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 CreativePepTM Peptide Library technology can be used for a number of different types of 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. The importance of choosing appropriate peptide length and offset number is illustrated with two extreme situations in Fig. 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, see below.
Fig. 1 Examples of strategies in selecting sequences for peptide libraries
Fig. 2 Schematic representation of the different strategies in constructing peptide libraries for sequence optimization
• 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 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.
• Truncation Library
In many cases, truncation library screening allows for the identification of peptides with enhanced proteolytic stability. It can serve as a tool to investigate the extent to which peptide drugs undergo metabolic degradation, which is a major consideration in bringing drugs to the market.
• 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.
• 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.
1. Lubner, J. M., Balsbaugh, J. L., Church, G. M., Chou, M. F., & Schwartz, D. (2018). Characterizing Protein Kinase Substrate Specificity Using the Proteomic Peptide Library (ProPeL) Approach. Current protocols in chemical biology, 10(2), e38.
2. Kara, E., Manna, D., Løset, G. Å., Schneider, E. L., Craik, C. S., & Kanse, S. (2017). Analysis of the substrate specificity of Factor VII activating protease (FSAP) and design of specific and sensitive peptide substrates. Thrombosis and haemostasis, 117(09), 1750-1760.