Fig. 1.

Strategies to isolate ubiquitylated substrates. A, protein-level enrichment of ubiquitylated proteins by purification of tagged ubiquitin (left), tandem ubiquitin binding domains (middle), or ubiquitin antibodies (right). B, schematic depicting peptide-level enrichment with diGLY remnant antibodies (diGPE). C, outline of SILAC-based proteomic approach for quantification of ubiquitylation sites using diGPE and subsequent analysis via MS. Text in the gray highlight technical improvements that have been shown to increase enrichment yield and specificity, including antibody usage (30) and fractionation of peptides either before or after immunoenrichment (26, 27, 29–32).