Peptide Library Technical Support

Peptide Library Design Dissolving Peptide Libraries

Peptide Library Design

Our technical and applications support team can offer advice on library design and experimental set up and analysis.  Relating to design, we can advise on the best overlap or offset of peptides to be used in your target application, and then using your full-length protein sequence, we can generate the list of peptides for your library.  Assistance with experimental set-up and analysis is available for applications such as ELISPOT, cell culture, intracellular cytokine staining and flow cytometry.

The Prospector® Peptide Library technology can be used for a number of different types of library:
> Overlapping peptide library
> Alanine Scanning Library
> Truncation Library
> Random Library
> Positional Scanning Library

Overlapping Peptide Library

The overlapping peptide library is most commonly used for linear, continuous epitope mapping, where the aim is to generate a library of overlapping peptide sequences of specific length and specific offset, to cover the entire native protein sequence. Choice of the appropriate peptide length and offset number depends on the application of the peptides and also affects both the cost of the peptide set and the usefulness of the data obtained from the experiment. Examples of design parameters for selected applications are shown below:

Application Length Offset
CD4+ and CD8+ T cell epitope mapping
CD8+ T cell epitope mapping
MHC-Peptide Binding Assays
CD4+ T cell epitope mapping
CD8+ T cell epitope mapping
B cell epitope mapping
(see also Truncation Library, below)
5-20 1

Summary of the potential effects of the different combinations between peptide length and offset number:

Offset Number Short Peptide Sequence Long Peptide Sequence
Short Offset Number 1. Requires synthesis of the most number of peptides
2. Shorter peptides are easier to synthesize, and often result in higher purity
3. More chances for multiple epitope hits
1. Requires synthesis of more peptides
2. Longer peptides are more difficult to synthesize, and may result in lower purity
3. Most chances for multiple epitope hits
Long Offset Number 1. Requires synthesis of fewer peptides
2. Shorter peptides are easier to synthesize, and often result in higher purity
3. Least chance for multiple epitope hits
1. Requires synthesis of the fewest number of peptides
2. Longer peptides are more difficult to synthesize, and may result in lower purity
3. Less chance for multiple epitope hits

The importance of choosing appropriate peptide length and offset number is illustrated with two extreme situations in figure 1.  If a 10mer with offset 2 is chosen, at least three peptide sequences would span the epitope (‘hits’), and 6 10mers need to be made.  If a 6mer with offset 4 is chosen, fewer peptides need to be synthesized, but the epitope could be missed outright, as would be the case if the hypothetical epitope spanned the sequence HIKLMN, see below.

Figure 1. Examples of strategies in selecting sequences for peptide libraries. Residues inside the dotted box are in the hypothetical active site or epitope.  The last peptide sequence must fulfil all three of the following requirements: (1) it must include the last residue in the native sequence, (2) it must have more amino acids than the “offset” number, and (3) it must have at least 6 residues, the minimum number that can potentially form an epitope.

Epitope identification is followed by studies to demonstrate structure and function relationships of peptide sequences, usually by peptide sequence optimization and structure stabilization. Synthesis of alternative types of peptide library can greatly assist the sequence optimization process.

Figure 2.
Schematic representation of the different strategies in constructing peptide libraries for sequence optimization.  The presumed essential positions are enclosed in the dotted box.

Figures 1 & 2 reproduced with permission from Sigma.

A. Alanine Scanning Library

Alanine is systematically substituted into each amino acid position in the identified epitope. This strategy identifies the amino acids in the native sequence that are essential for activity.  Substitution of an essential amino acid results in a reduction in peptide activity, and the degree of reduction in activity is usually taken as a relative measure of the importance of the amino acid being substituted.

B. Truncation Library

This strategy determines the minimum length required for optimum peptide activity by generating a set of peptides with systematic truncation of the flanking residues. If the essential amino acids are known, the direction of truncation can be selected around them, as opposed to systematic truncation from both ends of the peptide sequence.

C. Random Library

Selected residues in the peptide sequence (wobbles) are simultaneously substituted with a mixture of all 20 amino acids, or a mixture of specific amino acids. In practice, this strategy is usually used for preliminary identification of a group of active sequences that can then be re-synthesized to validate the initial results.

D. Positional Scanning Library

A selected position or positions in a peptide sequence are each systematically replaced with different amino acids in order to determine the preferred amino acid residues at these positions, measured by corresponding increases in activity.

Contact the technical support team to discuss your library design requirements; we are happy to generate the list of peptides for the library format you need.

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Recommendations for dissolving PEPscreen®: Custom Peptide Libraries

Complete solubilization of peptides is important for successful screening of peptide activities. Peptides can be fully active only if they are completely solubilized and are able to assume the correct conformation for binding to their receptors. As the number of peptides in a set increases, so does the potential solubility variation of the peptides within the set. Therefore, in order to obtain accurate and reliable peptide activity data, careful attention should be devoted to the process of dissolving peptide sets.

The strategy for dissolving the PEPscreen® peptide set, and any of the other Prospector® Libraries, is different from dissolving individual peptides. For individual peptides, conditions are chosen for optimum solubility based on the given peptide sequence.  However, for peptide sets, conditions are chosen in an effort to dissolve as many of the peptides in the set as possible in the first solubilization attempt. Suggested common strategies are schematically represented below:

Full details of the steps above: Guidelines for dissolving peptide sets (PDF)

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PEPscreen is manufactured by Sigma (formerly Sigma-Genosys) for ProImmune
PEPscreen is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.