Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry

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ADP-ribosylation is a posttranslational modification (PTM) that has crucial functions in a wide range of cellular processes. Although mass spectrometry (MS) in recent years has emerged as a valuable tool for profiling ADP-ribosylation on a system level, the use of conventional MS methods to profile ADP-ribosylation sites in an unbiased way remains a challenge. Here, we describe a protocol for identification of ADP-ribosylated proteins in vivo on a proteome-wide level, and localization of the amino acid side chains modified with this PTM. The method relies on the enrichment of ADP-ribosylated peptides using the Af1521 macrodomain (Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG, EMBO J 24:1911–1920, 2005), followed by liquid chromatography–high-resolution tandem MS (LC-MS/MS) with electron transfer dissociation-based peptide fragmentation methods, resulting in accurate localization of ADP-ribosylation sites. This protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture to data processing using the MaxQuant software suite.

Original languageEnglish
Title of host publicationPoly(ADP-Ribose) Polymerase : Methods and Protocols
EditorsAlexei V. Tulin
PublisherHumana Press
Publication date2023
ISBN (Print)978-1-0716-2893-5, 978-1-0716-2890-4
ISBN (Electronic)978-1-0716-2891-1
Publication statusPublished - 2023
SeriesMethods in Molecular Biology

Bibliographical note

Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

    Research areas

  • ADP-ribosylation, Af1521 macrodomain, Affinity purification, ETD, EThcD, Mass spectrometry, PARG, PARP, Proteomics, PTM

ID: 331584275