Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains in Cellulo

Abstract

Ubiquitin (Ub) has a broad functional range that has been ascribed to the formation of an array of polymeric ubiquitin chains. Understanding the precise roles of ubiquitin chains, however, is difficult due to their complex chain topologies. Branched ubiquitin chains are particularly challenging, as multiple modifications on a single ubiquitin preclude the use of standard bottom-up proteomic approaches. Developing methods to overcome these challenges is crucial considering evidence suggesting branched chains regulate the stability of proteins. In this study, we employ Ubiquitin Chain Enrichment Middle-down Mass Spectrometry (UbiChEM-MS) to identify branched chains that cannot be detected using bottom-up proteomic methods. Specifically, we employ tandem ubiquitin binding entities (TUBEs) and the K29-selective Npl4 Zinc Finger 1 (NZF1) domain from the deubiquitinase TRABID to enrich for chains from human cells. Minimal trypsinolysis followed by high resolution mass spectrometric analysis reveals that Ub chain branching can indeed be detected using both Ub binding domains (UBDs) tested at endogenous levels. We find that ∼1% of chains isolated with TUBEs contain Ub branch points, with this value rising to ∼4% after proteasome inhibition. Electron-transfer dissociation (ETD) analysis indicates the presence of K48 in these branched chains. The use of the NZF1 domain reveals that ∼4% of the isolated chains contain branch points with no apparent dependence on proteasome inhibition. Our results demonstrate an effective strategy for detecting and characterizing the dynamics of branched conjugates under different cellular conditions.

Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains

“…31 Building on this work, we sought to exploit the utility of Ub chain linkage specific antibodies, specifically the Lys11 linkage specific antibody ( α -Lys11-IgG). Data has shown α -Lys11-IgG exhibits a high affinity ( K d ~ 12 nM) for Lys11-linked chains.…”

“…The resin was pelleted at 1000 g for 5 min and then washed with 25 C.V. of a minimal buffer (25 mM Tris, 50 mM NaCl, pH 8) containing a lower concentration of salt than previously used. 31 The resin was then resuspended in minimal buffer to bring the final volume to 800 µL. 6× Laemmli loading buffer was added to aliquots of each step, and the enriched ubiquitinated proteins were separated on a 15% SDS-PAGE gel, and analyzed by Western blot with anti-Ub antibody (Figure S3).…”

“…Quantitative analysis of the relative abundance of the three Ub 1–74 species was performed as described previously. 30,31 The percent distribution was calculated from three biological replicates, each with three technical replicates; and data is represented as the mean ± standard error of the mean (SEM).…”

“…To overcome these issues, we recently described a method called UbiChEM-MS (ubiquitin chain enrichment middle-down mass spectrometry) that enables characterization of Ub chain architecture in enriched populations from cell extracts. 30,31 With this approach, Ub chains composed of a specific linkage are isolated using an immobilized Ub-binding domain. Trypsin-mediated minimal digestion is then performed to remove the C-terminal diGly motif of each Ub and afford a mixture of Ub species representative of mono-Ub or the end-caps of a chain (Ub 1–74 ), singly modified Ub or the linear portion of a chain ( GG Ub 1–74 ), and the doubly modified form or branch points ( 2×GG Ub 1–74 ).…”

Technical Report: Targeted Proteomic Analysis Reveals Enrichment of Atypical Ubiquitin Chains in Contractile Murine Tissues

“…This, additionally, highlights a role for K29- and/or K33-linked polyubiquitylation in non-degradative functions. Further analysis of the ubiquitylome and ubiquitin chain-linkage landscape, in combination with recently described methods such as Ub-clipping [48] and Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) [56], could reveal detailed information on atypical ubiquitylation and higher-order chain-linkage architecture in different tissues and pathological states.…”