del Rincon, SV;
Rogers, J;
Widschwendter, M;
Sun, DH;
Sieburg, HB;
Spruck, C;
(2010)
Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays.
PLOS ONE
, 5
(6)
, Article e11332. 10.1371/journal.pone.0011332.
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Abstract
Background: 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.Methodology/Principal Findings: 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.Conclusions/Significance: 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.
| Type: | Article |
|---|---|
| Title: | Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1371/journal.pone.0011332 |
| Publisher version: | http://dx.doi.org/10.1371/journal.pone.0011332 |
| Language: | English |
| Additional information: | © 2010 del Rincon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This work was supported by a grant from the Department of Defense Breast Cancer Research Program (DOD BCRP, W81XWH-07-1-0628) to CS. SVdR was supported by a postdoctoral fellowship from the DOD BCRP (W81XWH-06-1-0544) and an IDEA award from the California Breast Cancer Research Program (CBCRP, 15IB-0130). Materials and salary support for JR were provided by Life Technologies Corp. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. |
| Keywords: | UBIQUITIN-PROTEASOME PATHWAY, SACCHAROMYCES-CEREVISIAE, SUMOYLATED PROTEINS, PROLACTIN RECEPTOR, MASS-SPECTROMETRY, LIGASE, IDENTIFICATION, PROTEOMICS, CANCER, PHOSPHORYLATION |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL EGA Institute for Womens Health UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL EGA Institute for Womens Health > Womens Cancer |
| URI: | https://discovery.ucl.ac.uk/id/eprint/886671 |
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