Methods for the Efficient Identification of Ubiquitination Sites by Proteomics

Ubiquitination is a critical step in various cellular processes, including cell division, signal transduction, neurotransmission, and development, yet identifying ubiquitinated proteins and targets of specific ubiquitin ligases has not progressed because of the technical difficulties in recovering these proteins for complex cellular mixtures. Researchers in Dr. Samie Jaffrey's lab have developed an antibody reagent that can pluck out only peptides that had been ubiquitinated from trypsinized cellular mixtures, enabling identification of ubiquitinated proteins as well as the sites of ubiquitination. This is especially useful for a proteomic analysis using mass spectrometry. This antibody reagent (developed in both polyclonal and monoclonal formats) recognizes the trypsinized remnant of ubiquitination – a diglycine modified epsilon amine of lysine (see figure below). The antibody affinity does not depend on neighboring backbone amino acid sequences.

These scientists have shown this reagent enables high-throughput MS identification of ubiquitination sites, even though peptides with ubiquitination sites have very low abundance. An affinity separation with this antibody isolates only those peptides derived from the ubiquitinated portion of proteins, eliminating peptides without ubiquitination sites, as well as peptides from proteins which strongly interact with ubiquitin or ubiquitinated proteins, significantly reducing the complexity of the peptide mixture. This purified sample can be directly applied to tandem MS for efficient peptide sequence analysis and protein identification, easily revealing ubiquitinated proteins and ubiquitination sites. With this method the Jaffrey group has identified over 400 ubiquitinated human proteins, many of which were previously unknown.

In summary, this invention provides a simple immunopurification method for peptides derived from ubiquitinated proteins enabling the identification of the protein and its ubiquitination site by tandem MS. Its potential applications include substrate identification for specific ubiquitin ligases, analyzing drug efficacy for ubiquitination-related diseases, and early diagnostics of ubiquitination-related diseases.

Strategy for identifying ubiquitinated proteins by immunopurification of peptides with diglycine-modified lysines and tandem MS analysis.