The HARPER Lab

Department of Cell Biology

Quantitative and systematic analysis of the ubiquitin modified proteome

Work flow for ubiquitin capture proteomics

Together with the Gygi lab, we have used proteomic approaches to identify ~19,000 ubiquitination sites in ~5000 human proteins in multiple cell lines. The approach involves enrichment of “diGly renmants” using a monoclonal antibody that reacts with lysine residues carrying this modification, followed by mass spectrometry analysis. Using this approach, we have examined the temporal control of accumulation of proteins in response to proteasome inhibition, allowing us to classify various types of substrates. We have also examined the role of protein synthesis in defining the ubiquitin modified proteome, and have found that identification of a significant fraction of sites requires ongoing protein synthesis. Finally, we have used this approach to identify candidate substrates of cullin-RING ubiquitin ligases. These studies provide a resource for the identification and analysis of ubiquitinated proteins.

The database can be searched online at: https://gygi.med.harvard.edu/ggbase/