Development and validation of a method for profiling post-translational modification activities using protein microarrays.

<h4>Background</h4>Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary...

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Autores principales:	Sonia V Del Rincón, Jeff Rogers, Martin Widschwendter, Dahui Sun, Hans B Sieburg, Charles Spruck
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e4ee10f24fc348ad802a862252e12c53
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spelling	oai:doaj.org-article:e4ee10f24fc348ad802a862252e12c532021-12-02T20:20:28ZDevelopment and validation of a method for profiling post-translational modification activities using protein microarrays.1932-620310.1371/journal.pone.0011332https://doaj.org/article/e4ee10f24fc348ad802a862252e12c532010-06-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20596523/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens.<h4>Methodology/principal findings</h4>In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay.<h4>Conclusions/significance</h4>This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.Sonia V Del RincónJeff RogersMartin WidschwendterDahui SunHans B SieburgCharles SpruckPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 6, p e11332 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sonia V Del Rincón Jeff Rogers Martin Widschwendter Dahui Sun Hans B Sieburg Charles Spruck Development and validation of a method for profiling post-translational modification activities using protein microarrays.
description	<h4>Background</h4>Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens.<h4>Methodology/principal findings</h4>In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay.<h4>Conclusions/significance</h4>This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research.
format	article
author	Sonia V Del Rincón Jeff Rogers Martin Widschwendter Dahui Sun Hans B Sieburg Charles Spruck
author_facet	Sonia V Del Rincón Jeff Rogers Martin Widschwendter Dahui Sun Hans B Sieburg Charles Spruck
author_sort	Sonia V Del Rincón
title	Development and validation of a method for profiling post-translational modification activities using protein microarrays.
title_short	Development and validation of a method for profiling post-translational modification activities using protein microarrays.
title_full	Development and validation of a method for profiling post-translational modification activities using protein microarrays.
title_fullStr	Development and validation of a method for profiling post-translational modification activities using protein microarrays.
title_full_unstemmed	Development and validation of a method for profiling post-translational modification activities using protein microarrays.
title_sort	development and validation of a method for profiling post-translational modification activities using protein microarrays.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/e4ee10f24fc348ad802a862252e12c53
work_keys_str_mv	AT soniavdelrincon developmentandvalidationofamethodforprofilingposttranslationalmodificationactivitiesusingproteinmicroarrays AT jeffrogers developmentandvalidationofamethodforprofilingposttranslationalmodificationactivitiesusingproteinmicroarrays AT martinwidschwendter developmentandvalidationofamethodforprofilingposttranslationalmodificationactivitiesusingproteinmicroarrays AT dahuisun developmentandvalidationofamethodforprofilingposttranslationalmodificationactivitiesusingproteinmicroarrays AT hansbsieburg developmentandvalidationofamethodforprofilingposttranslationalmodificationactivitiesusingproteinmicroarrays AT charlesspruck developmentandvalidationofamethodforprofilingposttranslationalmodificationactivitiesusingproteinmicroarrays
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Development and validation of a method for profiling post-translational modification activities using protein microarrays.

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