title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, Maryland 21205, United States.
authors.forename: Casey M
authors.initials: CM
authors.lastname: Daniels
authors.forename: Shao-En
authors.initials: SE
authors.lastname: Ong
authors.forename: Anthony K L
authors.initials: AK
authors.lastname: Leung
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002853:Chromatography, Liquid; D003435:Crotalid Venoms / Q000201:enzymology*; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D015394:Molecular Structure; D043264:Phosphodiesterase I / Q000302:isolation & purification / Q000378:metabolism*; D011064:Poly Adenosine Diphosphate Ribose / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D003435:Crotalid Venoms; D017931:DNA Primers; D000246:Adenosine Diphosphate Ribose; D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; D043264:Phosphodiesterase I