TY - JOUR
T1 - ELTA
T2 - Enzymatic Labeling of Terminal ADP-Ribose
AU - Ando, Yoshinari
AU - Elkayam, Elad
AU - McPherson, Robert Lyle
AU - Dasovich, Morgan
AU - Cheng, Shang Jung
AU - Voorneveld, Jim
AU - Filippov, Dmitri V.
AU - Ong, Shao En
AU - Joshua-Tor, Leemor
AU - Leung, Anthony K.L.
N1 - Funding Information:
We thank Paul Chang, Joel Moss, Marc Greenberg and the members of the Leung lab for their critiques of the manuscript, Vinay Ayyappan for maintaining ADPriboDB used in this analysis, and Mohsen Badiee for drawing the schematics of Figure 1 A. We thank Timothy Mitchison, Edwin Tan, and Kristin Krukenberg for sharing in vitro purified PAR and their advice for synthesizing and purifying PAR to defined chain length. We thank Ivan Ahel for the plasmid encoding ha PARP, Wenqing Xu for iso- ADP-ribose, and Hans Kistemaker for the ADP-ribosylated peptide HK533. We thank Bob Cole in the Mass Spectrometry and Proteomics Facility at the Johns Hopkins School of Medicine for assisting mass spectrometry analyses and Arthur Makarenko from the CSHL mass spectrometry core facility for his help with PAR purifications using HPLC. We thank Anna Kondratova for pointing out a previous observation by Cayley and Kerr on the addition of oligoadenylate on ADP-ribose by OAS1 ( Cayley and Kerr, 1982 ). This work was supported by a Johns Hopkins Discovery Award (A.K.L.L.), the Proteomics Core Coins Award from the Johns Hopkins School of Medicine (A.K.L.L. and R.L.M.), W. W. Smith Charitable Trust Medical Research Award (A.K.L.L.), and research grants Research Scholar Award ( RSG-16-062-01-RMC ) (A.K.L.L.) from American Cancer Society , R01GM104135 (A.K.L.L.), T32CA009110 (R.L.M.), T32GM080189 (M.D.), and R01AR065459 (S.-E.O.) from the NIH. Part of this work utilized an EASY-nLC1200 UHPLC and Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer purchased with funding from an NIH SIG grant S10OD021502 (S.-E.O.). L.J.-T. is an Investigator of the Howard Hughes Medical Institute.
Publisher Copyright:
© 2018 Elsevier Inc.
PY - 2019/2/21
Y1 - 2019/2/21
N2 - ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2′-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates. Ando et al. describe a simple, efficient, and versatile platform technology called ELTA to label free or protein-conjugated ADP-ribose monomers and polymers with dATP analogs (radioactive, fluorescent, biotin, clickable tags, etc.). With these functionalized tags, ELTA simplifies the measurement, detection, and enrichment of various forms of ADP-ribose.
AB - ADP-ribosylation refers to the addition of one or more ADP-ribose groups onto proteins. The attached ADP-ribose monomers or polymers, commonly known as poly(ADP-ribose) (PAR), modulate the activities of the modified substrates or their binding affinities to other proteins. However, progress in this area is hindered by a lack of tools to investigate this protein modification. Here, we describe a new method named ELTA (enzymatic labeling of terminal ADP-ribose) for labeling free or protein-conjugated ADP-ribose monomers and polymers at their 2′-OH termini using the enzyme OAS1 and dATP. When coupled with various dATP analogs (e.g., radioactive, fluorescent, affinity tags), ELTA can be used to explore PAR biology with techniques routinely used to investigate DNA or RNA function. We demonstrate that ELTA enables the biophysical measurements of protein binding to PAR of a defined length, detection of PAR length from proteins and cells, and enrichment of sub-femtomole amounts of ADP-ribosylated peptides from cell lysates. Ando et al. describe a simple, efficient, and versatile platform technology called ELTA to label free or protein-conjugated ADP-ribose monomers and polymers with dATP analogs (radioactive, fluorescent, biotin, clickable tags, etc.). With these functionalized tags, ELTA simplifies the measurement, detection, and enrichment of various forms of ADP-ribose.
KW - ADP-ribose
KW - ADP-ribosylated protein
KW - ADP-ribosylation
KW - ADP-ribosyltransferase
KW - enzymatic labeling
KW - mono(ADP-ribosyl)ated protein
KW - oligoadenylate synthetase
KW - poly(ADP-ribose)
KW - poly(ADP-ribose) polymerase
KW - poly(ADP-ribosyl)ated protein
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U2 - 10.1016/j.molcel.2018.12.022
DO - 10.1016/j.molcel.2018.12.022
M3 - Article
C2 - 30712989
AN - SCOPUS:85061545874
SN - 1097-2765
VL - 73
SP - 845-856.e5
JO - Molecular cell
JF - Molecular cell
IS - 4
ER -