Cited by 119Advanced Proteomics Techniques and ApplicationsPARP inhibition in cancer therapyDNA Repair Mechanisms

Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells

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Daniels, Casey M., Ong, Shao-En, Leung, Anthony K. L.·2014·Journal of Proteome Research

Abstract

Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome.

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  • PARP1 transfers the first ADP-ribose moiety from NAD+ to specific residues on an acceptor protein and then adds multiple ADP-ribose units to form PAR chains.

    “Experimental evidence supports that PARP1 transfers the first ADP-ribose moiety from NAD+ to lysine, arginine, glutamate, aspartate, and serine residues on an acceptor protein, followed by addition of multiple ADP-ribose units to the preceding ones, thereby forming poly(ADP-ribose) (PAR) chains (Bonfiglio et al., 2017; Daniels et al., 2014).”

    From NAD+ in Brain Aging and Neurodegenerative Disorders (2019) · cites this paper as Daniels et al., 2014

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Authors
Daniels, C. M., Ong, S.-E., & Leung, A. K. L.
Affiliations
  • Johns Hopkins University (US)
  • University of Washington (US)
Corresponding author
  • Ong, Shao-En
  • Leung, Anthony K. L.
Venue
Journal of Proteome Research
Published
2014-06-11
Volume
13
Issue
8
Pages
3510-3522
ISSN
  • 1535-3893 (ISSN-L)
  • 1535-3907
OpenAlex
W2332306552
Cited by
119
References
50
Citations by year
2026: 5 · 2025: 6 · 2024: 6 · 2023: 3 · 2022: 6 · 2021: 7 · 2020: 7 · 2019: 14 · 2018: 14 · 2017: 21 · 2016: 8 · 2015: 19 · 2014: 3
FWCI
7.28
Type
journal article
Language
en
Open access
Locations
  • Journal of Proteome Research (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • U.S. Department of Defense (BC101881)
  • National Institute on Drug Abuse (P30DA028846-01)
  • National Cancer Institute (5T32CA009110-35)
  • Company of Biologists (TF261)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Health Sciences › Medicine › Oncology › PARP inhibition in cancer therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
poly(ADP-ribosyl)ation, ADP-ribose, poly(ADP-ribose) polymerase, PARP1, phosphodiesterase, phosphoproteomics, macrodomain, post-translational modification
MeSH terms
Adenosine Diphosphate Ribose, Amino Acids, Binding Sites, Chromatography, Liquid, Crotalid Venoms, HeLa Cells, Humans, Poly Adenosine Diphosphate Ribose, Poly(ADP-ribose) Polymerases, Molecular Structure, DNA Primers, Proteomics, Phosphodiesterase I, Tandem Mass Spectrometry

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