ADP-Ribosylated Peptide Enrichment and Site Identification: The Phosphodiesterase-Based Method

Abstract

Protein ADP-ribosylation is a post-translational modification (PTM) that plays an important role in all major cellular processes, including DNA repair, cellular signaling, and RNA metabolism. Site identification for this PTM has recently become possible through the development of several mass spectrometry based methods, a critical step in understanding the regulatory role played by mono(ADP-ribose) (MAR), poly(ADP-ribose) (PAR), and the enzymes which make these modifications: Poly(ADP-ribose) Polymerases (PARPs), best known for their role in DNA repair and as targets for chemotherapeutic PARP inhibitors. Here, we have described our method for enriching and identifying ADP-ribosylation events through the use of a phosphodiesterase to digest protein-conjugated ADP-ribose down to its attachment structure, phosphoribose. We also include here a guide to choosing between Collision-induced dissociation (CID), Higher-energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) based peptide fragmentation for the identification of phosphoribosylated peptides.

The Role of Electron Transfer Dissociation in Modern Proteomics

“…We direct readers to recent reviews that cover broader technologies for analysis of phosphorylation, 102 − 104 glycosylation, 105 − 107 and PTMs in general 100 , 101 for further insights. We also note that ETD has been valuable for PTM analysis beyond the scope discussed here, including ubiquitylation, 108 − 110 ADP-ribosylation, 111 − 113 and arginine methylation. 114 − 118 …”

Development of a mass-spectrometry based method for the identification of thein vivowhole blood and plasma ADP-ribosylomes

“…Nevertheless, it remained unclear if the demodifications observed here were mediated by classic ADP-ribosylhydrolases that could be present in the blood or other enzymes, like phosphodiesterases (PDEs), that have been shown to be abundant in the blood and able to reduce ADP-ribose to phosphoribose 37, 38 . To investigate this, we repeated the experiment described above in the presence of cAMP, a metabolite that can compete with ADP-ribose as a PDE substrate.…”

The multifaceted role of PARP1 in RNA biogenesis

“…Only a few of the PARPs (PARP1, 2, 5A, 5B) are capable of true polymerization (PARylation) while the majority, transfer only a single ADPr (Barkauskaite et al, 2015; Leung, 2014; Vyas & Chang, 2014) (MARylation) onto target molecules. For many years it was thought that ADPr transfer (MARylation and PARylation) occurred only on proteins via ester linkage at 7 amino acid residues (asparagine, aspartic acid, glutamic acid, arginine, lysine, serine, and cysteine) (Bonfiglio, Colby, & Matic, 2017; Daniels, Ong, & Leung, 2017; Palazzo et al, 2018; Vyas & Chang, 2014). However, recent studies now show MARylation and PARylation occurring on DNA (Munnur, Bartlett, et al, 2019; Munnur, Somers, et al, 2019; Talhaoui et al, 2016), RNA (Munnur, Bartlett, et al, 2019), antibiotics (reviewed in Palazzo, Mikoc, and Ahel (2017)), and other small molecules (Kirby & Cohen, 2019).…”