A microarray-based approach to evaluate the functional significance of protein-binding motifs
Publication year
2016Source
Analytical and Bioanalytical Chemistry, 408, 12, (2016), pp. 3177-84ISSN
Publication type
Article / Letter to editor

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Organization
Biochemistry (UMC)
Journal title
Analytical and Bioanalytical Chemistry
Volume
vol. 408
Issue
iss. 12
Page start
p. 3177
Page end
p. 84
Subject
Radboudumc 19: Nanomedicine RIMLS: Radboud Institute for Molecular Life Sciences; Radboudumc 3: Disorders of movement DCMN: Donders Center for Medical NeuroscienceAbstract
Intracellular proteins comprise numerous peptide motifs that interact with protein-binding domains. However, using sequence information alone, the identification of functionally relevant interaction motifs remains a challenge. Here, we present a microarray-based approach for the evaluation of peptides as protein-binding motifs. To this end, peptides corresponding to protein interaction motifs were spotted as a microarray. First, peptides were titrated with a pan-specific binder and the apparent K d value of this binder for each peptide was determined. For phosphotyrosine-containing peptides, an anti-phosphotyrosine antibody was employed. Then, in the presence of the pan-specific binder, arrays were competitively titrated with cell lysate and competition constants were determined. Using the Cheng-Prusoff equation, binding constants for the pan-specific binder and inhibition constants for the lysates were converted into affinity constants for the lysate. We experimentally validate this method using a phosphotyrosine-binding SH2 domain as a further reference. Furthermore, strong binders correlated with binding motifs engaging in numerous interactions as predicted by Scansite. This method provides a highly parallel and robust approach to identify peptides corresponding to interaction motifs with strong binding capacity for proteins in the cell lysate. Graphical Abstract Using an antibody as a pan-specific binder the capacity of interaction motifs to bind to proteins from cell lysates can be probed. Competition of the antibody is observed for only those peptides to which a lysate protein binds.
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