A peptide microarray (also commonly known as peptide chip or peptide epitope microarray) is a collection of peptides displayed on a solid surface, usually a glass or plastic chip. Peptide chips are used by scientists in biology, medicine and pharmacology to study binding properties and functionality and kinetics of protein-protein interactions in general. In basic research, peptide microarrays are often used to profile an enzyme (like kinase, phosphatase, protease, acetyltransferase, histone deacetylase etc.), to map an antibody epitope or to find key residues for protein binding. Practical applications are seromarker discovery, profiling of changing humoral immune responses of individual patients during disease progression, monitoring of therapeutic interventions, patient stratification and development of diagnostic tools and vaccines.
The assay principle of peptide microarrays is similar to an ELISA protocol. The peptides (up to tens of thousands in several copies) are linked to the surface of a glass chip typically the size and shape of a microscope slide. This peptide chip can directly be incubated with a variety of different biological samples like purified enzymes or antibodies, patient or animal sera, cell lysates etc. After several washing steps a secondary antibody with the needed specificity (e.g. anti IgG human/mouse or anti phosphotyrosine or anti myc) is applied. Usually, the secondary antibody is tagged by a fluorescence label that can be detected by a fluorescence scanner. Other detection methods are chemiluminescence, colorimetric or autoradiography.
Analysis and evaluation of results
Data analysis and evaluation of results is the most important part of every microarray experiment. After scanning the microarray slides, the scanner records a 20-bit, 16-bit or 8-bit numeric image in tagged image file format (*.tif). The .tif-image enables interpretation and quantification of each fluorescent spot on the scanned microarray slide. This quantitative data is the basis for performing statistical analysis on measured binding events or peptide modifications on the microarray slide. For evaluation and interpretation of detected signals an allocation of the peptide spot (visible in the image) and the corresponding peptide sequence has to be performed. The data for allocation is usually saved in the GenePix Array List (.gal) file and supplied together with the peptide microarray. The .gal-file (a tab-separated text file) can be opened using microarray quantification software-modules or processed with a text editor (e.g. notepad) or Microsoft Excel. This "gal" file is most often provided by the microarray manufacturer and is generated by input txt files and tracking software built into the robots that do the microarray manufacturing.
Major differences between peptide microarrays and protein microarrays
Peptide microarrays show several advantages over protein microarrays:
- Detection of binding events on epitope level, enabling study of i.e. epitope spreading
- Flexible design (i.e. posttranslational modifications, sequence diversity, non-natural amino acids ...)
- Assays rely on detection of robust linear epitope interactions
- Extended shelf stability
- Cost efficiency
- High batch-to-batch reproducibility
Applications of peptide microarrays
Peptide microarrays can be used to study all kinds of protein-protein interactions. Most publications can be found in the context of immune monitoring and enzyme profiling.
- Mapping of immunodominant regions in antigens or whole proteomes
- Seromarker discovery
- Monitoring of clinical trials
- Profiling of antibody signatures
- Finding neutralizing antibodies
- Identification of substrates for orphan enzymes
- Optimization of known enzyme substrates
- Elucidation of signal transduction pathways
- Detection of contaminating enzyme activities
- Consensus sequence and key residues determination
- Identifying sites for protein-protein interactions within a complex
Production of a peptide microarray
A peptide microarray is a planar slide with peptides spotted onto it or assembled directly on the surface by in-situ synthesis. Whereas peptides spotted can undergo quality controls that include mass spectrometer analysis and concentration normalization before spotting and result from a single synthetic batch, peptides synthesized directly on the surface may suffer from batch-to-batch variation and limited quality control options. Peptides are ideally covalently linked through an chemoselective bond leading to peptides with the same orientation for interaction profiling. Alternative procedures describe unspecific covalent binding and adhesive immobilization.
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