Details

Autor(en) / Beteiligte

• Sinzinger, Michael D.
• Chung, Yi-Da
• Adjobo-Hermans, Merel J. W.
• Brock, Roland

Titel

A microarray-based approach to evaluate the functional significance of protein-binding motifs

Ist Teil von

• Analytical and bioanalytical chemistry, 2016-05, Vol.408 (12), p.3177-3184

Ort / Verlag

Berlin/Heidelberg: Springer Berlin Heidelberg

Erscheinungsjahr

2016

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• 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