Peptide microarray

Peptide microarrayOther names| Peptide chip, peptide array

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Uses| To study binding properties, specificity and functionality and kinetics of protein-peptide or protein-protein interactions

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.

An example of a peptide array used for epitope identification and specificity mapping.[1]

## Contents

* 1 Principle
* 2 Production of a peptide microarray
* 3 Applications of peptide microarrays
* 3.1 Immunology
* 3.2 Enzyme profiling
* 4 Analysis and evaluation of results
* 5 References

## 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 and then be detected through a label-dependent fashion, for example, by a primary antibody that targets the bound protein or modified substrates. 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.[2] Other label-dependent detection methods includes chemiluminescence, colorimetric or autoradiography.

Label-dependent assays are rapid and convenient to perform, but risk giving rise to false positive and negative results.[3] More recently, label-free detection including surface plasmon resonance (SPR) spectroscopy, mass spectrometry (MS) and many other optical biosensors[4][5][6][7] have been employed to measuring a broad range of enzyme activities.[8]

Peptide microarrays show several advantages over protein microarrays:

* Ease and cost of synthesis
* Extended shelf stability
* Detection of binding events on epitope level, enabling study of i.e. epitope spreading
* Flexible design for peptide sequence (i.e. posttranslational modifications, sequence diversity, non-natural amino acids ...) and immobilization chemistries
* Higher batch-to-batch reproducibility

## 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 tens of thousands of peptides providing larger peptide libraries paired with lower synthesis costs.[9] Peptides are ideally covalently linked through a chemoselective bond leading to peptides with the same orientation for interaction profiling. Some alternative procedures describe unspecific covalent binding and adhesive immobilization.

However, lithographic methods can be used to overcome the problem of excessive number of coupling cycles. Combinatorial synthesis of peptide arrays onto a microchip by laser printing has been described,[9][10] where a modified colour laser printer is used in combination with conventional solid-phase peptide synthesis chemistry.[11] Amino acids are immobilized within toner particles, and the peptides are printed onto the chip surface in consecutive, combinatorial layers. Melting of the toner upon the start of the coupling reaction ensures that delivery of the amino acids and the coupling reaction can be performed independently. Another advantage of this method is that each amino acid can be produced and purified separately, followed by embedding it into the toner particles, which allows long-term storage.

## 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 commonly, 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

* Mapping of immunodominant regions in antigens or whole proteomes[12][13][14][15]
* Seromarker discovery[16][17]
* Monitoring of clinical trials[18]
* Profiling of antibody signatures[19][20] and epitope mapping
* Finding neutralizing antibodies[21]

### Enzyme profiling

* Identification of substrates for orphan enzymes[22]
* Optimization of known enzyme substrates[23]
* Elucidation of signal transduction pathways[24]
* Detection of contaminating enzyme activities
* Consensus sequence and key residues determination[25]
* Identifying sites for protein-protein interactions within a complex[26]

## Analysis and evaluation of results

Data analysis and evaluation of results is the most important part of every microarray experiment.[27] 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. ↑ Hansen, Lajla Bruntse; Buus, Soren; Schafer-Nielsen, Claus (2013-07-23). "Identification and Mapping of Linear Antibody Epitopes in Human Serum Albumin Using High-Density Peptide Arrays". PLOS ONE 8 (7). doi:10.1371/journal.pone.0068902. ISSN 1932-6203. PMID 23894373. Bibcode: 2013PLoSO...868902H.
2. ↑ 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.
3. ↑ Kaeberlein, Matt; McDonagh, Thomas; Heltweg, Birgit; Hixon, Jeffrey; Westman, Eric A.; Caldwell, Seth D.; Napper, Andrew; Curtis, Rory et al. (2005-04-29). "Substrate-specific Activation of Sirtuins by Resveratrol". Journal of Biological Chemistry 280 (17): 17038–17045. doi:10.1074/jbc.M500655200. ISSN 0021-9258. PMID 15684413.
4. ↑ Fernández Gavela, Adrián; Grajales García, Daniel; Ramirez, Jhonattan C.; Lechuga, Laura M. (2016-02-24). "Last Advances in Silicon-Based Optical Biosensors". Sensors 16 (3): 285. doi:10.3390/s16030285. PMID 26927105. Bibcode: 2016Senso..16..285F.
5. ↑ Fang, Ye (2010). "Resonant Waveguide Grating Biosensor for Microarrays". Optical Guided-wave Chemical and Biosensors II. Springer Series on Chemical Sensors and Biosensors. 8. Springer, Berlin, Heidelberg. pp. 27–42. doi:10.1007/978-3-642-02827-4_2. ISBN 978-3-642-02826-7.
6. ↑ Picaud, Sarah; Filippakopoulos, Panagis (2015-08-17). "SPOTing Acetyl-Lysine Dependent Interactions". Microarrays 4 (3): 370–388. doi:10.3390/microarrays4030370. PMID 27600229.
7. ↑ Hundsberger, Harald; Önder, Kamil; Schuller-Götzburg, Peter; Virok, Dezso P.; Herzog, Julia; Rid, Raphaela (2017-06-08). "Assembly and use of high-density recombinant peptide chips for large-scale ligand screening is a practical alternative to synthetic peptide libraries". BMC Genomics 18 (1): 450. doi:10.1186/s12864-017-3814-3. ISSN 1471-2164. PMID 28595602.
8. ↑ Szymczak, Lindsey C.; Kuo, Hsin-Yu; Mrksich, Milan (2018-01-02). "Peptide Arrays: Development and Application". Analytical Chemistry 90 (1): 266–282. doi:10.1021/acs.analchem.7b04380. ISSN 0003-2700. PMID 29135227.
9. ↑ 9.0 9.1 Beyer, M; Nesterov, A; Block, I; König, K; Felgenhauer, T; Fernandez, S; Leibe, K; Torralba, G et al. (Dec 21, 2007). "Combinatorial synthesis of peptide arrays onto a microchip.". Science 318 (5858): 1888. doi:10.1126/science.1149751. PMID 18096799. Bibcode: 2007Sci...318.1888B.
10. ↑ Breitling, Frank; Felgenhauer, Thomas; Nesterov, Alexander; Lindenstruth, Volker; Stadler, Volker; Bischoff, F. Ralf (2009-03-23). "Particle-Based Synthesis of Peptide Arrays". ChemBioChem 10 (5): 803–808. doi:10.1002/cbic.200800735. ISSN 1439-7633. PMID 19191248.
11. ↑ Stadler, Volker; Felgenhauer, Thomas; Beyer, Mario; Fernandez, Simon; Leibe, Klaus; Güttler, Stefan; Gröning, Martin; König, Kai et al. (2008-09-01). "Combinatorial Synthesis of Peptide Arrays with a Laser Printer". Angewandte Chemie International Edition 47 (37): 7132–7135. doi:10.1002/anie.200801616. ISSN 1521-3773. PMID 18671222. Bibcode: 2008ACIE...47.7132S.
12. ↑ Zandian, Arash; Forsström, Björn; Häggmark-Månberg, Anna; Schwenk, Jochen M.; Uhlén, Mathias; Nilsson, Peter; Ayoglu, Burcu (9 February 2017). "Whole-Proteome Peptide Microarrays for Profiling Autoantibody Repertoires within Multiple Sclerosis and Narcolepsy". Journal of Proteome Research 16 (3): 1300–1314. doi:10.1021/acs.jproteome.6b00916. PMID 28121444.
13. ↑ 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. PMID 19577281.
14. ↑ 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.
15. ↑ Jaenisch, Thomas; Heiss, Kirsten; Fischer, Nico; Geiger, Carolin; Bischoff, F. Ralf; Moldenhauer, Gerhard; Rychlewski, Leszek; Sié, Ali et al. (April 2019). "High-density Peptide Arrays Help to Identify Linear Immunogenic B-cell Epitopes in Individuals Naturally Exposed to Malaria Infection". Molecular & Cellular Proteomics 18 (4): 642–656. doi:10.1074/mcp.RA118.000992. ISSN 1535-9484. PMID 30630936.
16. ↑ Callaway, Ewen (2011). "Clues emerge to explain first successful HIV vaccine trial". Nature. doi:10.1038/news.2011.541.
17. ↑ Heiss, Kirsten; Heidepriem, Jasmin; Fischer, Nico; Weber, Laura K.; Dahlke, Christine; Jaenisch, Thomas; Loeffler, Felix F. (September 2020). "Rapid Response to Pandemic Threats: Immunogenic Epitope Detection of Pandemic Pathogens for Diagnostics and Vaccine Development Using Peptide Microarrays". Journal of Proteome Research 19 (11): 4339–4354. doi:10.1021/acs.jproteome.0c00484. PMID 32892628.
18. ↑ 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.
19. ↑ 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. PMID 19864486.
20. ↑ Weber, Laura K.; Palermo, Andrea; Kügler, Jonas; Armant, Olivier; Isse, Awale; Rentschler, Simone; Jaenisch, Thomas; Hubbuch, Jürgen et al. (April 2017). "Single amino acid fingerprinting of the human antibody repertoire with high density peptide arrays". Journal of Immunological Methods 443: 45–54. doi:10.1016/j.jim.2017.01.012. ISSN 1872-7905. PMID 28167275.
21. ↑ 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. PMID 21849452.
22. ↑ 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). doi:10.1074/mcp.M111.015701. PMID 22205724.
23. ↑ Lizcano, J. M.; Deak, M; Morrice, N; Kieloch, A; Hastie, CJ; Dong, L; Schutkowski, M; Reimer, U et al. (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.
24. ↑ 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. et al. (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. PMID 18045915.
25. ↑ 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.
26. ↑ Parsons, LS; Wilkens, S (2012). "Probing subunit-subunit interactions in the yeast vacuolar ATPase by peptide arrays". PLOS ONE 7 (10). doi:10.1371/journal.pone.0046960. PMID 23071676. Bibcode: 2012PLoSO...746960P.
27. ↑ 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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