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

TY - JOUR

T1 - Data-independent acquisition (DIA) approach for comprehensive ubiquitinome profiling in targeted protein degradation

AU - Sathe, Gajanan

AU - Roth, Sascha

AU - Gelders, Géraldine

AU - Brewer, Abigail

AU - MacArtney, Thomas J.

AU - Wood, Nicola

AU - De Muynck, Louis

AU - Nakasone, Mark

AU - Phung, Toan

AU - Buist, Arjan

AU - Moechars, Diederik W.E.

AU - Sapkota, Gopal

PY - 2026/9/15

Y1 - 2026/9/15

N2 - Targeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitinproteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the ubiquitylation landscape is essential for the development of selective and efficient targeted protein degradation approaches. Recently, data-independent acquisition (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. Here, we report a robust workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. We identify over 40,000 diGly peptides 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 with various TPD approaches. By also leveraging the cutting-edge Orbitrap Astral technology, we resolve rapid PROTACdriven ubiquitylation on target proteins, as early as 5 minutes post-treatment. Therefore, our workflow holds tremendous potential for rapidly establishing mode-of-action for various TPD modalities, including PROTACs and molecular glues.

AB - Targeted protein degradation (TPD) has emerged as a highly promising therapeutic strategy for a wide range of diseases, including cancer and neurodegenerative disorders. The ubiquitinproteasome system, which is responsible for protein degradation, plays a critical role in this process. Gaining comprehensive insights into the ubiquitylation landscape is essential for the development of selective and efficient targeted protein degradation approaches. Recently, data-independent acquisition (DIA) has gained significant popularity as a robust and unbiased approach for quantitative proteomics. Here, we report a robust workflow that utilizes diGly antibody-based enrichment followed by an optimized Orbitrap-based DIA method for the identification of ubiquitylated peptides. We identify over 40,000 diGly peptides 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 with various TPD approaches. By also leveraging the cutting-edge Orbitrap Astral technology, we resolve rapid PROTACdriven ubiquitylation on target proteins, as early as 5 minutes post-treatment. Therefore, our workflow holds tremendous potential for rapidly establishing mode-of-action for various TPD modalities, including PROTACs and molecular glues.

U2 - 10.1038/s41598-026-69423-4

DO - 10.1038/s41598-026-69423-4

M3 - Article

SN - 2045-2322

JO - Scientific Reports

JF - Scientific Reports

ER -