Structural Characterization of Protein Glycosylation Utilizing Fragmentationfrom Gas Phase Ion-Electron Reactions and Vibrational Excitation.

[摘要] Glycosylation is one of the most common post-translational modifications found in eukaryotes.Glycan structural characterization is a difficult undertaking, because full characterization demands knowledge of saccharide linkage, branching, sequence, glycosylation location, heterogeneity, and occupancy.Mass spectrometry, with its sensitivity and tandem mass spectrometric capabilities, is an extremely valuable tool in this endeavor.In this thesis, the utility of gas-phase ion electron reactions, including electron capture dissociation (ECD) and electron detachment dissociation (EDD), for glycosylation structural characterization is explored.Both ECD and EDD have been shown to be valuable techniques for the characterization of post-translationally modified peptides, including both phospho- and glycopeptides.However, further applications of these techniques remain to be explored. The combination of vibrational excitation (here infrared multiphoton dissociation (IRMPD)) and ion-electron based fragmentation (ECD) for de novo sequencing of a lectin with rare carbohydrate binding specificity is demonstrated.Despite the disadvantages associated with IRMPD and ECD, when used in conjunction these techniques proved to be a powerful tool for sequencing purposes.Over 75% of the protein was sequenced with the combination of vibrational excitation and ion-electron based fragmentation. IRMPD and ECD are also utilized for the characterization of high-mannose type glycopeptides.This category of glycopeptides had not been previously examined with these techniques.IRMPD of high-mannose type glycopeptides is shown to often result in a mixture of glycan and peptide backbone cleavage, while ECD results in exclusively peptide cleavage (allowing for glycosylation localization). Ion-electron based fragmentation for oligosaccharide structural characterization is investigated.ECD of metal–adducted oligosaccharides is shown to result in complementary structural information compared to IRMPD, and provide more cross-ring fragmentation than ECD of protonated species.Neutral and sialylated oligosaccharides are also examined with vibrational excitation and EDD.EDD often results in more cross-ring fragmentation compared to vibrational excitation, thus providing additional structural information. Strategies for examining pancreatic cancer associated O-linked glycans are explored.A protocol is developed for examining oligosaccharides in conditioned media from pancreatic cancer cell lines.

[发布机构] University of Michigan

[效力级别] MS/MS

[关键词] FT-ICR Mass Spectrometry;MS/MS;Electron Capture Dissociation;Oligosaccharides;Electron Detachment Dissociation;Glycosylation;Chemistry;Science;Chemistry