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

The dynamics of cellular signaling events are tightly regulated by a diverse set of ubiquitin chains. Recent work has suggested that branched ubiquitin chains composed of Lys11 and Lys48 isopeptide linkages play a critical role in regulating cell cycle progression. Yet, endogenous Lys11/Lys48 branched chains could not be detected. By combining a Lys11 linkage specific antibody with high-resolution middle-down mass spectrometry (an approach termed UbiChEM-MS) we sought to identify endogenous Lys11/Lys48 branched ubiquitin chains in cells. Using asynchronous cells, we find that Lys11-linked branched chains can only be detected upon cotreatment with a proteasome and nonselective deubiquitinase inhibitor. Releasing cells from mitotic arrest results in a marked accumulation of Lys11/Lys48 branched chains in which branch points represent ∼3-4% of the total ubiquitin population. This report highlights the utility of UbiChEM-MS in characterizing the architecture of Lys11 Ub chains under various cellular conditions and corroborates the formation of Lys11/Lys48 branched chains during mitosis.