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SNAP-25 contains two of the four helixes that form the SNARE complex, critical for neuronal exocytosis. The helixes are linked together with a cysteine-rich domain, which is the site of various post-translational modifications such as palmitoylation and possibly oxidation. SNAP-25's linker contains 4 cysteines and thus could be both palmitoylated and oxidized. Palmitoylation anchors SNAP-25 to the membrane, while the level of oxidation may regulate transmitter release.Using standard MS/MS collision-induced dissociation (CID) in an Ion Trap mass spectrometer we analyzed peptide fragmentation patterns to determine fragmentation events, and observed single and double backbone cleavage along with heterolytic cleavage of disulfide bonds. We modeled these same events in the doubly disulfide linked SNAP25B peptide and used a cumulative hypergeometric distribution with top-down scoring to first identify, and then to differentiate all three bonding patterns. This is the first study to assign all double disulfide bonding patterns in a protein, and uses methodology fully compatible with standard large scale shotgun proteomics.In addition, the structural stability of oxidized and reduced SNAP-25 was determined with Circular Dichroism (CD). We observe that oxidized protein is less stable (lower melting temperature) than reduced protein. Additional experiments focus on the stability of the SNARE complex with oxidized samples and on the extent of SNAP-25 oxidation in brain tissue, with the ultimate goal to elucidate the effects of palmitoylation and oxidative stress on SNARE complex formation and vesicle fusion.

SNAP-25 plays a critical role in neuronal exocytosis. The SNAP-25 linker region contains a cysteine-rich domain, which is the site of various post-translational modifications such as palmitoylation and possibly oxidation. Palmitoylation anchors SNAP-25 to the membrane, while the level of oxidation may regulate the extent of transmitter release. Using a biotinylation/chemiluminescence assay, within a reconstituted system, and mass spectrometry (LTQ Orbitrap) we characterize the modification of the four cysteine residues in the SNAP-25B linker. We show that the palmitoyl transferase DHHC17 increases the extent of SNAP-25B palmitoylation in vitro. We verified that oxidation of SNAP-25B leads to disulfide bond formation within the cysteine-rich domain, the predominant form containing two disulfide bonds. As expected, oxidation of cysteines prevented palmitoylation. Through additional experimentation we hope to elucidate the effects of palmitoylation and oxidative stress on SNARE complex formation and vesicle fusion.