Peptide collision cross sections of 22 post-translational modifications

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Ion mobility is increasingly used as an additional separation dimension in mass spectrometry (MS)-based proteomics.
• It separates ions based on size and shape in the gas phase.
• Understanding how post-translational modifications (PTMs) affect ion mobility is crucial for advancing proteomic analysis.

Purpose of the Study:

• To investigate the impact of 22 different PTMs on the collision cross section (CCS) of peptides.
• To quantify the changes in CCS for modified versus unmodified peptides.
• To explore the relationship between PTMs, peptide sequence, and gas-phase structure.

Main Methods:

• Analysis of approximately 4300 pairs of modified and unmodified peptide ion species using trapped ion mobility spectrometry (TIMS).
• Utilized spike-in reference peptides for linear alignment to ensure reproducible CCS measurements (median coefficient of variation of 0.26%).
• Compared CCS values between modified and unmodified peptides across different charge states.

Main Results:

• Observed redistribution in m/z vs. ion mobility space for modified peptides, influenced by charge state changes.
• Identified median CCS shifts ranging from -1.4% (arginine citrullination) to +4.5% (O-GlcNAcylation) between modified and unmodified peptides of the same charge state.
• Found that while increased peptide mass generally correlated with higher CCS, the actual CCS change for a given PTM was PTM- and sequence-specific, partly depending on the unmodified peptide's gas-phase structure.

Conclusions:

• PTMs exert significant and specific effects on peptide collision cross sections.
• These PTM- and sequence-specific CCS alterations can be exploited for improved proteome-wide PTM detection and characterization.
• The findings provide a foundation for leveraging ion mobility data to better understand the functional implications of PTMs in biological systems.