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Ubiquitinome Profiling: Optimizing the enrichment and Data-Independent Analysis for Deeper Insights into Targeted Protein Degradation

ABSTRACT: Targeted protein degradation 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 ubiquitination events is essential for the development of effective targeted protein degradation approaches. In recent years, data-independent analysis (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. By leveraging on this method, we have developed a cutting-edge workflow that combines diGly antibody-based enrichment with an optimized Orbitrap-based data-independent acquisition method for the identification of ubiquitylated peptides.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney Epithelial Cell

SUBMITTER: Gajanan Sathe

LAB HEAD: Prof Gopal Sapkota

PROVIDER: PXD077594 | Pride | 2026-06-09

REPOSITORIES: Pride

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			Action	DRS
	206_2024_GS-5min-R1-E.raw	Raw
	206_2024_GS-5min-R2-E.raw	Raw
	206_2024_GS-5min-R3-E.raw	Raw
	227_2024_GS-DMSO-R1-E.raw	Raw
	227_2024_GS-DMSO-R2-E.raw	Raw

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Ubiquitinome Profiling: Optimizing the enrichment and Data-Independent Analysis for Deeper Insights into Targeted Protein Degradation

Project description:Targeted protein degradation 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 ubiquitination events is essential for the development of effective targeted protein degradation approaches. In recent years, data-independent analysis (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. By leveraging on this method, we have developed a cutting-edge workflow that combines diGly antibody-based enrichment with an optimized Orbitrap-based data-independent acquisition method for the identification of ubiquitylated peptides.

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Project description:Small molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and the potential to tackle previously undrugged targets. Thermodynamically stable and kinetically long-lived degrader-mediated ternary complexes can drive faster, more profound and durable target degradation, however the mechanistic features by which they impact on target ubiquitination remain elusive. Here, we solve cryo-EM structures of the VHL Cullin 2 RING E3 ligase complexed with degrader MZ1, target protein Brd4BD2 and primed for catalysis with its cognate E2-ubiquitin bound. We find that Brd4BD2 adopts a favourable orientation towards the E2 active site. Mass spectrometry illuminates a patch of favourably ubiquitinable lysines on one face of in vitro ubiquitinated Brd4BD2, with Lys456 showing optimal distance and geometry for nucleophilic attack highlighted by cryo-EM. In vitro ubiquitination coupled with cellular degradation and ubiquitinomics confirm the importance of Lys456 and the ‘ubiquitination zone’ lysines. Our results demonstrate the proficiency of MZ1 in directing the substrate towards catalysis, explains the favourability of Brd4BD2 for ubiquitination bu UBE2R1, and reveals the flexibility of the enzyme in capturing sub-optimal lysines. We propose a model for ubiquitinability of degrader-recruited targets that provides a mechanistic blueprint for further rational drug design and optimization.

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