To quantify peptide translocation across vesicle membranes. Fluorescently labeled lipid vesicles are generated with encapsulated trypsin. A trypsin inhibitor is added to inhibit the activity of any exogenous trypsin leaving active trypsin in the vesicle lumen.
Only when fluorescently labeled peptides are added to the vesicles, their fluorescent tags act as fret donors and excite the fluorescent molecules on the vesicle. However, when proteins traverse the membrane, they are digested by trypsin and the fret signal is lost. Spectrometric analysis reveals the extent of peptide translocation, which is seen as a decrease in the fret between the vesicles and the peptide over time.
The main advantage of this technique over other methods, including other lipid vesicle techniques and methods to measure peptide entry into cells, is that it gives information about the kinetics of membrane entry, and it does not require the addition of a fluorescent label to the peptides if they contain a tryptophan. This method can help answer key questions in the design and engineering of membrane active peptides, such as which characteristics are necessary for effective membrane translocation. The first step of this procedure is to prepare large unal lipid vesicles or UUVs to serve as cell membrane mimics for the assay.
Begin by combining phosphatidylcholine, phosphatidyl, glycerol, and five dimethyl amino naphthalene. One sul phyl phosphatidyl ethanolamine in chloroform. In a 50 to 45 to five ratio, 0.376 milligrams of total lipid is usually sufficient to make enough vesicles for a single experimental or control trial.
Prepare one vial as the experimental sample and one as control evaporate off the chloroform using a nitrogen gas stream. The dried lipids that remain after this step are referred to as lipid cakes. Dry the lipid cakes for nine to 14 hours in a vacuum desiccate.
Next, prepare a 10 millimolar HEI stock solution, A 0.4 millimolar porcine trips in stock and 4.0 millimolar Bowman Burke trypsin inhibitor stock details for preparation can be found at the accompanying document. Add an equal volume of 0.4 millimolar trips in stock and 10 millimolar heis buffer solution to the dried lipid cakes. Then vortex the vial to reconstitute the multilam molar vesicles or mvs to reconstitute the control mvs at an equal volume of 0.4 millimolar trips in stock and 4.0 millimolar tripsin inhibitor to the dried lipid cake vortex As before.
Once the mvs are reconstituted, transfer the MLV solutions from glass vials to 1.7 milliliter micro centrifuge tubes. Then subject MLV solutions to five freeze thaw cycles, alternating between liquid nitrogen and a 30 degree Celsius water bath. Next, prepare a lipid extruder by moistening two filter supports in Heiss Buffer.
Then place each of them on a moistened Teflon extruder piece and place a nucleo pore track etched membrane with 0.1 micron pores between the two Teflon extruder pieces. Now place the extruder pieces inside a metal extruder canister and place a donut spacer over the two extruder pieces. Tighten the metal top of the extruder canister and then place it in the holder using a 250 microliter glass syringe.
Fill the extruder void with 500 microliters of 10 millimolar. He piece buffer to prevent any loss of the vesicle sample. Transfer the sample to a 10 milliliter beaker.
Then load each MLV sample into the extruder and push the sample through the membrane at least 21 times. To ensure uniform Ella vesicle size following extrusion, transfer the large ELLA lipid vesicles or L UUVs to a 1.7 milliliter micro centrifuge tube. Store the UUVs at four degrees Celsius and use them within 48 hours.
To assess LUV concentration, the total phosphorus content in the sample is determined. The following should be prepared according to the specifications in the accompanying written protocol. 8.9 normal H two s oh four, 2.5%weight per volume ammonium maib date, six 10%weight per volume, ascorbic acid and 0.65 millimolar phosphate buffer solution.
Prepare six phosphorus standards as shown here. Then prepare an LUV blank with the same volume of 0.2 millimolar trypsin and 2.0 millimolar Bowman BIR inhibitor solution as used for the controls. This is done to correct for any dried phosphorus salts in the Bowman B inhibitor powder.
Next pipette about 0.1 micromoles usually about 25 microliters of UUVs into disposable glass culture tubes in triplicate. Next, add 450 microliters of 8.9 normal H two SO four into each of the sample standard and blank tubes. Then place the samples standards and blanks in a 175 to 210 degree Celsius oven for 25 minutes.
After 25 minutes have passed, remove all of the tubes from the oven and allow them to cool. Add 150 microliters of 30%H2O tube to each of the tubes. Then heat the samples standards and blanks for another 30 minutes at 175 to 210 degrees Celsius.
After 30 minutes, remove the sample standards and blanks from heat. If any of the solutions are not yet colorless, add 50 microliters of H2O tube to all the tubes and heat for an additional 15 minutes at 175 to 210 degrees Celsius. Once the solutions are colorless, add 3.9 milliliters, nano pure H2O 0.5 milliliters, ammonium moate solution, and 0.5 milliliters ascorbic acid solution to each tube.
Then heat all of the tubes for five to seven minutes. In a boiling water bath, measure the absorbance of each standard and sample at 820 nanometers using an Agilent 8 4 5 3 UV visible spectrophotometer. Experimental and control LUV concentrations must be measured separately.
The tube containing 0.0 millimoles phosphorus should be used as the blank for all standards and experimental LUV samples. The tube containing 0.2 millimolar trypsin 2.0 millimolar bone and bir inhibitor solution should be used as the blank for the control LUV sample. The linear absorbance versus phosphorus content standard curve produced should be used to calculate the total phosphorus content and LUV concentration of the samples.
To prepare the peptide solutions dissolve the peptide in Oppu H2O peptides used in this assay must contain one tryptophan residue. Measure the absorbance of the peptide solution at 282 nanometers in triplicate. Calculate peptide concentration using the molar extinction coefficient for tryptophan of 5, 700.
Inverse molar inverse centimeters dilute the peptide in nano pure water to a final concentration of 30 micromolar store at minus 20 degrees Celsius. To quantify translocation, prepare experimental and control samples in 1.7 milliliter micro centrifuge tubes. Add enough UUVs to each solution for a final concentration of 250 micromolar.
Add enough Bowman BIR inhibitor solution so that the inhibitor is a final concentration, 10 x greater than the tryin concentration. Add enough 10 millimolar heis buffer to bring the final solution volume to 450 microliters. Place 50 microliters of peptide solution into a stana micro chords fluorimeter cell and place it in the spectrophotometer program.
A fluorescent spectrophotometer to perform a 25 minute fluorescent kinetics experiment in which a fluorescence reading is recorded at least once per second with a 280 nanometer excitation wavelength and a 525 nanometer emission wavelength at 25 degrees Celsius. With the PMT sensitivity to high, add the experimental or control sample to the peptide solution in the qve. This should bring the final peptide concentration to three micromolar immediately begin the fluorescent kinetics experiment.
Once both the experimental and control samples have run, calculate the translocation ratios as described in the accompanying document. The ratio betweens and peptide is high enough to ensure that virtually all of the intact peptides are in close enough proximity to a dansel group to give a fret signal, even if none of them have translocated into the vesicle. This is consistent with the similar fret observed in control conditions for peptides that do and do not translocate into vesicles.
Translocation assays were performed as described in this video. This figure shows the results from a representative peptide that showed robust translocation. The signal in this experiment represented by the black trace shows a marked drop in fret signal.
Over time, higher translocation ratios are indicative of peptides that trans efficiently. The translocation ratio for the cell penetrating peptides shown here is 1.19 weekly translating peptides have translocation ratios closer to one to control for the potential loss of fret signal due to incomplete trips and inhibition or other factors unrelated to translocation ability. The fret signal between peptide and UUVs containing both trypsin and Bowman BRK trypsin inhibitor is measured.
This is shown as the gray trace. The importance of the control experiment using UUVs containing trypsin inhibitor is highlighted by the data shown here. In this case, the peptide signal decreased a similar amount to that in the previous figure in the experimental sample represented by the black trace.
However, the control sample shows a more rapid decrease for this peptide, so its net translocation is lower. After watching this video, you should have a good idea of how to make lipid vesicles and to measure translocation of tryptophan containing peptides. While Attempting this procedure, it's important to remember to begin collecting data immediately after peptide and vesicles are mixed.
