Improved Protein and PTM Characterization with a Practical Electron-Based Fragmentation on Q-TOF Instruments

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Electron-based dissociation (ExD) offers advantages for intact protein analysis, including uncluttered spectra and preservation of post-translational modifications.
• Current ExD technology is limited to a few high-end mass spectrometers, posing accessibility challenges.
• Technical hurdles have restricted the widespread adoption of ExD for comprehensive protein characterization.

Purpose of the Study:

• To develop an efficient and retrofittable ExD cell for broader accessibility in mass spectrometry.
• To create supporting software for acquiring, processing, and annotating ExD fragmentation data.
• To enhance the capabilities of ExD for analyzing intact proteins and their modifications.

Main Methods:

• Development and integration of a novel ExD cell into existing LC/Q-TOF instruments.
• Creation of specialized software for ExD data handling and analysis.
• Utilizing combined collisional activation and electron fragmentation for enhanced peptide and protein analysis.

Main Results:

• The developed ExD cell is easily retrofitted into current LC/Q-TOF instruments.
• ExD spectra provide complementary fragmentation, distinguishing isobaric amino acid pairs and preserving labile modifications like phosphorylation and glycosylation.
• The method enables efficient fragmentation of longer peptides, revealing PTM cross-talk and cleaving disulfide bonds in complex proteins.

Conclusions:

• The retrofittable ExD cell significantly expands the accessibility of advanced protein analysis techniques.
• Enhanced fragmentation capabilities improve sequence coverage and analytical certainty for post-translational modification characterization.
• This technology facilitates deeper insights into complex proteoforms and signaling pathways.