Peptide arrays

In this instance, adamantane was present to promote interaction of peptides through its hydrophobicity, but its attachment did not hinder fibril formation. It might be possible to chemically or biologically derivatise this group before being introduced to the peptide, or to select another hydrophobic component that could be suitably modified and attached to the peptide. This research also highlights the feasibility of creating peptide arrays comprised of several different sequences. 

The earliest applications of the array format have been for peptide arrays. For example, the pin method for peptide synthesis involves the... 

The SPOT-synthesis method also employs Fmoc chemistry but uses hydroxyl groups present on cellulose filter paper to derivatize and thereby immobilize (3-alanine groups onto the paper. After deprotection, the 13-alanine groups can be used as platforms for the synthesis of peptide arrays (Fig. 7.5) (Frank, 1992 Gausepohl et al., 1992). This method has been widely used for mapping antigen-antibody interactions as well as protein-DNA, protein-metal and other protein-protein interactions (Reineke et al., 2001). 

The most frequent application of SPOT-synthesis has been in the preparation of peptide arrays for the identification of linear B-cell epitopes. If the protein antigen is known, a set of overlapping peptides that encompass the entire sequence can be readily synthesized and assayed for binding of antibody (Reineke et al., 1999). The individual residues critical for binding can then be determined by SPOT-synthesis of peptides containing amino acid substitutions. 

Figure 7.5. Peptide array construction by SPOT-synthesis. fl-alanine groups (b-A) interact with the cellulose filter that serves as a planar support. Peptide synthesis then proceeds using Fmoc chemistries using the fl-alanine group as a starting point. The peptide is attached to the filter via its carboxy-terminus. In this case, lysine is added at the second position and various amino acids are present at the amino terminus of the peptide.

Reineke, U., Volkmer-Engert, R., and Schneider-Mergener, J. (2001). Applications of peptide arrays prepared by the SPOT-technology. Curr. Opin. Biotechnol. 12, 59-64. 

Usui, K., Ojima, T., Takahashi, M., Nokihara, K. and Mihara, H. (2004a). Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns. Biopolymers 76, 129-139. 

Gannot G, Tangrea MA, Erickson HS, et al. Layered peptide array for multiplex immunohistochemistry./. Mol. Diagn. 2007 9 297-304. 

Sompuram S, Vani K, Bogen S. A molecular model of antigen retrieval using a peptide array. Am. J. Clin. Pathol. 2006 125 91-98. 

Figure 16.5 Immunostained peptide arrays after various treatments of fixation, protein cross-linking, and antigen retrieval, as indicated at the top. Each row has a different peptide that is immunoreactive for the antibody denoted to the left. Column A represents the baseline condition, without any treatment whatsoever. Column B shows immunoreactivity of each peptide after overnight formalin fixation. Column C shows the immunoreactivity after first coating the array with an irrelevant protein (casein) followed by overnight formalin fixation. Column D illustrates the immunoreactivity of the peptides after the treatment of column C, and then antigen retrieval. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

Figure 16.6 illustrates the proposed sequence of events during immuno-histochemistry staining with the peptide array. Amino acids are represented as circles. Each peptide is a string of amino acids that are covalently... 

A reasonable objection to any in vitro model is whether it accurately mirrors the actual process. A strength of this model is that the peptides in the array, mounted on the microscope glass slide, are the very same as the antibody epitopes in the native proteins. Therefore, the types of formaldehyde-induced chemical reactions at or near the epitope are the same as would likely occur in a tissue sample. An additional strength of the model is that the experimental data using the peptide array completely account for the loss of immunoreactivity after formalin fixation and the recovery of immunoreactivity after antigen retrieval (Fig. 16.5). Nonetheless, our data do not prove that the model accurately represents formaldehyde reactions in tissue specimens. For example, our data do not exclude other causes of steric interference. 

Figure 16.8 Intensity of immunohistochemical staining as a function of the length of antigen retrieval time. All values represent the mean of triplicate measurements. The staining intensity of a peptide array that was not formalin fixed is shown at the far right of the graph, as a control. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

G. J. Wegner, H. J. Lee, and R. M. Com, "Characterization and Optimization of Peptide Arrays for the Study of Epitopeantihody Interactions Using Surface Plasmon Resonance imaging," Analytical Chemistry 74, 5161-5168 (2002). 

Melnyk, O., Duburcq, X., Olivier, C., Urbes, R, Auriault, C., and Gras-Masse, H., Peptide arrays for highly sensitive and specific antibody-binding fluorescence assays. Bioconjugate Chem., 13, 713-720, 2002. 

Human Proteinpedia provides a list of phosphopeptides identified in mass spectrometry-based phosphoproteomic studies. In addition, phosphorylation and dephosphorylation data curated from the literature are mapped to corresponding site and residue of sequences provided in HPRD. Using PhosphoMotif Finder one can analyze the presence of phosphorylation-based motifs, which are derived from the literature, in any protein of interest. This is a valuable data for biomedical investigators in the development of phospho-spedfic antibodies and peptide arrays. Availability of many raw MS datasets deposited in Human Proteinpedia... 

Frank, R. (2002) The SPOT-synthesis technique. Synthetic peptide arrays on membrane supports—principles and applications. J. Immunol. Methods 267, 13-26. 

Wegner, G.J., Lee, H.J., Corn, R.M., Characterization and optimization of peptide arrays for the study of epitope-antibody interactions using surface plasmon resonance imaging. Anal. Chem. 2003, 74, 5161-5168. 

Kato R, Kaga C, Kunimatsu M et al (2006) Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design. J Biosci Bioeng 101(6) 485—495... 

John D. Wade Howard Florey Institute and School of Chemistry, University of Melbourne, Parkville, Victoria, Australia Jan-Christoph Westermann Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia Dirk F.H. Winkler Peptide Array Facility of the Brain Research Centre, University of British Columbia, Vancouver, British Columbia, Canada Weiguang Zeng Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia... 

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