dELTA-MS: A Mass Spectrometry-Based Proteomics Approach for Identifying ADP-Ribosylation Sites and Forms

Abstract

ADP-ribosylation, characterized by the addition of adenosine diphosphate ribose, can occur in both monomeric (MARylation) and polymeric (PARylation) forms. Little is known about the specific contributions of MARylation and PARylation to cellular processes due to a lack of tools for jointly investigating these individual forms. We present a novel mass spectrometry (MS)-based proteomics approach that preserves information about the native ADP-ribosylation form associated with the modification site within a single proteomics experiment. Our workflow enables the simplified, binary identification of ADP-ribosylation forms, avoiding some challenges typically presented by PARylated peptides during MS analysis. Our method uses the coronaviral enzyme NS2 to reverse our previous labeling approach, ELTA, which enzymatically labels the terminal ADP-ribose. NS2 deconjugates ELTA-labeled free or peptide-conjugated ADP-ribose monomers and polymers (thereby termed dELTA), leaving behind a signature phosphate. Our dELTA-MS workflow involves ELTA labeling, dELTA deconjugation, and further processing using Deinococcus radiodurans poly(ADP-ribose) glycohydrolase (DrPARG), resulting in two distinct mass shifts for MARylation and PARylation sites. We demonstrate the feasibility of this workflow for proteomics analyses using proof-of-principle peptide standards. dELTA-MS thus creates possibilities to reveal the fundamental biology of ADP-ribosylation and explore its dysregulation, in terms of both sites and forms, associated with disease progression.

Keywords: ADP-ribosylation; PARPs; mono-ADP-ribosylation; poly-ADP-ribosylation; post-translational modification; proteomics.

Figure 1.. NS2 deconjugates ELTA-labeled ADP-ribose.

(A) Comparison of ELTA-MS with dELTA-MS. Both workflows use the same click chemistry-based enrichment system which enzymatically labels the terminal ADP-ribose with an azido-based enrichment tag that can be clicked to an alkyne-based resin. However, the workflows differ in the release step. ELTA-MS uses NudT16 which cleaves phosphodiester bonds within ADP-ribose, leaving behind a phospho-ribose mass tag. In contrast, dELTA-MS uses NS2 which cleaves the linkage between the terminal ADP-ribose and the azido enrichment tag, thus releasing peptides while preserving the integrity of the ADP-ribosylation. HPLC analysis of ADP-ribose ELTA-labeled by OAS1 with 1 mM dATP (B), 500 μM dATP-azide (C), and 125 μM dATP-biotin (D). These OAS1 products were then treated with NS2.

Figure 2.. NS2 leaves behind a signature phosphate on the terminal ADP-ribose.

(A) MALDI-TOF mass spectra of ELTA-labeled JV-099 treated with NS2. (B) MALDI-TOF mass spectra of an ELTA-labeled 7mer PAR chain treated with NS2. (C) 15% urea-PAGE analysis of 7mer PAR ELTA-labeled with [α-³²P]-dATP and then treated with NS2. Dotted lines indicate two lanes that were removed for clarity. (D) MALDI-TOF mass spectra of the flowthrough for the controls of the click chemistry-based pulldown of JV-099. (E) MALDI-TOF mass spectra of the flowthrough after release of JV-099 via incubation of DBCO-agarose with NS2 and the positive control NudT16.

Figure 3.. PARG treatment after OAS1 and NS2 treatment preserves the phosphate on a MARylated peptide.

(A) Workflow of enzymatic reactions. (B) Autoradiogram of enzymatic workflow on JV-099 labeled with [α-³²P]-dATP. (C) Autoradiogram of enzymatic workflow on a 6mer PAR chain labeled with [α-³²P]-dATP. (D) HCD MS2 spectrum of JV-099 with ADPr-P modification. (E) HCD MS2 spectrum of JV-099 with ADPr modification.

Figure 4.. dELTA-MS workflow pulls down standard MARylated and PARylated peptides with unique mass tags.

(A) Schematic of dELTA-MS workflow for proof-of-principle experiments to identify JV-522 and H3(1-20)S10arn. In one experiment, JV-522 was spiked into H₂O₂-treated HeLa cell lysate prior to digestion. In another experiment, H3(1-20)S10arn was spiked into H₂O₂-treated HeLa cell lysate peptides right before dELTA-MS. (B) EThcD MS2 spectrum of JV-522 that was identified with the ADPr-P modification expected for a MAR site after processing with the dELTA-MS workflow. (C) EThcD MS2 spectrum of H3(1-20)S10arn that was identified with the ADPr modification expected for a PAR site after processing with the dELTA-MS workflow.