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 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.[1] Other detection methods are chemiluminescence, colorimetric or autoradiography.

Peptide microarrays show several advantages over protein microarrays:

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. However, peptide synthesis on chip allows the parallel synthesis of tenths of thousands of peptides providing larger peptide libraries paired with lower synthesis costs.[2] 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.

Applications of peptide microarrays

Peptide microarrays can be used to study different kinds of protein-protein interactions, specially those involving modular protein substructures called peptide recognition modules or, most communly, protein interaction domains. The reason for this is that such protein substructures recognize short linear motifs often exposed in natively unstructured regions of the binding partner, such that the interaction can be modelled in vitro by peptides as probes and the peptide recognition module as analyte. Most publications can be found in the context of immune monitoring and enzyme profiling.

Immunology

Enzyme profiling

Analysis and evaluation of results

Data analysis and evaluation of results is the most important part of every microarray experiment.[14] 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.

References

  1. ↑ Panse, S; Dong, L; Burian, A; Carus, R; Schutkowski, M; Reimer, U; Schneider-Mergener, J (2004). "Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays". Molecular diversity. 8 (3): 291–9. doi:10.1023/B:MODI.0000036240.39384.eb. PMID 15384422.
  2. ↑ Beyer, M; Nesterov, A; Block, I; König, K; Felgenhauer, T; Fernandez, S; Leibe, K; Torralba, G; Hausmann, M; Trunk, U; Lindenstruth, V; Bischoff, FR; Stadler, V; Breitling, F (Dec 21, 2007). "Combinatorial synthesis of peptide arrays onto a microchip.". Science. 318 (5858): 1888. doi:10.1126/science.1149751. PMID 18096799.
  3. ↑ Lin, Jing; Bardina, Ludmilla; Shreffler, Wayne G.; Andreae, Doerthe A.; Ge, Yongchao; Wang, Julie; Bruni, Francesca M.; Fu, Zhiyan; et al. (2009). "Development of a novel peptide microarray for large-scale epitope mapping of food allergens". Journal of Allergy and Clinical Immunology. 124 (2): 315–22, 322.e1–3. doi:10.1016/j.jaci.2009.05.024. PMC 2757036Freely accessible. PMID 19577281.
  4. ↑ Linnebacher, M; Lorenz, P; Koy, C; Jahnke, A; Born, N; Steinbeck, F; Wollbold, J; Latzkow, T; et al. (2012). "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". Analytical and Bioanalytical Chemistry. 403 (1): 227–38. doi:10.1007/s00216-012-5781-5. PMID 22349330.
  5. ↑ Callaway, Ewen (2011). "Clues emerge to explain first successful HIV vaccine trial". Nature. doi:10.1038/news.2011.541.
  6. ↑ Garren, H; Robinson, WH; Krasulová, E; Havrdová, E; Nadj, C; Selmaj, K; Losy, J; Nadj, I; et al. (2008). "Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis". Annals of Neurology. 63 (5): 611–20. doi:10.1002/ana.21370. PMID 18481290.
  7. ↑ Gaseitsiwe, S.; Valentini, D.; Mahdavifar, S.; Reilly, M.; Ehrnst, A.; Maeurer, M. (2009). "Peptide Microarray-Based Identification of Mycobacterium tuberculosis Epitope Binding to HLA-DRB1*0101, DRB1*1501, and DRB1*0401". Clinical and Vaccine Immunology. 17 (1): 168–75. doi:10.1128/CVI.00208-09. PMC 2812096Freely accessible. PMID 19864486.
  8. ↑ Tomaras, GD; Binley, JM; Gray, ES; Crooks, ET; Osawa, K; Moore, PL; Tumba, N; Tong, T; et al. (2011). "Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1-infected individuals". Journal of Virology. 85 (21): 11502–19. doi:10.1128/JVI.05363-11. PMC 3194956Freely accessible. PMID 21849452.
  9. ↑ Kindrachuk, J; Arsenault, R; Kusalik, T; Kindrachuk, KN; Trost, B; Napper, S; Jahrling, PB; Blaney, JE (2011). "Systems kinomics demonstrates congo basin monkeypox virus infection selectively modulates host cell signaling responses as compared to West African monkeypox virus". Molecular & Cellular Proteomics. 11 (6): M111.015701. doi:10.1074/mcp.M111.015701. PMID 22205724.
  10. ↑ Lizcano, J. M.; Deak, M; Morrice, N; Kieloch, A; Hastie, CJ; Dong, L; Schutkowski, M; Reimer, U; Alessi, DR (2002). "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". Journal of Biological Chemistry. 277 (31): 27839–49. doi:10.1074/jbc.M202042200. PMID 12023960.
  11. ↑ Delgado, J. Y.; Coba, M.; Anderson, C. N. G.; Thompson, K. R.; Gray, E. E.; Heusner, C. L.; Martin, K. C.; Grant, S. G. N.; O'Dell, T. J. (2007). "NMDA Receptor Activation Dephosphorylates AMPA Receptor Glutamate Receptor 1 Subunits at Threonine 840". Journal of Neuroscience. 27 (48): 13210–21. doi:10.1523/JNEUROSCI.3056-07.2007. PMC 2851143Freely accessible. PMID 18045915.
  12. ↑ Thiele, A; Krentzlin, K; Erdmann, F; Rauh, D; Hause, G; Zerweck, J; Kilka, S; Pösel, S; et al. (2011). "Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity". Journal of Molecular Biology. 411 (4): 896–909. doi:10.1016/j.jmb.2011.06.040. PMID 21756916.
  13. ↑ Parsons, LS; Wilkens, S (2012). "Probing subunit-subunit interactions in the yeast vacuolar ATPase by peptide arrays". PLoS ONE. 7 (10): e46960. doi:10.1371/journal.pone.0046960. PMC 3470569Freely accessible. PMID 23071676.
  14. ↑ Hecker, M; Lorenz, P; Steinbeck, F; Hong, L; Riemekasten, G; Li, Y; Zettl, UK; Thiesen, HJ (2012). "Computational analysis of high-density peptide microarray data with application from systemic sclerosis to multiple sclerosis". Autoimmunity reviews. 11 (3): 180–90. doi:10.1016/j.autrev.2011.05.010. PMID 21621003.
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