Synthesis and Structure Determination of µ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

This method can help answer key questions in the peptide synthesis field such as synthesizing disulfide rich peptides and determining their corresponding disulfide bridge pattern. The main advantage of this technique is that it allows for the selective synthesis of different disulfide bondend peptide isomers and their subsequent chemical and structural characterization. Generally individuals new to this method will struggle because each disulfide rich peptide behaves differently.

Synthesis and analysis may have to be partially optimized for individual peptide or protein. Transfer the reagents listed in the text protocol to the corresponding vessels and place them in the appropriate rackets of the solid phase peptide synthesizer. Then, add 100 milligrams of dry resin to the reaction columns and put them in the racket of the synthesizer.

Start the solid-phase peptide synthesis as detailed in the text protocol. After the synthesis is complete, lyophilized the resin overnight. Now cleave the peptide from the resin and perform deep protection of the side chains.

To do so combine the dried resin in a 12 milliliter tube and cool it to zero degrees Celsius on ice. First, add 150 microliters of a scavenger mixture, then add one milliliter of 95%trifluoroacetic acid per 100 milligrams of resin. Leave the mixture gently shaking for three hours at room temperature.

Now, filter the mixture through a glass frit. Collect the filtrate in tubes, individually filled with ice-cold diethyl ether at one milliliter of cleavage mixture per eight to 10 milliliters of diethyl ether. The peptide precipitates as a white solid.

After rinsing and centrifuging the tubes as described in the text protocol, freeze-dry the peptides. To purify the linear precursor with semi preparative chromatography add approximately 70 milligrams of the crude peptide to a 15 milliliter tube. Then dissolve the crude peptide in 0.1%TFA and one-to-one Acetonitrile to water.

Separate the peptide mixture using a gradient of zero to 50%Eluent B over 120 minutes at a flow rate of 10 milliliters per minute. Collect the fractions in individual tubes as they appear and prepare selected fractions for Mass Spectrometry and HPLC analysis as described in the text protocol. Freeze-dry the fractions and combine the peer fractions.

Start the Selective formation of disulfide bonds by performing the first oxidation. To do so, dissolve 15 milligrams of the freeze-dried linear peptide in 105 milliliters of an isopropanol water mixture. Leave the mixture stirring on air under basic conditions for 12 to 48 hours.

The stepwise oxidation procedure is very critical. Reaction controls are taken in all three oxidation steps to ensure individual disulfide rich formation. The third oxidation is the most crucial because disulfide scrambling can occur when the optimal reaction time is exceeded.

Now perform the second oxidation. Dissolve 15 milligrams of the freeze-dried peptide in 105 milliliters of an isopropanol water, one molar hydrochloric acid mixture. Add 158 microliters of a 0.1 molar iodine solution in methanol to the solution.

Stir the reaction at room temperature until the oxidation is completed. Then stop the reaction by adding 79 microliters of one molar ascorbic acid in water. Finally perform the third oxidation.

Dissolve 15 milligrams of the freeze-dried peptide in 5.5 milliliters of TFA containing a scavenger mixture. Precipitate the peptide into tubes containing cold diethyl ether at one milliliter of reaction solution per eight to ten milliliters of diethyl ether. To perform peptide purification at 15 milligrams of the freeze-dried crude product to a 15 milliliter tube.

Dissolve the crude product in the volume of the HPLC sample loop using a mixture of 0.1%TFA and one to one Acetonitrile to water. After vortexing until complete dissolution, centrifuge the solution from one minute at 3400 times G.Draw up the 3.6 milliliter mixture in a 5 milliliter syringe and inject the sample without any air bubbles into the injection loop. Start the injection into the HPLC system.

Purify the peptide mixture using a gradient of zero to 50%Eluent B over 120 minutes at a flow rate of 10 milliliters per minute. Collect the fractions in individual tubes as they appear. After the run is complete, prepare selected fractions for MS and HPLC analysis as described in the text.

Freeze-dry all fractions and store them at minus 20 degrees Celsius. To perform analytical HPLC, transfer samples of the peptide fractions or reaction controls into an HPLC vial and dissolve it in the mixture of 0.1%TFA in one to one Acetonitrile to water. Place the HPLC vial into the auto-sampler of the analytical reverse phase HPLC set to inject 250 microliters of each sample.

Use a gradient elution system of 0.1%TFA in water as Eluent A in 0.1%TFA in Acetonitrile as a Eluent B.Analyze the peptide using a gradient of 10%to 40%Eluent B over 30 minutes at a flow rate of 1.0 milliliter per minute. To perform amino acid analysis, transfer 100 micrograms of the pure peptide to a 1.5 milliliter micro-centrifuge tube and dissolve the powder in 200 microliters of six molar HCL. After transferring the solution to a glass ampule, close the ampule by heating the neck with a bunsen burner flame.

Then place a glass tube holding the ampule in a heating block for 24 hours at 110 degrees Celsius for hydrolysis. After 24 hours, open the ampule and transfer the solution into a two milliliter micro-centrifuge tube. Centrifuge the solution for six hours at sixty degrees Celsius and 210 times G in a rotational vacuum concentrator.

Then dissolve the hydrolyzed product in 192 microliters of the sampled dilution buffer and transfer the solution into a micro centrifugal filter. After centrifuging the sample for one minute at 2300 times G, transfer 100 microliters of the filtrate into an amino acid analysis sample tube. Place the tube into the amino acid analyzer and start the analysis.

During this past reduction protocol, it is crucial to take several reaction controls in order to obtain twice or four times kappa mido methylated species which are indispensable for subsequent disulfide analysis via mass spectrometry. First dissolve 600 micrograms of the pure peptide in 1.2 milliliters of 0.05 molar citrate buffer containing TCEP. Incubate the mixture at room temperature taking several 100 microliter reaction control samples ranging from zero to 30 minutes.

Mix the samples in a 1.5 milliliter micro-centrifuge tube with 300 microliters of alkylation buffer to stop the reaction and perform carbonyl methylation of the free thiol groups. Stop the reaction after five minutes by adding 100 microliters of 10%TFA and water and store the samples on dry ice. Now perform HPLC on the samples as detailed in the text protocol.

Transfer a small amount of each collected fraction to a 1.5 milliliter micro-centrifuge tube for MS/MS analysis of the oxidized forms. Dissolve the remaining peptide in 0.1%TFA in water and add an appropriate volume over a 100 millimolar TCEP solution to get a final TCEP concentration of 10 millimolar. At this point, selective cysteine alkylation can be checked via peptide sequencing.

With the two peptides in the middle, a determination of the disulfide bridge pattern is possible. NMR analysis of the different isomers is carried out to reveal the individual peptide structures. The 20 structures with the lowest energies as well as the disulfide connectivity of the different isomers are shown.

Notably, a combination of reversed-phase HPLC MS/MS fragmentation and NMR analysis is required for unambiguous identification of the disulfide connectivity. The comparison of the root mean square deviation value clarified that a rigid peptide mostly leads to a better resolved NMR structure. This video should give you an example how to selectively synthesize peptides with a desired disulfide bond pattern.

It also shows the possibility to check the correct disulfide connectivity via tender mass spectrometry and to obtain a three-dimensional structure via NMR spectroscopy. After its development, this technique paved the way for researchers in the field of peptide synthesis to explore the importance of disulfide bond patterns for the three-dimensional structure and consequently the activity of such peptide isomers. Following this procedure, other methods like activity ASAS can be performed in order to get insight into structure activity relationships of different peptide isomers at the specific biological target.

Though this method can provide insight into the synthesis and analysis of conotoxins, it can also be applied to other disulfide bridge peptides and proteins such as the fenzens, disulfide-rich animal toxins and other multiple cysteine containing molecules.