Abstract
Protein ubiquitylation is a highly conserved, central mechanism to regulate cellular events in all eukaryotes, such as proteasomal degradation, protein trafficking, DNA repair, synaptic plasticity, and immune response. The consequence of protein ubiquitylation is modulated by the structure of ubiquitin (Ub) moiety attached on the substrates, including ubiquitin monomer and diverse polyubiquitin chains with different linkages (N-terminus, K6, K11, K27, K29, K33, K48, and K63). The development of ubiquitin-enrichment strategies coupled with sensitive mass spectrometry enables direct analysis of ubiquitylated proteins in cells, providing an invaluable tool for ubiquitin research. In this chapter, we describe recent technology updates for analyzing tissue-specific ubiquitin conjugates in transgenic models, as well as targeted proteomics methods for quantifying different polyubiquitin chain linkages in any type of -samples, including human tissues.