| Electron capture dissociation (ECD) is an important tandem mass spectrometry (MS) method, which features homolytic backbone fragmentation providing extensive sequence coverage and retention of post-translational modifications (PTMs). Two major applications of ECD in proteomics are protein sequencing and PTM characterizations using both top-down and bottom-up approaches. Among the several mechanisms proposed, the free-radical cascade (FRC) is the only one that adequately explains secondary fragmentations in ECD. To further test the FRC mechanism, ECD of fixed charge tag modified peptides was investigated. The data indicated that both the number and location of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. Secondary fragmentations of several synthetic peptides were also studied by both ECD and electron transfer dissociation (ETD). Charge remote fragmentations of z• ions were observed, which were more abundant in ECD than in ETD, resulting in partial/entire side-chain losses and/or formation of internal fragments. ECD was performed on peptide b ions as well, which identified both linear and macro-cyclic structures. A sound understanding of b ion structure is important for spectra interpretation and peptide sequencing. ECD is a particularly useful method for studying deamidation. Deamidation is a spontaneous nonenzymatic PTM of proteins, which is involved in many diseases and postulated to function as a molecular clock in aging. Deamidation of asparagine/glutamine (Asn/Gln) introduces a negative charge to peptides/proteins and increases the molecular mass by +0.984 Da, which can be detected by many MS methods; however, diagnostic ions of isoaspartic and aspartic acid can only be generated by ECD. In order to differentiate the deamidation that occurred during sample preparation process from the pre-existing deamidation of biological interest, H218O labeling combined with ECD was used to monitor the artificial Asn deamidation of several peptides released from trypsin digestion. Due to the much slower deamidation rate of Gln than Asn, few Gin deamidation studies have been reported. The ECD method was further applied to investigate the isomeric Gln deamidation products in peptides. Diagnostic ions specific to gammaGlu were identified, indicating that ECD is also applicable to the study of Gln deamidation. Through this thesis, a better understanding of the ECD mechanism was gained, which helped protein characterization and study of deamidation. |
