Phosphoproteomic Approach to Characterize Protein Mono- and Poly(ADP-ribosyl)ation Sites from Cells

Abstract

Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome.

PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes

“…Enzymatic cleavage Enzymatic cleavage of PAR and ADP-ribose to reduce complexity using enzymes, such as snake venom phosphodiesterase (SVP), ARH3, PARG, or NUDIX hydrolases, is becoming a popular approach as a prelude to MS (Messner et al 2010;Chapman et al 2013;Sharifi et al 2013;Daniels et al 2014Daniels et al , 2015bMartello et al 2016). A number of groups have used SVP, a pyrophosphatase that cleaves ADP-ribose subunits to phosphoribose (also known as ribose-5 ′ -phosphate), which remains covalently attached to the amino acid in the ADP-ribosylated protein, and 5 ′ -AMP, which is released (Chapman et al 2013;Daniels et al 2014).…”

“…A number of groups have used SVP, a pyrophosphatase that cleaves ADP-ribose subunits to phosphoribose (also known as ribose-5 ′ -phosphate), which remains covalently attached to the amino acid in the ADP-ribosylated protein, and 5 ′ -AMP, which is released (Chapman et al 2013;Daniels et al 2014). The phosphoribose moiety remaining at the site of modification can be identified by MS as a shift of 212.01 Da (Fig.…”

“…7A,C). Phosphoribosylated peptides can be enriched using techniques developed previously for phosphopeptides, such as immobilized metal affinity chromatography (IMAC) (Chapman et al 2013;Daniels et al 2014). This approach is not restricted to acidic residues, unlike hydroxylamine treatment, providing a major advantage for this approach (Table 2).…”

“…This approach is not restricted to acidic residues, unlike hydroxylamine treatment, providing a major advantage for this approach (Table 2). Moreover, the coenrichment of phosphorylated amino acids allows for the study of the dynamics between phosphorylation and ADP-ribosylation (Daniels et al 2014). This approach has been used to identify sites of ADP-ribosylation on automodified PARP-1, including glutamate, aspartate, and lysine residues (Chapman et al 2013), as well as endogenous ADP-ribosylation sites from whole cells (Daniels et al 2014).…”

“…In HeLa cells, sites of glutamate, lysine, arginine, and aspartate ADP-ribosylation were comparably enriched, while, in liver tissue, sites of arginine ADPribosylation were markedly enriched (Martello et al 2016). Although this technique allows for the identification of all amino acids that are ADP-ribosylated, bias for the macrodomain against ADP-ribosylated glutamic acid could lead to the loss of a number of targets (Daniels et al 2014).…”