Peptide Arrays in Proteomics and Drug Discovery

Reineke, Ulrich; Schneider‐Mergener, Jens; Schutkowski, Mike

doi:10.1007/978-0-387-25843-0_7

Cited by 11 publications

(11 citation statements)

References 553 publications

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“…15,19,30 It was reported that introducing PEG chains reduced the nonspecific binding of biomolecules to the surface. 12,27,31−34 To determine the susceptibility of studied surfaces to adsorption of proteins, the surfaces were incubated in BSA solution.…”

Section: Resultsmentioning

confidence: 99%

Relevance of the Poly(ethylene glycol) Linkers in Peptide Surfaces for Proteases Assays

et al. 2014

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Poly(ethylene glycol)s (PEGs) with different lengths were used as linkers during the preparation of peptide surfaces for protease detection. In the first approach, the PEG monolayers were prepared using a "grafting to" method on 3-aminopropyltrietoxysilane (APTES)-modified silicon wafers. Protected peptides with a fluorescent marker were synthesized by Fmoc solid phase synthesis. The protected peptide structures enabled their site-specific immobilization onto the PEG surfaces. Alternatively, the PEG-peptide surface was obtained by immobilizing a PEG-peptide conjugate directly onto the modified silicon wafer. The surfaces (composition, grafting density, hydrophilicity, and roughness) were characterized by time-of-flight-secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), contact angle (CA), and atomic force microscopy (AFM). Introducing the PEG linker between the peptide and surface increased their resistance toward nonspecific protein adsorption. The peptide surfaces were examined as analytical platforms to study the action of trypsin as a representative protease. The products of the enzymatic hydrolysis were analyzed by fluorescence spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), and ToF-SIMS. Conclusions about the optimal length of the PEG linker for the analytical application of PEG-peptide surfaces were drawn. This work demonstrates an effective synthetic procedure to obtain PEG-peptide surfaces as attractive platforms for the development of peptide microarrays.

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Section: Resultsmentioning

confidence: 99%

Relevance of the Poly(ethylene glycol) Linkers in Peptide Surfaces for Proteases Assays

et al. 2014

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“…This leads to avidity and rebinding effects and, therefore, enables the identification of even low affinity peptide-antibody interactions. A comprehensive review covering over 600 citations until 2006 summarises studies about mapping of linear and discontinuous epitopes using the SPOT TM technology for peptide synthesis [54].…”

Section: Discussionmentioning

confidence: 99%

Characterisation of the epitope for a herpes simplex virus glycoprotein B-specific monoclonal antibody with high protective capacity

Däumer

Schneider

Giesen

et al. 2010

Med Microbiol Immunol

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Monoclonal antibody (MAb) 2c, specific for glycoprotein B of herpes simplex virus (HSV), had been shown to mediate clearance of infection from the mucous membranes of mice, thereby completely inhibiting mucocutaneous inflammation and lethality, even in mice depleted of both CD4(+) and CD8(+) cells. Additionally, ganglionic infection was highly restricted. In vitro, MAb 2c exhibits a potent complement-independent neutralising activity against HSV type 1 and 2, completely inhibits the viral cell-to-cell spread as well as the syncytium formation induced by syncytial HSV strains (Eis-Hübinger et al. in Intervirology 32:351-360, 1991; Eis-Hübinger et al. in J Gen Virol 74:379-385, 1993). Here, we describe the mapping of the epitope for MAb 2c. The antibody was found to recognise a discontinuous epitope comprised of the HSV type 1 glycoprotein B residues 299 to 305 and one or more additional discontinuous regions that can be mimicked by the sequence FEDF. Identification of the epitope was confirmed by loss of antibody binding to mutated glycoprotein B with replacement of the epitopic key residues, expressed in COS-1 cells. Similarly, MAb 2c was not able to neutralise HSV mutants with altered key residues, and MAb 2c was ineffective in mice inoculated with such mutants. Interestingly, identification and fine-mapping of the discontinuous epitope was not achieved by binding studies with truncated glycoprotein B variants expressed in COS cells but by peptide scanning with synthetic overlapping peptides and peptide key motif analysis. Reactivity of MAb 2c was immensely increased towards a peptide composed of the glycoprotein B residues 299 to 305, a glycine linker, and a C-terminal FEDF motif. If it could be demonstrated that antibodies of the specificity and bioactivity of MAb 2c can be induced by the epitope or a peptide mimicking the epitope, strategies for active immunisation might be conceivable.

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“…[49] A different approach that proved to be very powerful for mapping antibody epitopes is a complete substitutional analysis [56] (for a review see ref. [18]). For example, substitutional analysis of a histone H3-derived peptide on cellulose membranes led to the discovery of DYRKtide, which is a very efficient peptide substrate for DYRK1a.…”

Section: Substrate Identificationmentioning

confidence: 93%

“…SPOT synthesis has been reviewed extensively. [8][9][10][11][12][13][14][15][16][17][18] In fact, due to the simplicity of the SPOT concept, all published applications of in situ synthesized peptide arrays for kinase profiling are based on SPOT synthesis on flexible membranes (see references in Table 1).…”

Section: Array Production and Assay Principlesmentioning

confidence: 99%