Updated publication reference for PubMed record(s): 30110646. ADP-ribosylation has thus far been studied on the proteomic level mainly in cell lines and in combination with genotoxic stress. The clostridium-like ADP-ribosyltransferases (i.e. ARTC) are GPI-anchored or secreted proteins that are expressed in a highly tissue specific manner. Transcriptomic data revealed that ARTC1 is expressed in skeletal muscle and heart tissue. Although ARTC1 is highly active in vitro, identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level has been challenging and only a few ADP-ribosylated targets are known. Using a new MS-based workflow, we provide the thus far most extensive identification of endogenous ADP-ribosylomes with hundreds of ARTC1-ADP-ribosylated proteins in C2C12 myotubes and in skeletal muscle and heart tissues from wild type and newly generated ARTC1 knockout mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport and muscle function. Together we provide the first comprehensive systems-level analysis of endogenous ADP-ribosylation in two different muscle tissues revealing a widespread function of ARTC1 and its targets.