(489.18) Profiling the ubiquitinated proteome in human cells

Poster Board Number: A88

Stephanie Diaz (Purdue University), Xing Liu (Purdue University)

Ubiquitination is a post-translational modification that determines the half-life of many cellular proteins by conjugating a chain of the small protein ubiquitin to a target protein. The ubiquitin chain is then recognized by the 26S proteasome, and subsequently, the ubiquitinated protein is degraded. This ubiquitin-proteosome system (UPS) contains various components that function cooperatively to determine the ubiquitination status of cellular proteins that play key roles in biological events. It has become clear that mis-regulation of protein ubiquitination can lead to diverse human diseases, and multiple UPS components have been identified as promising drug targets. To facilitate studies that define roles of the UPS components in protein ubiquitination, a method that probes the ubiquitination status of the whole proteome in human cells is necessary. Tandem ubiquitin binding entities (TUBEs) is a synthetic protein that binds poly-ubiquitin chains with a dissociation constant in the nanomolar range. Therefore, TUBEs can recognize the poly-ubiquitinated protein and enrich them from human cell lysate. Combining the TUBE-dependent affinity precipitation, stable isotope labeling by amino acids in cell culture (SILAC), and mass spectrometry, we are establishing a method for quantitatively accessing the ubiquitinated proteome in human cells. To preserve the poly-ubiquitinated protein, HEK293 cells were treated with bortezomib, a proteosome inhibitor, to prevent the ubiquitinated proteins from degradation.  To perform the affinity precipitation, the recombinant TUBE was conjugated to biotin and immobilized on resin via the Streptavidin-biotin interaction. The lysate of the bortezomib-treated cells was incubated with the immobilized BiotinTUBE to enrich poly-ubiquitinated proteins, which were then digested by the trypsin protease. Following the trypsin digestion, ubiquitinated proteins generated peptides with the Lys-ɛ-Gly-Gly (K-ɛ-GG) remnant, which was further immunoprecipitated by antibodies specifically recognizing the K-ɛ-GG remnant and identified by mass spectrometry. To probe proteins with different ubiquitination status under different conditions, SILAC was used to grow one cell sample in normal (light) media and the other cell sample in (heavy) media containing stable isotope labeled amino acids. By comparing the abundance of the light versus heavy peptides from the same ubiquitinated protein, changes in the ubiquitination status of each detected protein can be determined. We anticipate that this method will help profile changes in the ubiquitinated proteome when the UPS components are mutated or interfered by drugs.

Support or Funding Information

This work was supported by National Institutes of Health R35 grant (to X.L.), American Heart Association Career Development Award (to X.L.), and the Bird Stair Fellowship (to S.D.).