The timsTOF Ultra 2 enables systematic understanding of ubiquitination and degradation kinetics of degrader drug targets using slice-PASEF acquisition schemes

Importance of the Topic

Targeted protein degradation via small‐molecule degraders such as PROTACs and molecular glues is transforming therapeutic strategies by harnessing the ubiquitin–proteasome system to eliminate disease-causing proteins. A critical challenge remains the unbiased identification of both on-target and off-target ubiquitination events and degradation kinetics at early time points. Integrating deep proteomics with global ubiquitinomics delivers a comprehensive view of compound-induced protein fate, providing insights essential for degrader design, specificity assessment, and safety profiling.

Objectives and Study Overview

This study employed the timsTOF Ultra 2 platform with advanced slice-PASEF acquisition to map ubiquitination and degradation kinetics of PROTAC and molecular glue treatments in HEK293 cells and primary human PBMCs. Using a time-course design (1–60 min), the authors aimed to:

• Track the immediate ubiquitination of target proteins
• Discriminate primary targets from secondary/off-target events
• Demonstrate applicability to low-input, high-throughput formats

Methodology and Instrumentation

HEK293 cells and human PBMCs were treated in quadruplicates with the VHL-recruiting PROTAC ACBI2 or molecular glue Pomalidomide. After defined incubation intervals, cells were lysed, trypsin-digested via an automated platform, and desalted. Proteome profiling used data-independent DIA-PASEF on a timsTOF HT system, while ubiquitin remnant (K-GG) peptides were enriched and analyzed on the timsTOF Ultra 2 with an optimized slice-PASEF scheme. Raw data were processed with DIA-NN v2.1 enterprise and an in-house analysis pipeline.

Main Results and Discussion

• Slice-PASEF markedly outperformed conventional dia-PASEF, identifying more K-GG peptides with lower coefficients of variation.
• Primary degradation targets (SMARCA2/4, PBRM1) showed rapid ubiquitination within 1 min, whereas off-targets (RAD51, FBXO22, SS18L2) displayed delayed kinetics and were validated by proteome downregulation at 6 h.
• In human PBMCs, both ACBI1 and Pomalidomide induced time-dependent ubiquitination of known targets (IKZF1, IKZF3, RAB28, ZFP91), confirming platform applicability to clinically relevant samples.

Benefits and Practical Applications

The high sensitivity and quantification precision of slice-PASEF enable detection of low-abundance ubiquitination events in small sample inputs (48-well/96-well formats), supporting high-throughput degrader screening. Integration with an automated sample preparation pipeline streamlines discovery, validation, and optimization of targeted protein degraders for industrial and clinical research.

Future Trends and Potential Applications

Advances in trapped ion mobility and acquisition schemes will further enhance multiplexed ubiquitin profiling. Integration with single-cell proteomics and real-time data analysis has the potential to accelerate degrader development, personalized medicine, and in-depth mechanistic studies of proteostasis.

Conclusion

The timsTOF Ultra 2 combined with slice-PASEF acquisition provides a powerful workflow for simultaneous proteome and ubiquitinome time-course analysis, enabling precise characterization of degrader drug targets and off-target effects even in low-input formats. This platform accelerates the rational design and safety assessment of targeted protein degraders.

References

1. Steger M, Ohmayer U, Schwalb B, et al. Systematic understanding of ubiquitination and degradation kinetics of degrader drug targets using slice-PASEF acquisition schemes. bioRxiv. 2024;doi:10.1101/2024.10.18.618633
2. Szyrwiel P, et al. Optimized slice-PASEF for precise ubiquitinomics profiling. bioRxiv. 2022;doi:10.1101/2022.10.31.514544