Synthesis of Multiple Peptides on Plastic Pins

Stuart J. Rodda1

1 Chiron Technologies Pty. Ltd., Victoria, Australia
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 9.7
DOI:  10.1002/0471142735.im0907s22
Online Posting Date:  May, 2001
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Scanning protein sequences by bioassay for smaller bioactive peptide sequences requires a source of many peptides homologous with the parent protein sequence. This unit deals with one of the synthetic methods for making such sets of peptides. The strategy of the multiple peptide approach to biological scanning is discussed, along with the synthetic protocols, and the handling of peptides after synthesis, i.e., cleavage, preliminary purification, storage, and analysis. It is specific for the multipin technique using equipment obtained from Chiron Technologies, although some of the approaches are applicable to other multiple synthesis techniques. Procedures for multipin equipment obtained from other suppliers may differ from the procedures described here, and the manufacturer's literature should be consulted. This unit also includes protocols for preparing Fmoc‐amino acid solutions and for acetylating or biotinylating synthesized peptides.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Multipin Synthesis of Peptides
  • Support Protocol 1: Preparing Activated Fmoc–Protected Amino Acid Solutions
  • Support Protocol 2: N‐Terminal Acetylation of Peptides
  • Support Protocol 3: N‐Terminal Biotinylation of Peptides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Multipin Synthesis of Peptides

  • recipe20% (v/v) piperidine/dimethylformamide (DMF; see recipe)
  • Dimethylformamide (DMF), analytical reagent grade
  • Methanol, analytical reagent grade
  • 100 mM activated 9‐fluorenylmethyloxycarbonyl (Fmoc)–protected amino acid solutions (see protocol 2)
  • recipeSide chain deprotecting (SCD) solution (see recipe)
  • Acidified methanol: 0.5% (v/v) glacial acetic acid/methanol
  • 1:2:0.003 (v/v/v) ether/petroleum ether/2‐mercaptoethanol (2‐ME)
  • 1:2 (v/v) ether/petroleum ether
  • 0.1 M NaOH
  • 0.1 M acetic acid
  • 0.1 M sodium phosphate buffer, pH 8.0
  • recipeSonication buffer (see recipe)
  • Peptide Synthesis Starter Kit (e.g., Chiron Technologies) of the desired type, containing:
    • Pepmaker computer program and ELISA reading and plotting programs
    • Manual
    • Pins with gears or macrocrowns
  • Storage boxes or sealable bags, polyethylene or polypropylene (ICN Biomedicals)
  • Pipettor tips, polyethylene or polypropylene (ICN Biomedicals)
  • 0.3‐ or 1.5‐ml reaction trays, polyethylene or polypropylene (Chiron Technologies, Nunc, or Beckman)
  • Sonicator with power output of ∼500 W
  • Dry nitrogen
  • Rack containing 96 1‐ml polypropylene tubes (Bio‐Rad)
  • 10‐ml capped conical polypropylene centrifuge tubes
  • Additional reagents and equipment for N‐terminal acetylation (see protocol 3; optional) or biotinylation (see protocol 4; optional)
CAUTION: Perform all chemistry steps in a well‐functioning chemical fume hood. Wear solvent‐resistant gloves, safety glasses, and protective clothing. The reagents can be flammable, toxic, and/or carcinogenic. Avoid sources of contamination which may affect the pins, including direct contact with the bench surface or exposure to vapors. The reagents for multipin synthesis can be handled in unsealed systems, but the amount of time that these reagents are left exposed to the open air should be minimized by using capped containers for liquids or polyethylene bags for pins wherever practical. Local regulations for safe disposal of solvents and used reagents must be followed.

Support Protocol 1: Preparing Activated Fmoc–Protected Amino Acid Solutions

  • 9‐fluorenylmethyloxycarbonyl (Fmoc)–protected amino acids with side‐chain‐protecting groups (Sigma, Bachem, Novachem, or Chiron Technologies), stored at 4°C
  • Catalyst: 1‐hydroxybenzotriazole (HOBt)
  • Activating agent: diisopropylcarbodiimide (DIC)
  • Dimethylformamide (DMF), amine‐free
  • Ethanol, analytical reagent grade
  • 5‐ or 10‐ml glass, polyethylene, or polypropylene bottles with inert (e.g., polythylene, Teflon) lids and liners

Support Protocol 2: N‐Terminal Acetylation of Peptides

  • recipeAcetylation solution (see recipe), prepared just before use
  • Pins with completed peptides (see protocol 1)

Support Protocol 3: N‐Terminal Biotinylation of Peptides

  • Biotin or long‐chain biotin
  • Dimethylformamide (DMF), amine free
  • Diisopropylcarbodiimide (DIC)
  • Pins with completed peptides (see protocol 1)
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Literature Cited

Literature Cited
   Burrows, S.R., Gardner, J., Khanna, R., Steward, T., Moss, D.J., Rodda, S., and Suhrbier, A. 1994. Five new cytotoxic T cell epitopes identified within Epstein‐Barr virus nuclear antigen 3 J. Gen. Virol. 75:2489‐2493.
   Carter, J.M., VanAlbert, S., Lee, J., Lyons, J., and Deal, C. 1992. Shedding light on peptide synthesis. Bio/Technology 10:509‐513.
   Fauchere, J.L. and Pliska, V. 1983. Hydrophobic parameters of amino acid side chains from the partitioning of N‐acetyl‐amino‐acid amides. Eur. J. Med. Chem. 18:369‐375.
   Geysen, H.M., Meloen, R.H., and Barteling, S.J. 1984. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. U.S.A. 81:3998‐4002.
   Geysen, H.M., Rodda, S.J., Mason, T.J., Tribbick, G., and Schoofs, P.G. 1987. Strategies for epitope analysis using peptide synthesis. J. Immunol. Methods 102:259‐274.
   Maeji, N.J., Bray, A.M., and Geysen, H.M. 1990. Multi‐pin peptide synthesis strategy for T cell determinant analysis. J. Immunol. Methods 134:23‐33.
   Mutch, D.A., Rodda, S.J., Benstead, M., Valerio, R.M., and Geysen, H.M. 1991. Effects of end groups on the stimulatory capacity of minimal length T cell determinant peptides. Pept. Res. 4:132‐137.
   Reece, J.C., Geysen, H.M., and Rodda, S.J. 1993. Mapping the major human T helper epitopes of tetanus toxin: The emerging picture. J.Immunol. 151:6175‐6184.
   Reece, J.C., McGregor, D.L., Geysen, H.M., and Rodda, S.J. 1994. Scanning for T helper epitopes with human PBMC using pools of short synthetic peptides. J. Immunol. Methods 172:241‐254.
   Rink, H. 1987. Solid‐phase synthesis of protected peptide fragments using a trialkoxydiphenylmethylester resin. Tetrahedron Lett. 28:3787‐3790.
   Stewart, J.M. and Young, J.D. 1984. Solid Phase Peptide Synthesis, 2nd ed. Pierce Chemical Co., Rockford, Ill.
   Valerio, R.M., Bray, A.M., Campbell, R.A., Dipasquale, A., Margellis, C., Rodda, S.J., Geysen, H.M., and Maeji, N.J. 1993. Multipin peptide synthesis at the micromole scale using 2‐hydroxyethyl methacrylate grafted polyethylene supports. Int. J. Pept. Protein Res. 42:1‐9.
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