Peptide microarray
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.
Principle
The assay principle of peptide microarrays is similar to an ELISA protocol. The peptides (up to thousands in several copies) are linked to the surface of a glass chip. 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 [1]. Other detection methods are chemiluminescence or autoradiography.
Analysis and Evaluation of Results
Data analysis and evaluation of results is the most important part of every microarray experiment [2]. After scanning the microarray slides, the scanner records a 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 evaluation software-modules or processed with a text editor (e.g. notepad) or Microsoft Excel.
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.
Immunology
- Mapping of immunodominant regions in antigens or whole proteomes [3] [4]
- Seromarker discovery [5]
- Monitoring of clinical trials [6]
- Profiling of antibody signatures [7]
- Finding neutralizing antibodies [8]
Enzyme Profiling
- Identification of substrates for orphan enzymes [9]
- Optimization of known enzyme substrates [10]
- Elucidation of signal transduction pathways [11]
- Detection of contaminating enzyme activities
- Consensus sequence and key residues determination [12]
Production of a Peptide Microarray
A peptide microarray is a planar slide with peptides spotted onto it or assembled directly on the surface. Whereas peptides spotted can undergo quality controls 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.
References
- ^ Molecular Diversity (2004-08). "Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays. - Panse et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Autoimmunity Reviews (2012-01). "Computational analysis of high-density peptide microarray data with application from systemic sclerosis to multiple sclerosis. - Hecker et al.".
{{cite web}}: Check date values in:|date=(help) - ^ The Journal of Allergy and Clinical Immunology (2009-08). "Development of a novel peptide microarray for large-scale epitope mapping of food allergens - Lin et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Analytical and Bioanalytical Chemistry (2012-02). "Clonality Characterization of Natural Epitope-Specific Antibodies Against the Tumor-Related Antigen Topoisomerase IIa by Peptide Chip and Proteome Analysis: a Pilot Study with Colorectal Carcinoma Patient Samples - Linnebacher et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Nature (2011-09-16). "Clues emerge to explain first successful HIV vaccine trial - Callaway".
- ^ Annals of Neurology (2008-05). "Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis. - Garren et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Clinical and Vaccine Immunology (2010-01). "Peptide Microarray – Based Identification of Mycobacterium Tuberculosis Epitope Binding to HLADRB1*0101, DRB1*1501 and DRB1*0401 - Gaseitsiwe et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Journal of Virology (2011-09). "Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals. - Tomaras et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Molecular & Cellular Proteomics (2011-12). "Systems kinomics demonstrates congo basin monkeypox virus infection selectively modulates host cell signaling responses as compared to West African monkeypox virus.- Kindrachuk et al." (PDF).
{{cite web}}: Check date values in:|date=(help) - ^ Journal of Biological Chemistry (2002-08). "Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6). Evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo. - Lizcano et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Journal of Neuroscience (2007-11). "NMDA receptor activation dephosphorylates AMPA receptor glutamate receptor 1 subunits at threonine 840. - Delgado et al.".
{{cite web}}: Check date values in:|date=(help) - ^ Journal of Molecular Biology (2011-08). "Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity. - Thiele et al.".
{{cite web}}: Check date values in:|date=(help)