Trapped Ion Mobility Spectrometry and PASEF Enables In depth Characterization of Protein Ubiquitination

Significance of the Topic

Ubiquitination is a key post-translational modification regulating protein turnover, immune signaling, DNA repair and other critical cellular functions. Comprehensive mapping of ubiquitination sites enhances understanding of disease mechanisms and supports biomarker discovery and drug development.

Study Objectives and Overview

This study demonstrates an in-depth profiling workflow for global protein ubiquitination using high-affinity antibody enrichment combined with trapped ion mobility spectrometry (TIMS) and parallel accumulation–serial fragmentation (PASEF). Applying this approach to HeLa and 293T cell lysates, the researchers aim to maximize site coverage, improve quantification reproducibility and resolve isobaric modification isomers.

Methodology

Ubiquitinated peptides were isolated from 2 mg of cell lysate using a high-affinity enrichment kit. Enriched peptides underwent reversed-phase separation on a 25 cm C18 column with a 50 min gradient (2–37% acetonitrile, 0.1% formic acid) at 450 nL/min and 50 °C. Data were acquired on a timsTOF Pro mass spectrometer with PASEF and processed using MaxQuant against the human SwissProt database (FDR 1%, minimum modified peptide score 40).

Used Instrumentation

• nanoElute LC system (Bruker Daltonics)
• timsTOF Pro Q-TOF mass spectrometer equipped with trapped ion mobility and PASEF
• In-house packed 25 cm × 100 µm C18 column (1.9 µm particle size)

Key Results and Discussion

The optimized workflow achieved highly reproducible label-free quantification of ubiquitinated peptides (Pearson R = 0.97). From two biological cell lines (HeLa and 293T), over 8 700 and 10 000 unique ubiquitination sites were identified per replicate, respectively. In total, more than 17 000 ubiquitination sites across 5 093 proteins were mapped using a 50-min LC gradient. Importantly, the additional ion mobility dimension resolved co-eluting isobaric peptides, enabling site-specific identification of modification isomers.

Benefits and Practical Applications

• High sensitivity and depth of coverage for global ubiquitinome profiling with limited sample input.
• Exceptional reproducibility supports robust comparative analyses in cell biology and clinical research.
• Ion mobility adds an orthogonal separation dimension, improving confidence in site localization and resolving isobaric species.

Future Trends and Opportunities

Advances in TIMS-PASEF technology and enrichment chemistries will further increase throughput and sensitivity. Integration with DIA workflows, single-cell proteomics and multi-omics platforms may enable comprehensive characterization of ubiquitination dynamics in heterogeneous samples and disease models.

Conclusion

The combination of high-affinity enrichment, TIMS separation and PASEF acquisition on the timsTOF Pro enables deep, reproducible and site-specific profiling of protein ubiquitination. This streamlined workflow provides a powerful tool for PTM research and biomarker discovery.

Reference

1. He et al., Plant Journal, 2020, 101(6):1430–1447
2. Meier F et al., Molecular & Cellular Proteomics, 2018, 17(12):2534–2545