Trapped Ion Mobility Spectrometry Reduces Spectral Complexity in Mass Spectrometry-Based Proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biophysics

Background:

• Bottom-up mass spectrometry struggles with deep proteome coverage due to high cofragmentation rates.
• Cofragmentation, where multiple peptides fragment together, complicates data analysis and limits identification.
• Ion mobility (IM) spectrometry is a promising technique for peptide separation based on collisional cross section.

Purpose of the Study:

• To computationally investigate the peptide separation capabilities of trapped ion mobility spectrometry (TIMS).
• To quantify the separation power of TIMS within a parallel accumulation-serial fragmentation (PASEF) workflow.
• To assess the impact of TIMS separation on spectral complexity and peptide identification.

Main Methods:

• Utilized a computational model to simulate TIMS separation of peptide ions.
• Quantified the reduction in cofragmentation and spectral complexity.
• Analyzed the effect of TIMS separation on data-dependent and data-independent acquisition PASEF workflows.

Main Results:

• TIMS separation increased interference-free MS1 peptide features 9.2-fold.
• Average peptide density in precursor spectra decreased 6.5-fold.
• In DDA-PASEF, IM separation increased spectra without cofragmentation 4.1-fold and high-quality spectra 17-fold.
• IM separation in DIA-PASEF reduced spectral complexity equivalent to a 4-fold decrease in isolation window width.

Conclusions:

• TIMS separation effectively reduces spectral complexity by minimizing cofragmentation.
• This reduction in complexity enhances the likelihood of peptide spectral matches and improves peptide identification rates.
• TIMS separation is a key factor contributing to the high identification rates observed in PASEF workflows.