Ubiquitination is a process that involves the covalent attachment of the 
76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine 
(K) residues on substrate proteins. This post-translational modification elicits 
a wide range of functional consequences including targeting proteins for 
proteasomal degradation, altering subcellular trafficking events, and 
facilitating protein-protein interactions. A number of methods exist for 
identifying the sites of ubiquitination on proteins of interest, including 
site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). 
Recent publications have also highlighted the use of peptide-level 
immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for 
global characterization of ubiquitination sites. Here we investigated the 
utility of this technique for focused mapping of ubiquitination sites on 
individual proteins. For a series of membrane-associated and cytoplasmic 
substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α 
(TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently 
yielded additional ubiquitination sites beyond those identified in protein level 
AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were 
prepared and used to compare the abundances of individual K-GG peptides from 
samples prepared in parallel. Consistently, K-GG peptide immunoaffinity 
enrichment yielded greater than fourfold higher levels of modified peptides than 
AP-MS approaches. Using this approach, we went on to characterize inducible 
ubiquitination on multiple members of the T-cell receptor complex that are 
functionally affected by endoplasmic reticulum (ER) stress. Together, these data 
demonstrate the utility of immunoaffinity peptide enrichment for single protein 
ubiquitination site analysis and provide insights into the ubiquitination of 
HER2, DVL2, and proteins in the T-cell receptor complex.