ELTA: Enzymatic Labeling of Terminal ADP-Ribose

Abstract

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.

Keywords: ADP-ribose; ADP-ribosylated protein; ADP-ribosylation; ADP-ribosyltransferase; enzymatic labeling; mono(ADP-ribosyl)ated protein; oligoadenylate synthetase; poly(ADP-ribose); poly(ADP-ribose) polymerase; poly(ADP-ribosyl)ated protein.

Figure 1.. ELTA labels free or protein-conjugated ADP-ribose monomers and polymers.

(a) Schematics of ELTA. Free or protein-conjugated ADP-ribose can be labeled by incubating with OAS1 and dATP, where the 2’-OH terminus is indicated in red. Colored box indicates various dATP analogs that can also be used in the ELTA reactions, including radioactive (³²P), fluorescent (Cy3, Cy5), biotinylated or clickable analogs. (b) 15% urea-PAGE analyses of the addition of ³²P-dAMP onto ADP-ribose monomers and polymers using ELTA and visualized by autoradiograph. (c) MALDI-TOF analyses of the reaction of ADP-ribose with dATP, and with or without OAS1. (d-e) Analyses of the ELTA labeling reaction of (d) MARylated PARP10 catalytic domain (mod-PARP10^cd) and (e) PARylated haPARP (mod-haPARP) using ³²P-dATP. Shown are a coomassie gel (left), an autoradiograph (middle), and a western blot probed with pan-ADP-ribose reagent (right). As negative controls, modified proteins were treated with the phosphodiesterase hsNudT16 to remove the 2’-OH termini of the ADP-ribose groups prior to ELTA labeling. For panel d, * indicates PARP10; OAS1 was ADP-ribosylated by PARP10 with the remnant of NAD⁺, and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1. For panel e, * indicates haPARP and ^§ indicates haPARP fragments that were also ADP-ribosylated and, therefore, detected by pan-ADP-ribose reagent and labeled by OAS1.

Figure 2.. Biophysical measurement of interaction between PAR-binding protein domain WWE and PAR of defined chain length.

(a) Workflow used for ELTA-modified PAR of defined length for biophysical measurement. The details of HPLC run are illustrated in Fig. S1c. (b) Filter binding assay of RNF146 WWE domain binding to 10- and 20-mer PAR, which were radiolabeled using ELTA and ³²P-dATP. (c) MST analysis of RNF146 WWE domain binding to 20-mer PAR, which was labeled using ELTA and Cy5-dATP.

Figure 3.. Detection of ADP-ribose length from individual proteins and cells using ELTA.

(a) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose monomer (lane 1) and PAR of mixed length (lane 2), as well as ADP-ribose monomers and polymers isolated from PARP1 automodification reactions with 1 mM NAD⁺ for 0 (lane 3), 10 (lane 4), or 30 min (lane 5) that were labeled by OAS1 and ³²P-dATP. As a comparison, the ADP-ribose isolated from PARP1 automodification reaction in the same time frame with 1 mM NAD⁺ with a trace of ³²P-NAD⁺ were loaded in lanes 6–8. We note that 50-fold less of the reaction were loaded in lanes 3–5 compared with lanes 6–8. (b) 15% urea-PAGE analyses of ELTA labeling reaction of ADP-ribose monomers and polymers isolated from automodification of PARP1 along with either BSA and HPF1. The first lane contained ELTA-labeling of an equal mole of 5-, 10- and 20-mer PAR. The reaction in the PARP1+BSA lane was diluted 15 times in water prior to ELTA labeling. (c) 15% urea-PAGE analyses of the ELTA labeling reaction of ADP-ribose isolated from in vitro modified haPARP (lane 1), from untreated HaCaT cells (lane 2), from HaCaT cells treated with 1 mM H₂O₂ for 10 min (lane 3), from HaCaT cells treated with 1 mM H₂O₂ for 10 min, but pre-treated the cells with 20 μM PARP inhibitor Olaparib for 2 h (lane 4), or pre-treated with 1 μM PARG inhibitor PDD00017273 for 2 h (lane 5). Corresponding lysates of cells from lanes 2–5 were probed with β-actin.

Figure 4.. Enrichment of ELTA-labeled ADP-ribosylated peptides from complex mixtures.

(a) Schematics of the pipeline to selectively label and enrich ADP-ribosylated peptides. (b) MALDI-TOF analyses of (1) the reaction mixtures after ELTA but prior to enrichment, (2) the flowthrough from the enrichment matrix, (3) the eluant with the phosphodiesterase hsNudT16. The m/z traces shown are the overlay of two experiments, where red lines indicate samples with OAS1 and black lines indicate samples without OAS1. (c) The total intensity chromatograph (TIC) of the input sample (left), which includes 1 nmol of ADP-ribosylated peptide HK533 in 1 mg cell lysate peptides, and of the enriched sample after elution (middle) and the mass spectrometry scan analyses at the retention time of 6.53 min (right). (d) Quantification of the peptide-spectrum matches (PSM) to the ADP-ribosylated peptide HK533 enriched from 1 mg cell lysate peptides, which are dosed with either 1 nmol, 1 pmol, or 1 fmol HK533. (e) Identified MS/MS spectra of a trypic peptide identified from endogenous hnRNPU, with the modification site signified by the addition of a phosphoribose group (red).