Abstract
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 language | English |
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Title of host publication | Poly(ADP-Ribose) Polymerase : Methods and Protocols |
Editors | Alexei V. Tulin |
Publisher | Humana Press |
Publication date | 2023 |
Pages | 251-270 |
ISBN (Print) | 978-1-0716-2893-5, 978-1-0716-2890-4 |
ISBN (Electronic) | 978-1-0716-2891-1 |
DOIs | |
Publication status | Published - 2023 |
Series | Methods in Molecular Biology |
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Volume | 2609 |
ISSN | 1064-3745 |
Bibliographical note
Publisher Copyright:© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
Keywords
- ADP-ribosylation
- Af1521 macrodomain
- Affinity purification
- ETD
- EThcD
- Mass spectrometry
- PARG
- PARP
- Proteomics
- PTM