High sensitivity lysine acetylation profiling with Trapped Ion Mobility Spectrometry and PASEF

Significance of the Topic

Lysine acetylation is a critical post-translational modification that regulates transcription, cell cycle progression, apoptosis, and other cellular processes. However, its low abundance and wide dynamic range demand highly sensitive and selective analytical techniques to achieve comprehensive profiling.

Objectives and Study Overview

The study aimed to develop and demonstrate a high-sensitivity workflow for global lysine acetylation profiling in complex biological samples. By integrating a high-affinity enrichment strategy with trapped ion mobility spectrometry (TIMS) and Parallel Accumulation–Serial Fragmentation (PASEF) on the timsTOF Pro platform, the authors assessed performance using rice leaf and mouse liver tissues.

Methodology and Instrumental Setup

• Sample Preparation
  
  • Enrichment of acetylated peptides using a lysine acetylation kit (PTM-104).
  • Trypsin digestion of extracted proteins from rice leaves and mouse liver.
• Chromatography and Mass Spectrometry
  
  • nanoElute UHPLC with a home-packed 25 cm × 100 µm I.D. C18 column at 450 nL/min and 50 °C.
  • Linear gradient from 5 % to 22 % acetonitrile over 50 min.
  • Bruker timsTOF Pro equipped with TIMS and PASEF for enhanced sensitivity and sequencing speed.
• Data Processing
  
  • MaxQuant and Mascot searches against SwissProt (mouse) and Oryza_sativa databases.
  • False discovery rate controlled below 1 % for peptide spectrum matches and protein groups.

Key Results and Discussion

• Depth of Coverage
  
  • Rice leaves: Identification of nearly 18 000 acetylation sites across more than 6 100 protein groups from nine samples.
  • Mouse liver: Detection of ~1 800 acetylated peptides from only 100 µg of starting material in a single 50 min run.
• Quantitative Reproducibility
  
  • High consistency in label-free quantification with Pearson correlation coefficients above 0.94.
• Benefit of TIMS Separation
  
  • Resolution of co-eluting peptides with identical m/z and retention time, enabling accurate acetylation site localization and non-chimeric MS/MS spectra.

Benefits and Practical Applications

This integrated TIMS-PASEF workflow offers:

• Superior sensitivity for low-abundance PTMs with reduced sample requirements.
• Rapid analysis with short LC gradients without sacrificing depth.
• Robust qualitative and quantitative data conducive to large-scale proteomics, QA/QC, and systems biology.

Future Trends and Opportunities

Ongoing advancements are expected in combining ion mobility with multi-omic platforms, real-time data analytics, and automated workflows. These developments will broaden applications in clinical diagnostics, drug discovery, and industrial quality control.

Conclusion

The presented TIMS-PASEF approach enables high-coverage, sensitive profiling of lysine acetylation in complex tissues. By leveraging targeted enrichment, ion mobility separation, and rapid fragmentation, the method delivers extensive site identification and accurate localization, supporting advanced proteomic research and its translational applications.

References

• Meier F. et al., Journal of Proteome Research, 2015.