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A microarray-based approach to evaluate the functional significance of protein-binding motifs
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 peptid...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830892/ https://www.ncbi.nlm.nih.gov/pubmed/26892640 http://dx.doi.org/10.1007/s00216-016-9382-6 |
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author | Sinzinger, Michael D. Chung, Yi-Da Adjobo-Hermans, Merel J. W. Brock, Roland |
author_facet | Sinzinger, Michael D. Chung, Yi-Da Adjobo-Hermans, Merel J. W. Brock, Roland |
author_sort | Sinzinger, Michael D. |
collection | PubMed |
description | 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. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-016-9382-6) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-4830892 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-48308922016-04-22 A microarray-based approach to evaluate the functional significance of protein-binding motifs Sinzinger, Michael D. Chung, Yi-Da Adjobo-Hermans, Merel J. W. Brock, Roland Anal Bioanal Chem Research Paper 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. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-016-9382-6) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2016-02-18 2016 /pmc/articles/PMC4830892/ /pubmed/26892640 http://dx.doi.org/10.1007/s00216-016-9382-6 Text en © The Author(s) 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Research Paper Sinzinger, Michael D. Chung, Yi-Da Adjobo-Hermans, Merel J. W. Brock, Roland A microarray-based approach to evaluate the functional significance of protein-binding motifs |
title | A microarray-based approach to evaluate the functional significance of protein-binding motifs |
title_full | A microarray-based approach to evaluate the functional significance of protein-binding motifs |
title_fullStr | A microarray-based approach to evaluate the functional significance of protein-binding motifs |
title_full_unstemmed | A microarray-based approach to evaluate the functional significance of protein-binding motifs |
title_short | A microarray-based approach to evaluate the functional significance of protein-binding motifs |
title_sort | microarray-based approach to evaluate the functional significance of protein-binding motifs |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830892/ https://www.ncbi.nlm.nih.gov/pubmed/26892640 http://dx.doi.org/10.1007/s00216-016-9382-6 |
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