Mass spectrometry insights into a tandem ubiquitinâ??binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin

ABSTRACT: Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been described. We describe the design of a linker-optimised ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains. Utilising native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48-linked ubiquitin dimers. Furthermore MS/MS analyses indicate that the t-UBD, when applied as an affinity enrichment reagent, can be used to favour the purification of endogenous unanchored Lys48-linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain-selective binding in non-biological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the â??ubiquitomeâ??.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Dr Rob Layfield 

PROVIDER: MSV000079990 | MassIVE | Fri Jul 22 16:06:00 BST 2016

SECONDARY ACCESSION(S): PXD004059

REPOSITORIES: MassIVE