Middle-Down Mass Spectrometry Enables Characterization of Branched Ubiquitin Chains

ABSTRACT: Protein ubiquitylation, one of the most prevalent post-translational modifications in eukaryotes, is involved in regulating nearly every cellular signaling pathway. The vast functional range of ubiquitylation has largely been attributed to the formation of a diverse array of polymeric ubiquitin (polyUb) chains. Methods that enable the characterization of these diverse chains are necessary to fully understand how differences in structure relate to function. Here, we describe a method for the detection of enzymatically derived branched polyUb conjugates in which a single Ub subunit is modified by two Ub molecules at distinct lysine residues. Using a middle-down mass spectrometry approach in which restricted trypsin-mediated digestion is coupled with mass spectrometric analysis, we characterize the polyUb chains produced by bacterial effector E3 ligases NleL (non-Lee-encoded effector ligase from enterohemorrhagic Escherichia coli O157:H7) and IpaH9.8 (from Shigella f lexneri). Because Ub is largely intact after minimal trypsinolysis, multiple modifications on a single Ub moiety can be detected. Analysis of NleL- an