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Recent years have seen a growing demand for synthetic peptides in the biosciences. A major reason for this has been the realization that synthetic peptides represent extremely powerful probes for many biochemical studies. In particular, the analysis of protein:protein interactions that are central to many physiological processes have become the focus of many investigations where synthetic peptides can be utilized. Binding of extracellular receptor domains to ligands, intracellular receptor motifs to signaling molecules, antibodies to antigens, and enzymes to substrates are all governed by specific interactions of amino acids and other moieties that are components of proteins. Although these interactions often involve amino acid residues from distant parts of the protein (discontiguous epitopes), there are many examples where short stretches of amino acids govern highly specific contacts (contiguous epitopes).
Peptides have become invaluable tools in elucidating important contiguous epitopes that are essential for functional and structural domains in proteins. In particular, the systematic screening with series of overlapping fragments or substitution analogues needs short peptides in large numbers, although in general, micromolar quantities are sufficient. In this regard, the value of using synthetic peptides as probes in biochemistry and pharmacology is comparable to the value of oligonucleotides in molecular biology. The systematic use of peptides, however, has been limited by the cost, time and special expertise or equipment required to provide a significant number of peptides for evaluation. Therefore, over the past several years a number of methods that are capable of the simultaneous synthesis of many peptides in a reasonable amount of time have been developed (1-5).
In our endeavor to meet the increasing demand for synthetic peptides, we have added a multiple peptide synthesizer service to our facility. The instrument that we are using (Model MPS 350 from Advanced Chemtech, Louisville, KY) is a fully automated robotic instrument capable of the simultaneous small-scale synthesis of up to 96 different peptides (4). This instrument is now available as an upgraded model (MPS 396) which has automatic cleavage capabilities. Other automated multiple peptide synthesizers that are commercially available are the Gilson AMS 422, the Compas Corp. Compas 242, the Protein Technologies Symphony/Multiplex and the Shimadzu PSSM-8 systems. These instruments are capable of the simultaneous synthesis of 48, 24, 12 and 8 peptides respectively. Not mentioned here are devices for manual simultaneous synthesis of multiple peptides.
The Advanced Chemtech MPS 350 instrument is essentially comprised of a pipetting robotic workstation capable of delivering and aspirating the various amino acid solutions, reagents and solvents that are required for solid phase peptide synthesis on resin beads. Wash stations ensure that no cross-contamination between different amino acid containers and reaction vessel tubes takes place. Synthesis chemistries and parameters can be manipulated over a wide range and resin amounts of up to 125 mg can be accommodated by the 2 ml reaction vessels.
Table I outlines synthesis conditions that we have used with the MPS 350 to synthesize over 1,000 peptides over the past two years. Other synthesis parameters such as the number of washes and reaction times are as recommended by the manufacturer (4).
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Preparation for setting up the instrument includes the weighing of resins and the protected amino acid derivatives that are to be dissolved in DMF or NMP as well as the mixing of reagents (i.e., piperidine and diisopropylcarbodiimide with DMF). Amounts and volumes for all amino acids and reagents required for a synthesis are calculated by the computer based on the peptide sequences that are to be synthesized and the synthesis parameters. Reaction times and steps are essentially as recommended by the manufacturer but can be modified. After the synthesis has been completed the tubes containing peptide resins are washed with methanol (by the instrument) and then dried in a speed-vac. Cleavage is currently carried out manually in the reaction vessels containing the dried resins and after cleavage the mix is filtered through a 45 um PTFE filter into a 15 ml polypropylene tube. Peptides are precipitated by the addition of 12 ml ether, after which they are kept at -20deg.C for 30 min prior to centrifuging at 3,000 rpm for 5 min. The peptide pellets are then washed twice with cold ether, air dried in a fume hood and dissolved in 50% acetic acid. Depending on the application, workup of the crude peptides is carried out by either desalting through a Sep-Pak column or reversed phase HPLC. For most screening applications (see below) it is sufficient, however, that the desired peptide is the major product in the mix and extensive HPLC purification is therefore not necessary. Analysis of many peptides by HPLC and mass-spectrometry has revealed comparable quality of the peptides generated by the multiple peptide synthesizer with peptides that are made by conventional synthesizers (Fig. 1).
Depending on the synthesis parameters and workup conditions, the typical total cost per amino acid coupling is between $1-2 which makes the multiple peptide synthesis approach considerably cheaper than conventional peptide synthesis instrumentation. Yields for an average 20-mer peptide are typically in the range of 40-60% but obviously depend on the peptide sequence. In general, we have found the quality of most peptides that extend up to about 25 amino acids in length to be comparable when they are made via either a multiple or traditional peptide synthesis strategy. The only side product that seems to occur at unusually high levels in peptides made via the multiple synthesis strategy is oxidized methionine which is presumably caused by air exposure of the amino acid solutions.
Some of the ways in which our multiple peptides have been used are in screening experiments, where the peptides have been used to probe for protein-protein interactions, and as substrates in determining the specificities of enzyme reactions.
In one set of studies phosphotyrosine-containing peptides were synthesized and subsequently used in an in vitro kinase assay to test for the association of cytoplasmic signaling molecules with activated growth factor receptor tyrosine kinases (6). In one case the goal was to determine the phosphotyrosine residue that is responsible for the association of GTPase Activating Protein (GAP) with the mouse PDGF-receptor. Sequence analysis of the cytoplasmic tail of the receptor revealed 29 tyrosine residues that were potential association sites after they had become phosphorylated. We therefore synthesized 29 peptides containing phosphotyrosine and additional amino acids derived from the receptor. Only one peptide showed specific inhibition of binding of GAP to the activated PDGF receptor. Confirmation that tyrosine 739 was indeed the association site for GAP on the PDGF receptor was subsequently obtained by mutating this tyrosine to phenylalanine. Unlike the wild type receptor, the resulting PDGF receptor mutant did not associate with GAP after ligand stimulation, thus indicating that phosphotyrosine 739 represents the site of binding of GAP to the PDGF receptor.
In other, more standard applications multiple peptides were used to delineate important T cell epitopes that are recognized on antigens derived from Borrelia burgdorferi, a tick-borne spirochete causing Lyme disease (7). For this purpose a series of overlapping 18 amino acid long peptides was synthesized encompassing the entire sequences of four spirochete antigens. T cell proliferation assays using these peptides revealed the region of the different antigens that were specifically recognized by the T cells from individuals infected with the spirochete.
Another application deals with using multiple peptides for the analysis of substrate specificity of enzymes, in this case proteases (8). Mixtures containing 5 to 15 peptide nucleophiles were incubated with a serine protease and an excess of an acyl donor ester. The decrease in each nucleophile concentration was monitored by HPLC analysis of the dansylated mixtures. Relative kinetic parameters which characterize the leaving group specificity of the enzyme were calculated from the resulting data sets. In all the above studies peptide amounts in the low milligram range were sufficient, which is well within the capacity of the multiple peptide synthesizer. Synthesis times of 5 days are standard for the preparation of up to 35 peptides applying the conditions listed in Table 1. Workup (i.e., cleavage, partial purification and Iyophilization) typically takes another 2 days. Synthesis of that many peptides by conventional instruments takes at least ten times as long and results in peptide amounts that exceed the amounts required for the above experiments by orders of magnitude. Multiple peptide synthesis is therefore ideally suited for many applications that are increasingly important in biological research.
References
1. Houghton, R.A. (1985) Proc. Natl. Acad. Sci. USA 82, 5131-5135.
2. Geysen, H.M., Rodda, S.J., Mason, T.J., Tribbick, G., and Schoofs, P.G. (1987) J. Immunol. Meth. 102, 259-274.
3. Fodor, S.P.A., Read, J.L., Pirrung, M.C., Stryer, L., Lu, A.T., and Solas, D. (1991) Science 251, 767-773.
4. Groginsky, C. (1990) Americ. Biotechnol Lab. October,.40-42.
5. Gausepohl, H., Boulin, C., Kraft, M., and Frank, R.W. (1992) Peptide Res. 5, 315-320.
6. Fantl, W.J., Escobedo, J.A., Martin, G.A., Turck, C.W., del Rosario, M., McCormick, F., and Williams, L.T. (1992) Cell 69, 413-423.
7. Lahesmaa, R., Shanafelt, M.-C., Allsup, A., Soderberg, C., Anzola, J., Turck, C.W., Steinmann, L., and Peltz, G. (1993) J. Immunol. 150, 4125-4135.
8. Schellenberger, V., Turck, C.W., Hedstrom, L., and Rutter, W.J. (1993) Biochem. 32, 4349-4353.
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