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An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

Karunakaran, Kalesh; Lukauskas, Saulius; Borg, Aaron J.; Snijders, Ambrosius P.; Ayyappan, Vinay; Leung, Anthony K.L.; Haskard, Dorian O.; DiMaggio, Peter A.

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Authors

Saulius Lukauskas

Aaron J. Borg

Ambrosius P. Snijders

Vinay Ayyappan

Anthony K.L. Leung

Dorian O. Haskard

Peter A. DiMaggio



Abstract

ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. We herein show that an adenosine analogue with a terminal alkyne functionality at position 2 of the adenine (2-alkyne adenosine or 2YnAd) is suitable for selective enrichment, fluorescence detection and mass spectrometry proteomics analysis of the candidate ADP-ribosylome in mammalian cells. Although similar labelling profiles were observed via fluorescence imaging for 2YnAd and 6YnAd, a previously reported clickable NAD+ precursor, quantitative mass spectrometry analysis of the two probes in MDA-MB-231 breast cancer cells revealed a significant increase in protein coverage of the 2YnAd probe. To facilitate global enrichment of ADP-ribosylated proteins, we developed a dual metabolic labelling approach that involves simultaneous treatment of live cells with both 2YnAd and 6YnAd. By combining this dual metabolic labelling strategy with highly sensitive tandem mass tag (TMT) isobaric mass spectrometry and hierarchical Bayesian analysis, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib.

Citation

Karunakaran, K., Lukauskas, S., Borg, A. J., Snijders, A. P., Ayyappan, V., Leung, A. K., …DiMaggio, P. A. (2019). An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation. Scientific Reports, 9, Article 6655. https://doi.org/10.1038/s41598-019-43154-1

Journal Article Type Article
Acceptance Date Apr 11, 2019
Online Publication Date Apr 30, 2019
Publication Date Apr 30, 2019
Deposit Date May 8, 2019
Publicly Available Date May 9, 2019
Journal Scientific Reports
Publisher Nature Research
Peer Reviewed Peer Reviewed
Volume 9
Article Number 6655
DOI https://doi.org/10.1038/s41598-019-43154-1

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http://creativecommons.org/licenses/by/4.0/

Copyright Statement
This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.




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