Use of a High-Affinity Ubiquitin-Binding Domain to Detect and Purify Ubiquitinated Substrates and Their Interacting Proteins.

Abstract

OtUBD is a high-affinity ubiquitin-binding domain (UBD) derived from a large protein produced by the microorganism Orientia tsutsugamushi. The following protocol describes a step-by-step process for the enrichment of ubiquitinated proteins from baker's yeast and mammalian cell lysates using OtUBD. The OtUBD affinity resin can strongly enrich both mono- and poly-ubiquitinated proteins from crude lysates. The protocol further describes the use of different buffer formulations to specifically enrich for proteins covalently modified by ubiquitin with or without proteins that associate with them. Combining different OtUBD-mediated enrichment protocols with liquid chromatography-tandem mass spectrometry (LC-MS/MS) helps distinguish the pool of covalently ubiquitinated proteins (the ubiquitinome) from ubiquitin- or ubiquitinated protein-interacting proteins (the ubiquitin interactome). The OtUBD tool described in the protocol has been used successfully with downstream applications such as immunoblotting and differential proteomics. It provides researchers with a versatile and economical tool for the study of ubiquitin biology. Key Features • The protocol offers a native workflow and a denaturing workflow for enrichment of ubiquitinated proteins with or without noncovalently associated proteins, respectively. • Included in the protocol are different resin compositions, lysate preparation methods, elution methods, and pulldown formats to suit different experimental needs. • The protocol has been used in various applications, including immunoblotting, proteomics, and UbiCREST (ubiquitin chain restriction), and works with all types of ubiquitin conjugates. • The protocol was developed and tested with budding yeast and mammalian cell lysates but can be adapted to other biological samples and organisms.