title: Resolving double disulfide bond patterns in SNAP25B using liquid chromatography-ion trap mass spectrometry.
abstract: Complex disulfide bond patterns in synaptosomal-associated protein of 25 kD B (SNAP25B) are thought to regulate neurotransmitter release in response to oxidative stress. However, the steric feasibility of each possible disulfide pattern in SNAP25B has not been assessed. To assess the steric feasibility of hypothesized closely spaced complex disulfide patterning in SNAP25B and also the feasibility of identifying complex disulfide bond patterns with MS, we have developed a novel probabilistic analysis to unambiguously resolve complex double disulfide bond patterns by using an ion trap mass spectrometer. We analyzed fragmentation patterns of singly linked peptides to determine likely fragmentation events in an ion trap mass spectrometer and observed double and single backbone cleavage along with heterolytic cleavage of the disulfide bond. We modeled these same events in the doubly disulfide linked SNAP25B peptide and used a cumulative hypergeometric distribution with top-down scoring to both identify and differentiate these bonding patterns. Because of the presence of unique MS/MS peaks, two of the bonding patterns were directly identified. The third was assigned on the basis of full chromatographic separation and confirmed by modeling triple breakage fragments. In total, this work demonstrates the feasibility--and also limitations--of identification of complex intradisulfide patterns by using ion trap-based collision-induced dissociation-based fragmentation methods.
authors.affiliation: Brigham Young University, Neuroscience Center and Department of Physiology and Developmental Biology, Provo, UT, USA.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Ogawa
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Taylor
authors.forename: Dixon J
authors.initials: DJ
authors.lastname: Woodbury
authors.forename: John T
authors.initials: JT
authors.lastname: Prince
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019295:Computational Biology; D003198:Computer Simulation; D004220:Disulfides / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D050825:Synaptosomal-Associated Protein 25 / Q000032:analysis / Q000737:chemistry*
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25