Ion Mobility-Assisted Free Radical-Initiated Peptide Sequencing

Area of Science:

• Analytical Chemistry
• Biochemistry
• Mass Spectrometry

Background:

• Free radical-initiated peptide sequencing (FRIPS) is a tandem mass spectrometry (MS/MS) technique for peptide backbone fragmentation.
• Traditional FRIPS often requires specialized instruments or complex MS3 workflows, leading to intricate spectra.
• Existing methods can struggle with sequencing post-translationally modified peptides.

Purpose of the Study:

• To investigate the initiation of FRIPS using collisional-induced dissociation within a trapped-ion mobility spectrometry (TIMS) device.
• To determine if ion mobility separation can enhance the analysis of FRIPS-generated product ions.
• To assess the impact on peptide sequence coverage and spectral complexity.

Main Methods:

• Peptide ions were activated within a TIMS device to induce homolytic cleavage of FRIPS precursors.
• Collisional-induced dissociation (CID) was applied within the TIMS device.
• Resultant product ions were separated based on their ion mobility.
• Mass spectrometry was used to analyze the separated product ions.

Main Results:

• Activation within the TIMS device successfully initiated FRIPS, promoting radical generation and peptide backbone fragmentation.
• Ion mobility separation of the radical-driven product ions facilitated their assignment.
• The combined approach led to increased peptide sequence coverage compared to standard MS/MS methods.
• Spectral complexity was reduced due to the mobility separation of fragment ions.

Conclusions:

• Collisional-induced dissociation within TIMS is an effective method for initiating FRIPS.
• Ion mobility separation significantly aids in the analysis of FRIPS-generated fragments.
• This integrated technique offers a promising strategy for improved peptide sequencing, particularly for complex samples.