Mapping physiological ADP-ribosylation using Activated Ion Electron Transfer Dissociation (AI-ETD)

Abstract

max. 150 words)ADP-ribosylation (ADPr) is a post-translational modification that plays pivotal roles in a wide range of cellular processes. Mass spectrometry (MS)-based analysis of ADPr under physiological conditions, without relying on genetic or chemical perturbation, has been hindered by technical limitations. Here, we describe the applicability of Activated Ion Electron Transfer Dissociation (AI-ETD) for MS-based proteomics analysis of physiological ADPr using our unbiased Af1521 enrichment strategy. To benchmark AI-ETD, we profiled 9,000 ADPr peptides mapping to >5,000 unique ADPr sites from a limited number of cells exposed to oxidative stress, corresponding to 120% and 28% more ADPr peptides compared to contemporary strategies using ETD and EThcD, respectively. Under physiological conditions AI-ETD identified 450 ADPr sites on low-abundant proteins, including in vivo cysteine automodifications on PARP8 and tyrosine auto-modifications on PARP14, hinting at specialist enzymatic functions for these enzymes. Collectively, our data provides new insights into the physiological regulation of ADP-ribosylation.

Chemical genetics and proteome-wide site mapping reveal cysteine MARylation by PARP-7 on immune-relevant protein targets

“…This led to the notion that the major ADPr acceptor residues are the acidic amino acids, glutamate (Glu) and aspartate (Asp), as well as the basic amino acid arginine (Arg). More recently, however, mass spectrometry-based proteomics has revealed that in addition to Glu/Asp and Arg, serine (Ser), tyrosine (Tyr), histidine (His), and cysteine (Cys) can also act as ADPr acceptors ( Buch-larsen, 2020 ; Larsen et al, 2018 ; Leslie Pedrioli et al, 2018 ; Palazzo et al, 2018 ; Leidecker et al, 2016 ). While many residues can act as ADPr acceptors, a major outstanding question in the field is whether or not individual PARP family members demonstrate selectivity toward ADP-ribosylation of specific residues in protein targets.…”

Progress and outlook in studying the substrate specificities of PARPs and related enzymes

“…With thousands of sites in the human cell, serine ADP-ribosylation appears to be a major contributor to the proteome complexity. Further promising candidates include cysteine [23,57,60] and tyrosine [23,51,58], which might be the targets of specific PARP family members; several other amino acids are also possible as acceptors. Moreover, it has emerged that PARPs and related ARTs can modify not only proteins, but also nucleic acids.…”

“…The identification of PARP1 itself as the main or at least a major PAR acceptor in vivo [19] was followed by the narrowing down of the modification sites to the 'automodification domain', a central region of the polymerase between the DNA-binding and catalytic segments [20]. For reasons that are explained below, PARP1 automodifies in cells at both glutamate/aspartate and serine residues, and major sites include glutamates 488 and 491 [21] and serines 499, 507 and 519 [22,23]. One key function of PARP automodification appears to be to release the enzyme from DNA lesions, presumably through electrostatic and steric repulsion [24][25][26].…”

“…Indeed, a broader approach to ADP-ribosylation localisation, accompanied by a more tailored mass spectrometry fragmentation procedure, allowed the identification of serine sites in the PARP1 automodification domain, histone tails and other known PARP substrates, both in vivo and in vitro [22,43,47]. Independent studies have confirmed serine as the most frequently detected ADP-ribose acceptor in mammalian cells, especially under genotoxic conditions [2,23,[48][49][50][51]. The identified serine sites are enriched for Lys-Ser and, to a lesser extent, Arg-Ser motifs [22,47,50].…”

“…The possible novel PARP acceptors do not stop at serine. Other amino acid residues that have been identified to be ADP-ribosylated in recent studies includein addition to glutamate, aspartate and lysinealso arginine, cysteine, histidine, threonine and tyrosine residues [2,23,51,57,58]. It is not known to which ADP-ribosylation writers these sites can be attributed, and some of them might be, in part or completely, accounted for by non-PARP ARTs; this applies in particular to arginine, which is known to be modified by human cholera toxin-like ectoenzymes [9].…”