PXD063877

PXD063877 is an original dataset announced via ProteomeXchange.

Title	Ubiquitination site analysis of eGFP by DARPin-based bioPROTACs analysed by LC-MS-MS
Description	We investigated design principles of DARPin-based bioPROTACs targeting the model substrate GS-eGFP (eGFP with an n-terminal tag, necessary for proteasomal unfolding). Using a highly active E3 ligase domain from CHIP and nine well-characterized DARPins, we evaluated their ability to induce proteasomal degradation. Seven of nine DARPin bioPROTACs successfully mediated catalytic degradation of GS-eGFP, with the binding epitope emerging as a key determinant of efficiency. LC-MS/MS analysis revealed that efficient degradation required ubiquitination at four specific lysine residues on eGFP. Notably, DP6-bioPROTAC promoted polyubiquitination, whereas DP9-bioPROTAC did not, likely due to differences in binding geometry. DP7-bioPROTAC appeared to hinder E2 enzyme access, further confirming the importance of structural orientation. Additionally, we observed that DARPins lacking an E3 domain can trigger non-catalytic degradation, potentially via recruitment of endogenous E3 ligases. Ubiquitination patterns of selected GS-eGFP complexes (DP1, DP9, and a lysine-free DP6 mutant) were also characterized by LC-MS/MS.
HostingRepository	PRIDE
AnnounceDate	2025-06-05
AnnouncementXML	Submission_2025-06-05_07:31:01.953.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Dorothea Winkelvoss
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	ubiquitination signature dipeptidyl lysine
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2025-05-13 00:48:00	ID requested
⏵ 1	2025-06-05 07:31:02	announced

Dataset with its publication pending

submitter keyword: Ubiquitination, DARPin, bioPROTAC, eGFP

Andreas Plückthun
contact affiliation	Plückthun lab, department of biochemistry, university of Zürich, Switzerland
contact email	plueckthun@bioc.uzh.ch
lab head
Dorothea Winkelvoss
contact affiliation	University of Zürich
contact email	d.winkelvoss@bioc.uzh.ch
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD063877
     1. Label: PRIDE project
     2. Name: Ubiquitination site analysis of eGFP by DARPin-based bioPROTACs analysed by LC-MS-MS