Posttranslational modifications of proteins increase the complexity of the cellular proteome andenable rapid regulation of protein functions in response to environmental changes. Proteinubiquitylation is a central regulatory posttranslational modification that controls numerousbiological processes including proteasomal degradation of proteins, DNA damage repair and innateimmune responses. Here we combine high-resolution mass spectrometry with single-stepimmunoenrichment of di-glycine modified peptides for mapping of endogenous putativeubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites onproteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates coresignaling pathways common for each of the studied tissues. In addition, we discover thatubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation siteswere obtained from brain highlighting the complexity and unique physiology of this organ. Wefurther demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequencepreferences, and that their complementary use increases the depth of ubiquitylation site analysis,thereby providing a more unbiased view of protein ubiquitylation.