Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry
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Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry. / Anagho, Holda A.; Elsborg, Jonas D.; Hendriks, Ivo A.; Buch-Larsen, Sara C.; Nielsen, Michael L.
Poly(ADP-Ribose) Polymerase: Methods and Protocols. ed. / Alexei V. Tulin. Humana Press, 2023. p. 251-270 (Methods in Molecular Biology, Vol. 2609).Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-review
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TY - CHAP
T1 - Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry
AU - Anagho, Holda A.
AU - Elsborg, Jonas D.
AU - Hendriks, Ivo A.
AU - Buch-Larsen, Sara C.
AU - Nielsen, Michael L.
N1 - Publisher Copyright: © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2023
Y1 - 2023
N2 - 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.
AB - 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.
KW - ADP-ribosylation
KW - Af1521 macrodomain
KW - Affinity purification
KW - ETD
KW - EThcD
KW - Mass spectrometry
KW - PARG
KW - PARP
KW - Proteomics
KW - PTM
U2 - 10.1007/978-1-0716-2891-1_15
DO - 10.1007/978-1-0716-2891-1_15
M3 - Book chapter
C2 - 36515840
AN - SCOPUS:85144586189
SN - 978-1-0716-2893-5
SN - 978-1-0716-2890-4
T3 - Methods in Molecular Biology
SP - 251
EP - 270
BT - Poly(ADP-Ribose) Polymerase
A2 - Tulin, Alexei V.
PB - Humana Press
ER -
ID: 331584275