AAPPTec Peptide Information, Catalog Peptides, Custom Peptides


Peptide Information
Before starting synthesis of a peptide or even planning the synthesis, the exact sequence of the peptide should be carefully considered. Early planning can avoid later problems and delays, such as insoluble or unstable peptide products or peptides without the expected level of activity. Careful selection of the peptide sequence can also minimize the problems that might be encountered during synthesis.
How the peptide will be utilized is one of the major factors in designing the peptide. If the goal is to synthetically replicate a naturally occurring peptide, then the peptide sequence and length are fixed. More often, though, the peptide to be prepared is a fragment of a larger peptide or protein, and it will be used in binding studies, epitope mapping, structure-function studies, fragment assembly or antibody production. The peptide should be long enough to include the residues responsible for activity, but very long sequences should be avoided for synthesis, and purification problems increase with increased peptide length and yield and purity decrease. Peptides used for epitope mapping or antibody production should generally be 10 - 15 residues.
Peptides that are segments of a larger peptide or protein may need modification at the N- and C- terminals to accurately model the binding of the parent molecule. A fragment derived from the N-terminal of a large peptide or protein could require an amide function at the C-terminal, a C-terminal fragment might require acylation of the N-terminal. A peptide modeling a segment from the interior portion of a protein or larger peptide may require modification of both N- and C-terminals.
Peptide synthesis of peptide with a high percentage of hydrophobic residues may be difficult due to aggregation of the resin bound peptide. Many modifications to the standard solid phase peptide synthesis protocols have been developed to overcome this problem, ranging from a simple change of solvent to special resins and reagents. Pseudoprolines formed from Ser or Thr have been quite useful in peptide synthesis of long or difficult peptides. The pseudoprolines prevent aggregation by introducing a "kink" into the peptide backbone, which disrupts the peptide hydrogen bonding that produces secondary structure. This also improves the solubility of protected peptide fragments used in fragment condesation peptide synthesis. Incorporated at the C-terminal of a peptide fragment, pseudoprolines have the added advantage of eliminating C-terminal epimerization in fragment condesation reactions. Pseudoprolines also accelerate peptide cyclizations by inducing a more favorable conformation of the peptide backbone. Since coupling to the nitrogen atom of pseudoprolines occurs in unsatisfactory yields, pseudoprolines are incorporated as dipeptide units. Upon treatment with TFA, the pseudoprolines are converted to unprotected Ser and Thr resides.
Another method that is often effective in the a difficult peptide synthesis is to perform the reactions at a higher temperature. The increased temperature reduces the extent of hydrogen bonding that causes aggregation, while freeing the peptide terminus for reaction. Typically a modest increase in temperature produces a significant improvement in peptide synthesis yield. Temperatures of 40°C to 60°C enables peptide synthesis of peptides that are intractible at room temperature. Sonication also promises to be useful in peptide synthesis. Sonication is reported to disrupt peptide aggregation and improve yield of the peptide during cleavage from the resin.
Even utilizing the modern peptide synthesis methods for preparing long or difficult peptides, the crude peptide may need purification. Typically, the analysis and purification of peptides is performed with RP-HPLC. Columns optimized for peptide purification are usually packed with C-18 coated particles with 130Å pores. For very large peptides and small proteins, packings with a 300Å pore size are usually used. The length of the column is less critical than in small organic molecule separations. Unlike small molecules which repeatedly partition between the mobile phase and the stationary phase as they pass through the column, peptides and proteins adsorb to the solid phase near the column inlet and remain there until the mobile phase reaches the right aqueous/organic composition and the peptide or protein deadsorbs and travels through the column with little interaction with the solid phase. Peptide columns can be relatively short without adversely affecting the separation. Likewise, the gradients for separating peptides are much shallower than the gradients used to separate small organic molecules.
Once the peptide has been purified, the pure HPLC fractions should be lyophilized as soon as possible for peptides can easily oxidize or decompose in solution. Lyophilization, or freeze drying, is the preferred method of isolating peptides from solution. Since the freeze drying process takes place while the sample is frozen, oxidation and decomposition are avoided. Additionally, the lyophilized peptide is a light fluffy powder that can be easily redissolved. Lyophilized peptides chould be stored at -40°C in tightly sealed containers. Lyophilized peptides can be stored for several months at these conditions.
Lyophilized peptides are usually soluble in water. If the peptide does not dissolve in water, it can be dissolved in a small amount of solvent and then diluted to the desired concentration. The solvent chosen should be compatible with the use of the peptide, e.g. the solvent should be non-toxic for in vivo use. Basic peptides can be dissolved in a small amount of 30% acetic acid, then diluted to the appropriate concentration. Acidic peptides can be dissolved in a small amount of 10% ammonium bicarbonate and diluted to the proper concentration. Hydrophobic peptides can be dissolved in a tiny amount of dimethylsulfoxide (DMSO) and then diluted with water. While diluting the peptide solution, check that the peptide does not precipitate. If the peptide begins to precipitate, a tiny amount of DMSO can be added. Peptides that aggregate can be dissolved by adding 6 M urea, 6 M urea with 20% acetic acid or 6 M guanidine hydrochloride salt, then diluting to the required concentration. Peptides should be dissolved just before they are used. In solution, peptides can oxidize and degrade quickly. Peptide solutions should not be stored longer than one or two days. If a peptide solution must be stored for a longer time, it should be frozen and stored under inert gas.