Poly [ADP-ribose] polymerase 1
NAD+-protein-tyrosine ADP-ribosyltransferase activity
Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site.
Despite recent mass spectrometry (MS)-based breakthroughs, comprehensive ADP-ribose (ADPr)-acceptor amino acid identification and ADPr-site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP-ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1-dependent <i>in vivo</i> ADPr-acceptor amino acid. MS analyses of <i>in vitro</i> ADP-ribosylated proteins confirmed tyrosine as an ADPr-acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that <i>trans</i>-modification of RPS3A is dependent on HPF1. We provide an ADPr-site Localization Spectra Database (ADPr-LSD), which contains 288 high-quality ADPr-modified peptide spectra, to serve as ADPr spectral references for correct ADPr-site localizations.