Peptide Arrays
Custom Peptide Array Synthesis Services

Peptide arrays are powerful tools for the investigation of protein-protein and drug-protein interactions. Screening peptides for potentially active compounds with peptide arrays is a very convenient method for basic and applied research such as drug development. We describe here some of the principles and applications of peptide macroarrays.

For peptide-based drug design, with peptide microarrays it is possible to screen a high number of peptides on a small chip. However, due to the miniscule amounts of peptides synthesized directly on chips, and because of interactions of the peptides with the chip surface, this approach has is sometimes be difficult and unreliable. These disadvantages can be overcome using peptide macroarrays on cellulose membranes. These are useful for screening on the solid phase as well as investigation off the peptides in solution-phase assays. Cellulose membranes are porous, hydrophilic, flexible and stable in organic as well as aqueous solvents. These properties make cellulose paper very useful for biochemical and biological studies in aqueous as well as in organic media and are a major reason why cellulose is still the most widely used material for macroarray membranes.

Array Formats

Depending on the purpose of the screening, different array formats can be generated. The maximum size of that array would be about 8 cm x 13 cm. The standard size of the spots containing peptides is between 1 and 2 mm in diameter (small spots). We offer the synthesis of up to 1400 peptides per membrane. For solution assays, we synthesize the peptides in spots with a diameter of 5-7 mm (large spots) with about 110 peptides per membrane. In this section, we present some array techniques to screen for active peptides on the membrane.

Epitope Mapping (Peptide Scan)

Epitope mapping is a very useful method for screening a known protein sequence for biologically active regions (e.g. epitopes for antibody binding or regions for kinase activity). The peptide sequences are generated by shifting a frame with a distinct peptide length over a protein sequence of interest. A peptide length between 10 and 15 amino acids is commonly used. Shifting of the frame between 1 and 3 amino acids is recommended; the smaller the shift the more precise will be localization of the binding region.

Substitution Analysis (Replacement Analysis, Mutational Analysis)

Substitution analyses are used for investigation of the importance of amino acids and their possible exchanges in a known peptide sequence. The sequence of peptides in this array will be generated by successive systematic substitution of each amino acid by other amino acids or building blocks of interest. Our standard substitution analysis will be performed by the systematic exchange of all positions in the known peptide sequence by all 20 common amino acids.

On demand, we would also perform substitution analyses by using non-natural amino acids or other organic building blocks. The number of peptides on the array is dependent on the starting length of the parent (wild-type) peptide, and the number of amino acids/building blocks used for the substitution.

Truncation Analysis (Length scan)

To investigate the minimum possible length of an active peptide while maintaining activity, variations of the peptide sequence are synthesized by systematic shortening by one amino acid residue at each step from the C-terminus, N-terminus, and both termini simultaneously. The number of peptides on the array is dependent on the starting length of the parent (wild-type) peptide.

Random Peptide Library

To screen for active peptides without prior knowledge of a starting sequence, a random peptide library can be used. This array contains randomized peptide sequences. Compared with the combinatorial peptide library, the advantage of the random peptide array is that the peptides on each spot are unique, providing the possibility of higher individual activity.
The disadvantage is in the relative low number of synthesized peptides available for screening. Our standard random peptide library contains about 1400 peptide sequences per membrane with a length of 12 or 15 amino acids. But a synthesis of random peptide libraries based on other parameters is feasible.

Combinatorial Peptide Library

A very powerful method for screening active peptides without knowing the actual sequence is the combinatorial peptide library. Using combinatorial libraries, screening begins in theory with the pool of all possible peptide sequences. Due to the impossibility of synthesizing all peptides as single sequences (for instance all possible 6-mers of the common 20 L-amino acids would result in 64,000,000 peptide sequences), a mixture of all possible amino acids and building blocks of interest would be used at most unknown positions. After the first synthesis and screening cycle there are two possibilities: First, one can combine different active sequence patterns of the library to a complete sequence. The second possibility is to select the sequence pattern with the strongest activity, keeping that amino acid motif, and perform a deconvolution of the other unknown positions over several synthesis and screening rounds until one obtains a complete sequence in which all amino acid positions are defined. The standard size of peptides in a combinatorial library is 6- to 8-mers, which would need 3 or 4 synthesis and screening rounds to achieve defined peptide sequences. The number of spots depends on the number of amino acids used for the combinations (for the 20 common amino acids an array contains 400 spots).

Cluster Peptide Library

To reduce the number of possibilities, combine several amino acids of similar properties in clusters (e.g. hydrophobicity – Ile, Leu, Val; or steric similarity – Ser, Cys, Abu). These cluster amino acid peptide libraries provide easier and faster screening of large peptide libraries. It is a very useful alternative screening method to combinatorial and random peptide libraries. It is of course necessary to resolve these clusters at the end of the screening process.

Loop Scan (Cysteine Scan)

To stabilize loop structures or to increase their resistance to proteolytic digestion, it is convenient to cyclize peptides. There are two main types of cyclizations – cyclization via cysteines forming a disulfide bridge and cyclization via a pair of peptide amino and carboxy groups to form an amide bond. In both cases, a pair of amino acids is involved. If they are not present in the original peptide sequence, they must be inserted or two existing amino acids should be replaced which could lead to a loss of activity. Therefore, it is necessary to investigate the effect of the insertion/exchange and cyclization on the activity of the peptide. Our standard loop scan is a cysteine loop scan that contains a set of all possible combinations of insertions or replacements using a pair of cysteines. If requested, we could also perform an amide loop scan. The number of peptides on the array depends on the length of the parent (wild-type) peptide.

Customized Peptide Library

We offer also to synthesize a peptide macroarray on cellulose with a set of peptide sequences developed by our customers.

Turnaround Time

The turnaround time for these services is estimated to be 3 to 4 weeks. However, this could varies slightly according to the size of the array and the demand of the service at the time when the order is placed. Each arrayed will be delivered with a comprehensive report.

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