There are a number of elements to consider when designing individual peptides, specifically amino acid composition, length, solubility and the application in which the peptides are to be used. See Peptide Library Design for design of large numbers of peptides in sets for high throughput synthesis.
As the length of the peptide increases, so the proportion of full-length peptide obtained from the synthesis will decrease. Optimal synthesis results are achieved for peptides up to 15 amino acids, and peptides 10-15 amino acids long are recommended for generation of peptide-antisera.
The amino acid content strongly influences the purification of the peptide and the resulting solubility. The ratio of charged amino acids to uncharged and hydrophobic residues is critical in determining the ease of solubility of a peptide, and therefore it’s usefulness in the downstream application.
A high content of hydrophobic residues will reduce the solubility of a peptide in aqueous solution. A design with at least one charged residue every five amino acids is recommended, otherwise it is recommended to replace hydrophobic amino acids with charged or polar residues where possible.
Additionally, there are certain amino acids that can cause other problems in synthesis, solubilization and storage of peptides.
Cysteine, Methionine and Tryptophan
There residues are prone to oxidation and their presence in the sequence can cause problems with cleavage and subsequent purification of peptides. To eliminate this issue, use Ser to replace Cys and use Norleucine (Nle) to replace Met. If multiple Cys residues are present, dsisulphide links may form in the presence of oxygen. To minimize this, use a buffer containing reducing agent for peptides containing free Cys, or replace Cys with Ser.
Gln will cyclize to form pyroglutamate when exposed to the acidic conditions of cleavage; to avoid this either acetylate the N-terminus, synthesize with pyroglutamate instead of Gln to stabilize the peptide, or remove or substitute the Gln.
Amino terminal Asparagine
The protecting group for Asp can be difficult to remove when at the N-terminus; to avoid problems remove Asn or substitute.
Peptides containing Asp can undergo hydrolysis. The peptide chain may be cleaved under acidic conditions when particular amino acid pairs are present. Avoid Asp-Gly, Asp-Pro and Asp-Ser pairs if possible.
Multiple Serine or Proline
Avoid adjacent Ser residues as synthesis frequently results in a product that is low in purity and that also may contain many deletions. Proline may undergo cis/trans isomerization in solution and subsequently show low purity.
The presence of beta-sheet can lead to deletion sequences in the final peptide. Multiple or series of Gln, Ile, Leu, Phe, Thr, Tyr or Val can lead to beta-sheet formation. If possible, break up these stretches of amino acids by making replacements, such as Asn for Gln or Ser for Thr, or add Pro or Gly every third residue.
The amino acid composition of the peptide affects the ease of solubility. When designing peptides it is advisable to have at least 20% charged residues to aid solubilization. See Peptide Design.
Recommendations for solubilization of individual custom peptides
Start with an appropriate choice of solvent that is both compatible with your experimental procedures and that does not react with or degrade the peptide. Determine whether the peptide is acidic, basic or neutral and proceed with solubilization using a small amount of the peptide. Acidic and basic peptides are more soluble at neutral pH than acidic pH.
Short non-hydrophobic peptides (less than 5 amino acids) and peptides containing >25% non-clustered, charged residues and <25% hydrophobic residues will typically dissolve in aqueous solutions.
For basic peptides with net charge of +1 or greater, an acidic solution is needed. The peptide is acidic when shipped due to the presence of trifluoroacetate as the counter ion (a result of the cleavage or purification process). Simply adding water may dissolve basic peptides. If it does not, first try a drop (10-20 µl) of glacial acetic acid and Sonicate or vortex. This may be increased up to 30% acetic acid by volume for problematic sequences. Addition of base can also promote oxidation of cysteines to the disulphide so deoxygenated buffers should always be used.
Acidic peptides with net charge of -1 or greater should be dissolved in a small amount of basic solvent such as 0.1% ammonium hydroxide or ammonium bicarbonate and diluted to the required stock concentration with water. The exception is peptides containing Cys, as disulphide bonds may form at alkaline pH.
Neutral or hydrophobic peptides with a net charge of zero can sometimes be brought into solution by addition of base, but often a gel forms. Generally this gel will only respond to dilution with higher amounts of distilled, deionized water, along with sonication and vortexing. Peptides that are >50% hydrophobic may be difficult to dissolve in water alone and should be dissolved in a small amount of organic solvent, for example acetonitrile, methanol, isopropanol, DMSO, DMF or other. This should be added drop wise, followed by sonication and vortexing after every drop until the peptide dissolves. The drop wise addition of the organic solvent can also be used for peptides that do not respond to pH adjustment.
Peptides that are >75% hydrophobic are unlikely to dissolve in aqueous solution alone and may require solubilization in a stronger solvent such as TFA or formic acid and at high concentration. The peptide may precipitate out when aqueous buffer is added. These conditions may not be compatible with some cell culture based experiments.
Organic solvents at certain concentrations are incompatible with some biochemical assays. A small amount of DMSO should be compatible with most immunological assays. However, some cell culture based assays may not react well to DMSO, so a different solvent should be considered. Avoid DMSO if the peptide contains Met, Cys or Trp due to suphoxide or disulphide formation. These peptides should be prepared using 1,2-ethanediol (EDT) or dithiothreitol (DTT) in order to prevent oxidation. Oxygen-free water or buffers, or DTT are recommended for solubilizaton.
Recommendations for dissolving Custom Peptide Libraries
See Custom Peptide Libraries technical support information page.
Peptide purity, measured by analytical reversed phase HPLC, is the percentage of the peptide compared to impurities that absorb at 210-220 nm, (peptide bond absorption wavelength). Impurities consist of deletion sequences, incompletely deprotected sequences, truncated sequences and side products of the synthesis process. Residual salt and water that do not absorb at this wavelength are not quantified. In experimental planning it is important to consider that some peptide or non-peptide impurities may be toxic to cells. This can generally be avoided by purifying the peptide, which will result in only trace amounts of toxic impurities.
Recommended purity based on application of peptide:
Lyophilized peptides contain impurities in the form of counterions and residual water. Net peptide content, measured by amino acid analysis, is the percentage of all peptides relative to these non-peptide impurities. It varies according to the purification and lyophilization procedures, and is affected by the amino acid composition, particularly the presence of hydrophilic amino acids in the sequence.
Peptide purity and net peptide content are the major factors affecting variability between batches of peptides. The lower the purity, the greater will be the variability between batches synthesized. Once the peptide has been solubilized, measure the peptide concentration in solution in order to be able to compare peptide activity in experimental assays between batches.
Peptides are shipped as a lyophilized powder and should be stored at -20°C upon receipt. Allow peptides to warm to room temperature before opening the vial to prevent condensation of atmospheric moisture onto the peptide, making it easier to handle and weigh. It is recommended to dissolve only the required amount into a buffer. The remainder of the stock should be stored dry at -20°C. Storage in buffer is not recommended. Peptides in solution are only stable for up to one week when stored at 4°C. If peptides are to be used frequently, solubilize and aliquot, then store frozen at -20°C to avoid freeze-thaw cycles. Typically the peptide will remain stable for several months. For short term storage (up to 2 months), freeze at -20°C; for long term storage -80°C is recommended.
Peptides containing Cys, Trp or Met are susceptible to oxidation. It is advisable to blanket the peptide with argon or nitrogen when the vial is opened. Buffers used to dissolve these peptides should be degassed, either by bubbling argon or nitrogen through the solution for 10 minutes, or by subjecting the solution to high vacuum for 10 minutes using a common ultrafiltration capsule. These peptides have limited stability; long-term storage is not recommended.
Hygroscopic peptides containing several charged residues (D, E, K, R, H) may take up water when exposed to air. To prevent this, argon or nitrogen may be used to blanket the peptide when the vial is opened. If inert gases are unavailable, then storage in a desiccator is a viable alternative. Before use, bring samples to room temperature in a desiccator.
Generation of anti-peptide antisera requires immunization of the host with peptide conjugated to a carrier protein in order to have a good chance of eliciting a strong immune response. Commonly used carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA). KLH is often favoured since there is no cross-reactivity with reagents used in subsequent ELISA or Western blot experiments.
The carrier protein may be conjugated using a variety of chemistries (MBS, EDC and activated EDC), the choice depending on the sequence of the peptide and the position of the peptide in the original protein sequence. It is recommended to conjugate the carrier protein at the N- or C-terminus of the peptide, rather than to an internal residue so that as much of the peptide sequence as possible is presented to the host immune system.
If the peptide originates from the N-terminal region of the protein sequence, coupling to the carrier protein should be at the C-terminus of the peptide. Conversely, if the peptide comes from the C-terminal region of the protein sequence, couple the carrier protein to the N-terminus of the peptide. If the peptide comes from an internal region of the protein the carrier protein may be added to either end of the peptide.
The MBS method couples the peptide and carrier protein via the thiol group of a Cys (C) residue. This method is not recommended if there is a Cys residue internally in the peptide sequence. The design process can incorporate a Cys at the N- or C-terminus of the peptide in order to use this method.
The EDC method couples the peptide and carrier protein via the carboxyl group of Asp (D), Glu (E) or the free carboxyl group of the C-terminal amino acid. This method is not recommended if there are Asp or Glu residues internally in the sequence.
The activated EDC method conjugates the carrier protein to the peptide via the amine group of Lys (K) or the free amine of the N-terminal amino acid. This method is not recommended if there is a Lys internally in the peptide sequence.
If the peptide sequence contains Cys, Asp, Glu and Lys internally it is not possible to conjugate the carrier protein specifically to the N- or C-terminus. It is recommended that the peptide sequence be redesigned to meet one of the above criteria for conjugation, in order to have the best chance of a strong immune response.
Epitope Specific Considerations
Prediction of the antigenicity of a peptide for antisera production can be attempted with a combination of predictive algorithms. Where possible the peptide should be conjugated to the carrier at the end that is furthest away from the peak of predicted antigenicity. If you have questions about predicting the antigenicity of peptides, please contact us.
It is also important to screen the peptide for homology with with other protein sequences, both for the species of the target tissue and the host immunized. An antibody generated against a peptide that has high homology with other proteins in the target species may be cross-reactive in that species, leading to false positive results. Where antibodies are specific for continuous peptide sequences they usually recognize epitopes between 5 and 15 amino acids in length. As a general rule, sequences that are identical for 6 consecutive residues have a high risk for antibody cross-reactivity, especially if both the sequence of the target peptide and those of the potentially cross-reactive peptides are located on the protein surface. For short, predicted epitope sequences, 5 to7 amino acids in length, it may be worth considering extending the sequence used for conjugation in order to provide sufficient spacing between the epitope and the carrier molecule, and also to reduce the potential for cross-reactivity with other proteins.
Peptides that have high homology with proteins of the host species to be immunized may not be very antigenic in that species. For proteins that are conserved in many mammalian species one should consider immunizing a phylogenetically more distant host species, such as chickens.
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