact with skin.
   2. Optional: To promote sodium adduct formation, supplement the solution of CHCA in chloroform-methanol with aqueous sodium bicarbonate (NaHCO₃) to a final concentration of 1 mM.
7. Spin the sample briefly. With a pipette, carefully transfer the liquid to a new low protein binding microcentrifuge tube. This solution will contain the bulk of the N-terminal lipopeptides.
   NOTE: The nitrocellulose may partially or fully dissolve in the chloroform-methanol solution. This will not negatively affect MS results, and may be used as an alternate method to increase lipopeptide return. PVDF membrane will not dissolve in the same manner.
8. Deposit 1 μL of the eluted lipopeptides with CHCA onto a polished steel MALDI target.
   NOTE: The chloroform-methanol will evaporate quickly, leaving behind crystallized CHCA and lipopeptides. An optional second 1 μL aliquot can be deposited onto the same spot to increase sample concentration. Chloroform-methanol may spread significantly after deposition onto the target and as such, care should be taken to avoid sample mixing on the target. It is recommended to deposit and shoot multiple spots of each sample.
9. Immediately proceed to mass spectrometry. Scan all areas of the spot for lipopeptide signal, especially if the spot contains two layers of sample, as the second layer may push the lipopeptides to the spot's outer rim.
   NOTE: Recommended starting laser intensity is 25%, though it may be necessary to increase intensity to acquire signal and sum multiple spectra (20 or more scans) to achieve adequate signal-to-noise ratio. Here, spectra were acquired on a MALDI-TOF-TOF instrument (see the Table of Materials) using a factory-configured instrument method for the reflector positive-ion detection over the 700-3500 m/z range. The instrument was calibrated with a bovine serum albumin (BSA) tryptic peptide mixture.