title: 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.
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.|Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
authors.forename: Sean O
authors.initials: SO
authors.lastname: Crowe
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.|Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
authors.forename: Ambar S J B
authors.initials: ASJB
authors.lastname: Rana
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: Kirandeep K
authors.initials: KK
authors.lastname: Deol
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.|Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.|Human Proteomics Program, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Ying
authors.identifier: 0000-0001-5211-6812
authors.initials: Y
authors.lastname: Ge
authors.affiliation: Department of Chemistry, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.|Department of Biochemistry and Molecular Biology, University of Massachusetts-Amherst , Amherst, Massachusetts 01003, United States.
authors.forename: Eric R
authors.identifier: 0000-0003-3447-3669
authors.initials: ER
authors.lastname: Strieter
mesh_terms: D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination; D016335:Zinc Fingers
chemical_list: D025801:Ubiquitin; D010450:Endopeptidases; C570449:ZRANB1 protein, human