Ubiquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LC)-MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.

Intracellular signaling via the covalent attachment of different ubiquitin linkages to protein substrates is fundamental to many cellular processes. Although linkage-selective ubiquitin interactors have been studied on a case-by-case basis, proteome-wide analyses have not been conducted yet. Here, we present ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), a quantitative interaction proteomics method that makes use of chemically synthesized diubiquitin to enrich and identify ubiquitin linkage interactors from crude cell lysates. UbIA-MS reveals linkage-selective diubiquitin interactions in multiple cell types. For example, we identify TAB2 and TAB3 as novel K6 diubiquitin interactors and characterize UCHL3 as a K27-linkage selective interactor that regulates K27 polyubiquitin chain formation in cells. Additionally, we show a class of monoubiquitin and K6 diubiquitin interactors whose binding is induced by DNA damage. We expect that our proteome-wide diubiquitin interaction landscape and established workflows will have broad applications in the ongoing efforts to decipher the complex language of ubiquitin signaling.

Deubiquitinating enzymes (DUBs) are proteases that fulfill crucial roles in the ubiquitin (Ub) system, by deconjugation of Ub from its targets and disassembly of polyUb chains. The specificity of a DUB towards one of the polyUb chain linkages largely determines the ultimate signaling function. We present a novel set of diubiquitin FRET probes, comprising all seven isopeptide linkages, for the absolute quantification of chain cleavage specificity of DUBs by means of Michaelis–Menten kinetics. Each probe is equipped with a FRET pair consisting of Rhodamine110 and tetramethylrhodamine to allow the fully synthetic preparation of the probes by SPPS and NCL. Our synthetic strategy includes the introduction of N,N′‐Boc‐protected 5‐carboxyrhodamine as a convenient building block in peptide chemistry. We demonstrate the value of our probes by quantifying the linkage specificities of a panel of nine DUBs in a high‐throughput manner.

The post-translational modification ubiquitin can be attached to the ɛ-amino group of lysine residues or to a protein's N-terminus as a mono ubiquitin moiety. Via its seven intrinsic lysine residues and its N-terminus, it can also form ubiquitin chains on substrates in many possible ways. To study ubiquitin signals, many synthetic and semi-synthetic routes have been developed for generation of ubiquitin-derived tools and conjugates. The strength of these methods lies in their ability to introduce chemo-selective ligation handles at sites that currently cannot be enzymatically modified. Here, we review the different synthetic and semi-synthetic methods available for ubiquitin conjugate synthesis and their contribution to how they have helped investigating conformational diversity of diubiquitin signals. Next, we discuss how these methods help understanding the ubiquitin conjugation-deconjugation system by recent advances in ubiquitin ligase probes and diubiquitin-based DUB probes. Lastly, we discuss how these methods help studying post-translational modification of ubiquitin itself.