Resolving double disulfide bond patterns in SNAP25B using liquid chromatography-ion trap mass spectrometry.

Area of Science:

• Biochemistry
• Proteomics
• Mass Spectrometry

Background:

• Complex disulfide bond patterns in SNAP25B are implicated in regulating neurotransmitter release.
• The steric feasibility and identification of these patterns, especially under oxidative stress, remain unassessed.

Purpose of the Study:

• To assess the steric feasibility of complex disulfide patterning in SNAP25B.
• To evaluate the feasibility of identifying complex disulfide bond patterns using mass spectrometry (MS).
• To develop a novel probabilistic analysis for resolving complex double disulfide bond patterns.

Main Methods:

• Developed a novel probabilistic analysis integrating cumulative hypergeometric distribution with top-down scoring.
• Analyzed fragmentation patterns of singly and doubly disulfide-linked SNAP25B peptides using an ion trap mass spectrometer.
• Modeled peptide fragmentation events, including backbone and disulfide bond cleavage.

Main Results:

• Successfully identified two complex disulfide bonding patterns in SNAP25B directly via unique MS/MS peaks.
• Assigned a third pattern based on chromatographic separation and confirmed through modeling of triple breakage fragments.
• Demonstrated the feasibility and limitations of identifying complex intradisulfide patterns using ion trap MS/MS.

Conclusions:

• The developed probabilistic analysis is effective for identifying complex disulfide bond patterns in SNAP25B.
• Ion trap mass spectrometry with collision-induced dissociation can identify complex intradisulfide patterns, though limitations exist.
• This work provides a foundation for understanding SNAP25B's role in oxidative stress response through disulfide bond regulation.