Solid Phase Synthesis of peptides includes Fmoc synthesis, peptide cleavage and precipitation. The synthesized peptides are subsequently purified using HPLC technique and sequenced using MALDI. After self-assembly of peptides,AFM is used for characterization.

1 Fmoc Synthesis

(a) Determinate the amount of resin required.

(b) Prepare:

  1. Fmoc-aa-OH 0.2× a (the required volume)mmol+0.2×5mmol (dead volume in the needles)

  2. HBTU/HOBt 0.2/1.1× a (the required volume ) mmol + 0.2×15 mmol (dead volume in the needles)

  3. DIPEA 0.5/1.1× a mL

(c) Weigh out the amino acid residues into appropriate sized centrifuge tubes, and add DMF to reach desired volume. Dissolve well.

(d) Transfer the amino acid solutions to the appropriately labeled monomer vial in peptide synthesize. The labels on the computer correspond to their physical location in the machine.

(e) Transfer HBTU/HOBt and DIPEA to solvent containers.

(f) Prepare appropriate amounts of 20% piperidine in DMF and DMF/DCM. (The DMF/DCM volume ratio is dependent on what resin is being used. Neutral buoyancy of the resin in the DMF/DCM is optimal. To determine the ratio, start with a 50/50 mixture of DMF/DCM and slowly add DCM till the resin beads are neutrally buoyant (suspended throughout the entire mixture). Typical DMF/DCM ratios range from 30/70 to 50/50.)

Material and Equipment:

  • Peptide synthesizer
  • Dichloromethane (DCM)
  • N-Dimethylformamide (DMF)
  • Piperidine
  • Diisopropylethylamine (DIPEA)
  • 2-(1H-Benzotriazol-1-yl)-1,1,3,3-Tetramethyluronium Hexafluorophosphate (HBTU)
  • 1-Hydroxybenzotriazole monohydrate (HOBT)
  • Fmoc Amino Acids
  • The hydroxymethyl-based Resin (wang)
  • Nitrogen

2 Cleavage

(a) Preparation for cleavage: Wash with DCM, the remove from synthesizer, subsequently vacuum dry the peptide.

(b) Cleavage:

  1. Slurry the resin in an appropriate cleavage cocktail.

  2. Swirl the mixture occasionally during the reaction time.(The reaction time will depend on the composition of the substrate. Normally, cleavage will take 1.5 to 2 hours.)

  3. Filter the resin and wash the resin 3 times with small portions of TFA.(Transfer the filtrate to a tube)

Cleave the peptide in high precentage of TFA to yeild full deprotect peptide. The most common cleavage cocktail are listed in below Table.

Reagent Recipe Hours Comments
B TFA/phenol/water/TIPS (88/5/5/2) 1-4 All peptides
K TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5) 1-4 All peptides
K’ TFA/phenol/water/thioanisole/1-dodecanethiol(82.5/5/5/5/2.5) 1-4 All peptides
L TFA/DTT/water/TIPS (88/5/5/2) 1-4 All peptides
P TFA/DTT/water/TIPS (88/5/5/2) 1-4 tBu group. Do not use with Trp, Met or Cys
P+ TFA/phenol/Methanesulfonic acid (95/2.5/2.5) 15 All peptides
R TFA/thioanisole/EDT/anisole (90/5/3/2) 1-8 All peptides
T TFA/TES (95/5) 1-4 Boc, tBu, Trt. Do not use with Arg or Trp
  TFA/TES (88/5/5/2) 1-4 Boc, tBu, Trt, Pbf. Do not use with Trp, Met or Cys
  TFA/DCM/indole (70/28/2) 1-4 Do not use with Arg

(c) Purifying with precipitation

  1. Evaporate TFA.(This step is not always performed, but it can lead to higher yield of peptide.)

  2. Precipitation with ether. For each 1mL of TFA solution, add 10mL of ether for complete precipitation. The peptide should have precipitated now. If not, add a few drops of water and cool the mixture on ice. The precipitated peptide can be filtered off using a fine pore filter, not the filter column! If necessary, keep the mixture at 4◦C. overnight to precipitate the peptide. Filter the peptide using a fine sintered glass funnel. Wash the crude peptide further with cold ether.

  3. Add 1mL of water to dissolve the peptide. 30% acetonitrile is required for hydrophobic sequences, very acidic peptide a drop of ammonia solution.

  4. Frezze the tube at an angle of 45 degree.(Increase the surface area for lyophilization.)(Note: The peptide precipitate may not be dried before adding water! The residual ether is easily removed after freezing.)

Material and Equipment:

  • Trifluoroacetic acid (TFA)
  • Phenol
  • Triisopropylsilane (TIS)
  • 1, 2 Ethanedithiol (EDT)
  • Ether, anhydrous
  • Ice
  • Dry Ice
  • Thioanisole (Depend on peptide)
  • Acetonitrile (Depend on peptide)
  • Ammonia (Depend on peptide)
  • Freeze Dryers (Lyophilizer)
  • Sintered glass funnel
  • Stirrer or stir bar
  • Filtering flask
  • Round bottom flask
  • Polypropylene frits
  • Filter column with plunger
  • An adapter with Teflon tube to fit the Luer tip of the filter column
  • Polypropylene vial with a screw cap and a sturdy holder
  • Rotary evaporator (optional)

3 Analysis of purity using HPLC

  • Sample Preparation: Dissolve 1 mg of sample in 1 mL of Buffer A. If there is some undissolved material, filter the sample through a 0.22μ m filter.

Material and Equipment:

  • HPLC solvent delivery system with binary gradient capability and a UV detector.
  • Reversed-phase octadecylsilica (C18) column (4.6 mm id (internal diameter) 250 mm length, 5μm particle size, 300 pore size.
  • C18 guard column.
  • Solvent filtration apparatus equipped with a 0.22μm Teflon filter.
  • Sample filters, 0.22μm porosity.
  • Buffer A: 0.1% (v/v) TFA in water.
  • Buffer B: 100% CH3CN containing 0.1% (v/v) TFA.

4 Peptide sequencing using MALDI

  1. Sample solvent: TA30 solvent (30:70 [v/v] acetonitrile : 0.1% TFA in water) or 0.1% TFA

  2. Matrix solubilization procedure:
    Prepare a saturated solution of alpha-Cyano-4- hydroxy cinnamic acid (HCCA) in TA30 solvent

  3. Sample preparation:

  • Mix 1 part saturated HCCA solution with 1 part sample solution.
  • Deposit 0.5μ L of the matrix/analyte mixture onto the MALDI target and allow to dry. The concentration of the peptide solution should be between 10 fmol - 1 pmol/μ L.

5 Peptide self-assembly

Self-assembly into beta-sheet structure

Materials and Equipments:

  • Pipette
  • PBS/ Aqueous NaCl/ CaCl2
  • Congo red dye
  • Deionized water

Procedure:

  1. Prepare aqueous solutions of 2–30 mM peptide.

  2. Pipette these solutions into either Dulbeccos phosphate-buffered saline (PBS), aqueous NaCl (1– 100 mM), or aqueous CaCl2 (1-100 mM).

  3. Each solution should contain 10μM Congo red, an indicator of aggregated beta-sheet structure.

Alternative technique for self-assembly of peptides into beta-sheet structure

Materials and Equipments:

  • Lyophilized peptides
  • Vortex mixer
  • NaCl solutions of varying concentrations (0 mM, 100 mM, 200 mM, etc.)
  • Deionized water

Procedure:

  1. If the peptides are lyophilized, dissolve the lyophilized powder in deionized water.

  2. Subsequently, the solution undergoes vortex treatment for 1 min.

  3. To study the ionic strength necessary for inducing self-assembly, prepare NaCl solutions (0 mM, 100 mM, 200 mM, etc.) and add directly to the lyophilized powder, followed by vortex treatment for 1 min.

6 Characterization with Atomic Force Microscope (AFM)

  1. Characterize peptide solutions using a NanoScope IIIa scanning probe work station equipped with a MultiMode head using an E-series piezoceramic scanner .

  2. Image the samples under dry helium.

  3. Spot 10 to 50μl of sample solution on freshly cleaved mica.

  4. Incubate at room temperature for 5 min, rinse with 0.02μ m filtered and deionized water.

  5. Blown dry with tetrafluoroethane.

  6. Image data can be acquired at scan rates between 1 and 2 Hz, keeping drive amplitude and contact force to a minimum.

References

  1. Flanagan, D., & Matsumoto, Y. (2008). The Ruby Programming Language. O’Reilly Media.
  2. Practical Synthesis Guide to Solid Phase Peptide Chemistry. \urlhttp://www.aapptec.com/solid-phase-peptide-synthesis-i-241.html. Retrieved from http://www.aapptec.com/solid-phase-peptide-synthesis-i-241.html
  3. Coin, I., Beyermann, M., & Bienert1, M. (2007). Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nature Protocols, 2, 3247–3256.
  4. Cleavage, Deprotection, and Isolation of Peptides after Fmoc Synthesis. Technical Bulletin.
  5. Disposable reaction vessels for peptide-resin cleavage. Retrieved from http://www.intavis.com/SupportArea/Automated_Peptide_Synthesis/Columns_for_resin_cleavage.pdf
  6. The Handbook of Analysis and Purification of Peptides and Proteins by Reversed-Phase HPLC. Retrieved from http://wolfson.huji.ac.il/purification/PDF/ReversePhase/VYDAChandbookRPC.pdf
  7. Frecklington, D. General Method for MALDI-MS Analysis of Proteins and Peptides. Retrieved from http://cshprotocols.cshlp.org/content/2007/3/pdb.prot4679.full
  8. Collier, J. H., & Messersmith, P. B. (2003). Enzymatic Modification of Self-Assembled Peptide Structures with Tissue Transglutaminase. Bioconjugate Chem., 14, 748–755.
  9. Collier, J. H., Hu, B. H., Ruberti, J. W., Zhang, J., Shum, P., Thompson, D. H., & Messersmith, P. B. (2001). Thermally and Photochemically Triggered Self-Assembly of Peptide Hydrogels. J. Am. Chem. Soc., 123, 9463–9464.
  10. Easterhoff, D., DiMaio, J. T. M., Doran, T. M., Dewhurst, S., & Nilsson, B. L. (2011). Enhancement of HIV-1 Infectivity by Simple, Self-Assembling Modular Peptides. Biophysical Journal, 100, 1325–1334.
  11. Stine, W. B., Dahlgren, K. N., Krafft, G. A., & LaDu, M. J. (2003). In Vitro Characterization of Conditions for Amyloid-beta Peptide Oligomerization and Fibrillogenesis . The Journal of Biological Chemistry, 278, 11612–11622.