Dataset Information

Data-independent acquisition approach for comprehensive ubiquitinome profiling in targeted protein degradation

ABSTRACT: Targeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitin-proteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the landscape of cellular ubiquitylation events is essential for the development of selective and efficient targeted protein degradation approaches. In recent years, data-independent acquisition (DIA) approach has gained significant popularity as a robust and unbiased approach for quantitative proteomics. By leveraging DIA, we have developed a cutting-edge workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. Through the implementation of our workflow, we have identified over 40,000 diGly precursors corresponding to more than 7,000 proteins in a single measurement from cells exposed to a proteasome inhibitor, highlighting an exceptional throughput. By applying our optimized workflow, we successfully identify ubiquitylation sites on substrate proteins in various TPD approaches, thereby helping establish mode of action for TPD. Our workflow therefore holds tremendous potential for rapidly establishing mode of action for various TPD modalities, such as for PROTACs and molecular glues.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Gajanan Sathe

LAB HEAD: Dr Prof Gopal Sapkota

PROVIDER: PXD060403 | Pride | 2026-06-09

REPOSITORIES: pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 35

Similar Datasets

Project description:Huntington’s disease is caused by a polyglutamine repeat expansion at the N-terminus of the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804 and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804 and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction while K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared to wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold-changes in the soluble fraction revealed that diGly site abundances of the majority of the proteins were not related to protein fold-changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold-change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington’s disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin as well as ubiquitin-regulated processes in Huntington’s disease.

			Action	DRS
	DMSO_GG_R1.raw	Raw
	DMSO_GG_R2.raw	Raw
	DMSO_GG_R3.raw	Raw
	DMSO_TP_R1.raw	Raw
	DMSO_TP_R2.raw	Raw

Dataset Information

Data-independent acquisition approach for comprehensive ubiquitinome profiling in targeted protein degradation

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets