Gas-phase peptide sequencing by TEMPO-mediated radical generation

Area of Science:

• Mass Spectrometry
• Chemical Biology
• Proteomics

Background:

• Electron capture dissociation (ECD) and electron transfer dissociation (ETD) are key methods for peptide sequencing.
• These methods rely on radical cations and are limited to doubly or highly protonated peptides.
• A need exists for versatile peptide sequencing techniques applicable to a broader range of peptide charges.

Purpose of the Study:

• To develop a novel method for peptide sequencing using collisional activation of radical cations.
• To investigate the applicability of this method to singly protonated peptides.
• To demonstrate the universality of the method across different tandem mass spectrometers.

Main Methods:

• Conjugation of a TEMPO-derived precursor (TEMPO-Bz) to peptide N-termini or lysine side chains.
• Preparation of peptide cations via attachment of the TEMPO precursor.
• Collisional activation of these peptide cations followed by tandem mass spectrometry.

Main Results:

• Collisional activation generated radical species from TEMPO-Bz-conjugated peptide cations.
• Tandem mass spectrometry revealed odd-electron peptide backbone dissociations (a-, x-, z-ions) similar to ECD/ETD.
• The disulfide bond was effectively cleaved.
• The method, termed TEMPO-FRIPS (free radical initiated peptide sequencing), successfully characterized singly protonated peptides.
• TEMPO-FRIPS demonstrated applicability to any type of tandem mass spectrometer.

Conclusions:

• TEMPO-FRIPS offers a new approach for peptide sequencing via radical cation dissociation.
• This method overcomes the charge limitations of ECD and ETD, enabling analysis of singly protonated peptides.
• The universality of TEMPO-FRIPS makes it a valuable tool for diverse mass spectrometry applications.