ADP-ribosylated peptide enrichment and site identification: The phosphodiesterase-based method

Casey M. Daniels, Shao En Ong, Anthony Kar Lun Leung

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Protein ADP-ribosylation is a posttranslational modification (PTM) that plays an important role in all major cellular processes, including DNA repair, cellular signaling, and RNA metabolism. Site identification for this PTM has recently become possible through the development of several mass spectrometry-based methods, a critical step in understanding the regulatory role played by mono(ADP-ribose) (MAR), poly(ADP-ribose) (PAR), and the enzymes which make these modifications: poly(ADP-ribose) polymerases (PARPs), best known for their role in DNA repair and as targets for chemotherapeutic PARP inhibitors. Here, we have described our method for enriching and identifying ADP-ribosylation events through the use of a phosphodiesterase to digest protein-conjugated ADP-ribose down to its attachment structure, phosphoribose. We also include here a guide to choosing between collision-induced dissociation (CID)-, higher-energy collisional dissociation (HCD)-, and electron-transfer dissociation (ETD)-based peptide fragmentation for the identification of phosphoribosylated peptides.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages79-93
Number of pages15
Volume1608
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1608
ISSN (Print)1064-3745

Keywords

  • ADP-ribosylation
  • ENPP1
  • IMAC
  • MAR
  • Mass spectrometry
  • MS/MS
  • Nudix hydrolase
  • NudT16
  • pADPr
  • PDE
  • Phosphodiesterase
  • Phosphoenrichment
  • Phosphoproteomics
  • Poly(ADP-ribose)
  • Posttranslational modifications
  • RppH
  • SVP

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Daniels, C. M., Ong, S. E., & Leung, A. K. L. (2017). ADP-ribosylated peptide enrichment and site identification: The phosphodiesterase-based method. In Methods in Molecular Biology (Vol. 1608, pp. 79-93). (Methods in Molecular Biology; Vol. 1608). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6993-7_7