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

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.