A dedicated ubiquitin-K48/63R variant-based affinity approach specifically identifies both high-risk and low-risk substrates of the HPV E6-E6AP ubiquitin ligase complex

To identify potential substrate proteins of both low risk and high risk E6 proteins, we established an affinity-based enrichment approach using a dedicated biotin-tagged ubiquitin variant that in the absence of the E6 proteins, cannot or only poorly be used by E6AP for ubiquitination. Enriched proteins were identified by high-resolution mass spectrometry (LC-MS/MS) and label-free quantification. E6-mediated ubiquitination/degradation of some of the proteins identified were validated in vitro and within cells by using recombinant proteins and in transient transfection experiments in cells demonstrating the potential of this approach.