PXD004059

PXD004059 is an original dataset announced via ProteomeXchange.

Title	Mass spectrometry insights into a tandem ubiquitin‐binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin
Description	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 ‘ubiquitome’.
HostingRepository	PRIDE
AnnounceDate	2016-05-18
AnnouncementXML	Submission_2016-05-18_01:50:59.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD004059
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Andrew Bottrill
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	Ammonia-loss; LeuArgGlyGly; Dehydrated; Oxidation; Acetyl; Carbamidomethyl; GlyGly
Instrument	LTQ Orbitrap Velos

Revision	Datetime	Status	ChangeLog Entry
0	2016-04-26 08:23:08	ID requested
⏵ 1	2016-05-18 01:51:00	announced

Scott D, Garner TP, Long J, Strachan J, Mistry SC, Bottrill AR, Tooth DJ, Searle MS, Oldham NJ, Layfield R, Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin. Proteomics, 16(14):1961-9(2016) [pubmed]

curator keyword: Biological

submitter keyword: Ubiquitin, polyubiquitin

Rob Layfield
contact affiliation	School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK
contact email	Robert.Layfield@nottingham.ac.uk
lab head
Andrew Bottrill
contact affiliation	University of Leicester
contact email	arb29@le.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD004059
     1. Label: PRIDE project
     2. Name: Mass spectrometry insights into a tandem ubiquitin‐binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin