PEPTIDE MICROARRAYS

Peptides are short chains of amino acids and are distinguished from proteins generally by size-with peptides having fewer than 50-60 amino acids. The use of peptides in microarrays has several advantages over arrays of full-length proteins: for small amounts as needed in microarrays, peptides can be chemically synthesized more economically and efficiently than purification of proteins from biological sources, and peptide arrays may have a longer shelf life that arrays of larger proteins. Furthermore, peptides offer a high degree of specificity, usually, a single epitope compared to larger proteins.

The two main applications of protein arrays are for monitoring immune response and profiling enzyme binding sites. Peptides are used to generate highly specific antibodies to discrete epitopes, enabling the distinction between similar or isotypic proteins. In a typical immune response, there are ‘immunodominant’ peptide sequences that may be part of larger proteins that can efficiently stimulate an immune response. Thus peptides are often screened in vaccine development studies of the immune response to pathogens (Gaseltsiwe et al., 2010) as well as identification of antibodies used for diagnosis of infection (Maksimov et al., 2012).  Another advantage of peptides is their use to identify specific sites of protein-protein interactions that can identify discrete protein structures (as in enzyme binding sites)  for drug targets. For both of these applications, peptide microarrays offer the ability to economically screen a large number of peptides in a single experiment.

Recently peptide arrays have been used to identify cell modulators (Khan et al., 2010). In these experiments, cells are incubated over a peptide microarray, and the array is analyzed for cell adherence or differentiation associated with specific spots. Biologically active peptides required for stem cell growth, differentiation, cell adhesion, or other activation of cell activity have been identified with this technique. Thus peptide arrays may help identify potential therapeutic peptides, a growing class of pharmaceuticals including hormones such as insulin (Lax and Meenan, 2012).

There are disadvantages to peptide microarrays when compared to full protein microarrays, mainly the relatively low affinity of peptide-protein interactions, potentially resulting in false-negative results and lower sensitivity for detection. Additionally, peptides alone do not bind well to glass or polymer substrates and so most peptide arrays are manufactured using a chemical linker to covalently bind the peptides to the substrate. Covalent attachment of the peptide limits the concentration of a peptide within a spot based on the density of the linkers, and may also interfere with its orientation and bio-recognition, again raising the potential for limited sensitivity and false-negative results.

Use of Grace Bio-Labs products in Peptide microarrays:

Incubation chambers from Grace Bio-Labs, including the HybriSlip and ProPlate, provide excellent results with peptide microarrays. The incubation chamber is an important parameter that is often overlooked when performing microarray experiments. The ideal chamber should allow for sufficient sample mixing during the assay incubation and wash steps and should minimize the volume of sample required. Active mixing has been shown to significantly affect assay signal and uniformity.

Coverslips generally allow for the lowest sample incubation volumes and, but do not allow sample mixing during incubation.  If the use of a coverslip is necessary for your particular assay, we recommend the use of Grace Bio-Labs HybriSlip™ over conventional glass coverslips. In addition, Grace Bio-Labs has developed incubation chambers that facilitate incubations for a wide range of sample volumes with various The Pro-Plate® chamber from Grace Bio-labs is excellent for most microarray applications on slide or plate format.

Nitrocellulose Films slides are not the best choice for peptide arrays due to the nature of their protein binding. The binding of biomolecules to nitrocellulose occurs through combined weak intermolecular forces, primarily hydrophobic and Van der Waals forces. Peptides likely do not provide enough opportunities for these interactions to create a strong bond, and thus are generally difficult to detect on nitrocellulose film. For proteins greater than 20 kD in molecular weight,  the advantages of porous nitrocellulose include non-covalent attachment that does not disrupt the three-dimensional structure and bioactivity of the protein. In addition, nitrocellulose film has tremendous binding capacity compared to other two- and three-dimensional surfaces (for a complete review, see the Oncyte Guide to Protein Arrays on this website).   This advantage is lost on small peptides.

Gaseltsiwe et al., Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1 0101, DRB1 1501 and DRB1 0401, Clinical and Vaccine Immunology, 2010: 17(1) 168-75. www.ncbi.nlm.nih.gov/pmc/articles/PMC2812096/

Maksimov et al., Peptide Microarray Analysis of In Silico-Predicted Epitopes for Serological Diagnosis of Toxoplasma gondii Infection in Humans, Clinical and Vaccine Immunology, 2012:  19(6), 865-74. www.ncbi.nlm.nih.gov/pmc/articles/PMC3370440/

F. Khan, et al., Strategies for cell manipulation and skeletal tissue engineering using high-throughput polymer blend formulation and microarray techniques. Biomaterials, 2010, 31, 2216-2228. http://www.sciencedirect.com/science/article/pii/S0142961209013453?via%3Dihub