The overall goal of this procedure is to isolate and digest bacterial cell walls to determine the identities and relative fractions of different neuropeptides. Using UPLC analysis, this is accomplished by first lysing bacterial samples by boiling in sodium ecol sulfate. After washing out SDS, the second step is to digest samples with pronase E to purify away the outer membrane of gram-negative bacteria.
Next, the samples are digested with mease to solubilize the peptidoglycan into individual neuropeptides. The final step is to reduce the neuropeptides and adjust pH to the neuropeptide isoelectric point. Ultimately, UPLC is used to separate neuropeptides according to size and hydrophobicity, facilitating the quantification of important cell wall characteristics, such as degree of cross linking and average glycan strand length.
This method can help to reveal the answers to key questions in the field of microbiology, such as the relationships among morphogenesis, cell wall architecture, immune system activation, and pathogenesis. Although this method has been developed to purify gram-negative Peptidyl glycan, it can also be applied to gram-positive bacteria with the addition of a few enzymes and chemical treatments To grow the bacterial cultures back dilute overnight cultures one to 100 into 250 milliliters of fresh media and grow to the desired optical density at 600 nanometers while diluted cultures are growing. Set up a boiling water bath on a hot plate in a one liter beaker.
Once the water is boiling, aliquot six milliliters of 6%sodium dyl sulfate or SDS into 50 milliliter polypropylene tubes. Add one small stir bar to each tube and finger tighten the tube lids securely. Place the tubes in the boiling water bath and stir at 500 RPM on the hot plate.
Harvest the 250 milliliter cultures by spinning at 5, 000 times G for 10 minutes at room temperature. Resuspend the pellets in three milliliters of media or one x phosphate buffered saline. Slowly pipette the cell suspensions into the 50 milliliter tubes with 6%boiling SDS to lice the cells while the tubes are submerged in the boiling water bath and reclose the lids to finger tight.
Cover the boiling water bath and allow cells to boil for three hours, checking the water level periodically and refilling the water bath when necessary. After three hours, turn off the heat on the hot plate and continue to stir overnight at 500 RPM. If SDS has precipitated in the 50 milliliter tubes overnight, set the water bath to boil for an additional one to two hours.
Shown here is how a normal culture should look after lysing overnight. In SDS, note the transparency as opposed to opaqueness that correlates with precipitation. Prepare the prone e buffer and activate prone E at 60 degrees Celsius in a heat block for at least 30 minutes.
Use an ultracentrifuge set at 400, 000 times G to spin samples for 20 minutes at room temperature in order to pellet the large peptol glycan or PG macro molecules and thereby purify them from other cellular components. Remove the supernatant carefully and then resuspend each pellet in room temperature. Ultrapure water resus suspension volume depends upon the volume of the ultracentrifuge tubes used.
Use a volume that fills the tubes at least halfway, but does not exceed the maximum volume of the tubes. Repeat centrifugation and washing until the water does not form bubbles during Resus suspension indicating that the SDS has been fully removed at this point. Resus suspend the samples in 900 microliters of tris HCL buffer and transfer to two milliliter tubes previously poked with holes in the tops.
Using a small needle, add 100 microliters of activated pronase E to each sample before incubating at 60 degrees Celsius for two hours. Stop the prone indigestion by adding 200 microliters of 6%SDS to each sample and boil the samples in the 100 degree Celsius heat block for 30 minutes. As before, use an ultracentrifuge set at 400, 000 times G to spin the samples for 20 minutes at room temperature and wash with room temperature ultrapure water until the SDS is fully removed on the last centrifugation washing step, resus suspend the samples in 200 microliters of 50 millimolar sodium phosphate buffer.
This volume can be adjusted according to the amount of Pepto glycan in the sample and may be species dependent. If the sample contains more pep glycan, increase the suspension volume. If the sample has little peptide glycan.
Reduce the resus suspension volume to a minimum of 50 microliters. Transfer the samples to 1.5 milliliter tubes and add one milligram per milliliter mease to give a final concentration of 40 micrograms per milliliter. Incubate for six to eight hours or overnight in a 37 degrees Celsius heat block.
To prepare samples for UPLC, turn on a heat block to 100 degrees Celsius. Boil the samples without SDS for five minutes to stop the mease digestion centrifuge samples for 10 minutes at 16, 000 times. G at room temperature.
The neuropeptides are now in the supernatant. Transfer the supernatant to 13 by 100 millimeter glass tubes. Try to recover as much supernatant as possible, getting very close to the palate without disturbing it.
Adjust the pH by adding 500 millimolar borate buffer to the sample for a final concentration of 100 millimolar borate buffer bore eight buffer is compatible with the reducing agent. Sodium boro hydride add several grains of sodium boro hydride to reduce each sample and let the reaction proceed for at least 30 minutes at room temperature. Next, adjust the samples to pH six as measured with pH indicator paper with ortho phosphoric acid using 20 microliter increments, the sample should bubble in response to addition of ortho phosphoric acid.
The sample typically stops bubbling when a pH of six has been reached. Then continue to adjust the samples to between pH three and four with ortho phosphoric acid using two microliter increments. Filter the sample through a 0.22 micron syringe.
Filter directly into A-U-P-L-C vial. If a precipitate has reformed in the samples once transferred into the UPLC vials, heat the vials with several passes through a flame. Place the UPLC vial into the auto sampler and inject 10 microliters of each sample onto A-U-P-L-C instrument.
Equipped with a C 18 reversed phase UPLC column and an absorbance detector set to monitor. 202 to 208 nanometers samples are injected sequentially. Set the flow to 0.25 milliliters per minute and use a linear gradient over 25 minutes to achieve 100%solvent B and sequential E of neuropeptides within 30 minutes.
If mass spectrometry will be used to characterize neuropeptides after UPLC, collect fractions of the peak of interest in a fraction collector, transfer the fractions to a 1.5 milliliter tube and then dry the fractions using a centrifugal evaporator fractions must be desalted prior to MS analysis. In this typical UPLC result. Detection via UV absorbance at 202 to 208 nanometers as a function of time establishes a particular neuropeptides retention time.
A clear resolution between most neuropeptide species and strong signal strength across the spectrum is observed, which enables the analysis of the degree of cross-linking average glycan strand length and the identity of neuropeptides and their concentrations. An example chromatogram reflecting C precipitation of neuropeptides is shown here. No peaks alluded resulting in the absence of any data on PG composition.
The absence of discernible peaks from UPLC analysis can occur as a result of super concentrating the sample or mis adjusting the pH to well below the isoelectric point of the neuropeptides. Once mastered, this technique can be done in three days, albeit hectic ones it performed properly Following this procedure. Other methods like mass spectrometry can be used to positively identify neuropeptide species based on mass After its development.
This technique paved the way for researchers in the field of microbiology to explore the biochemical function of key cell wall enzymes and the mode of action of chemical inhibitors in a wide variety of species and mutants.
