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Published on: April 17, 2026
Optimized detection of acidic ADP-ribosylation by PARP14 in vitro using multi-modal mass spectrometry reveals
Steph Nivaggioli1, Hannah H Nguyen1, Kiana K Harker1
1Department of Chemistry and Biochemistry, Santa Clara University, Santa Clara, CA, USA.
Abstract:
ADP-ribosylation is a highly dynamic process that is increasingly linked to a diverse set of cellular functions and pathophysiological outcomes. The modification of target substrates (e.g., proteins, DNA, RNA) involves ligation to an array of different chemical moieties. Recent work has shown that the ester bonds formed between ribose and Asp/Glu residues are particularly labile. A common strategy to overcome this lability is the replacement of the ADP-ribose (ADPr) by hydroxylamine. Attachment of hydroxamic acid (HA) permanently shifts the mass of labeled Asp/Glu residues by +15 Da. However, this method suffers from two notable limitations: HA treatment for long periods of time will label Asp/Glu sites that are solvent exposed, but not modified by ADPr; and the competing hydrolysis reaction still occurs in the conditions routinely used for HA labeling. Herein, we optimize a mass spectrometry (MS) workflow that overcomes these drawbacks. As a proof-of-principle, a truncated form of PARP14 was assessed for its ability to ADP-ribosylate peptides and itself. Conditions that prioritize HA attack over the background rate of hydrolysis were determined using an ultrathin matrix-assisted laser-desorption/ionization (TLC-MALDI) time-of-flight (TOF) method with ADPr-peptides. These efforts doubled HA labeling efficiency and completely removed ADPr within an hour. Next, a series of MS experiments identified the sites of PARP14 automodification using both standard HA labeling conditions (overnight, pH 7.0, 25 °C) and the optimized conditions. Of the identified 9 ADPr sites on PARP14, 5 are unique to the optimized conditions and 6 are newly identified. Mutagenesis revealed a set of sites that down-regulated PARP14 and another set that appears to activate PARP14. Taken together, these efforts have improved the true positive site identification rate using HA and have revealed a novel role for several automodification sites in PARP14 regulation.
