Large-scale collision cross-section profiling on a traveling wave ion mobility mass spectrometer

Area of Science:

• Proteomics
• Analytical Chemistry
• Biophysics

Background:

• Ion mobility (IM) separates ions by charge and collision cross-section (CCS).
• Traveling wave IM (TW IM) is used in mass spectrometry, often with high voltages.
• Polyalanine calibration for TW IM is typically validated at low voltages.

Purpose of the Study:

• To assess the accuracy of TW IM polyalanine calibration under high voltages used in HDMS(E).
• To evaluate potential errors introduced by liquid chromatography (LC)-HDMS(E) analysis.
• To demonstrate the utility of a large peptide CCS database built using this method.

Main Methods:

• Applied TW IM polyalanine calibration to shotgun proteomics.
• Characterized calibration accuracy and CCS measurement under high transmission voltages.
• Investigated peptide structural sub-families based on digestion and ion charge state.

Main Results:

• High transmission voltages did not significantly impact TW IM calibration accuracy.
• LC-HDMS(E) analysis introduced insignificant error, validating the calibration method.
• A large peptide CCS database was created, revealing structural differences based on lysine position.

Conclusions:

• The polyalanine calibration method is robust for TW IM in shotgun proteomics across a range of voltages.
• The validated method facilitates the construction of comprehensive peptide CCS databases.
• This approach enhances the capability of mass spectrometry for detailed proteomic analysis and structural characterization.