Gas-phase Ion-electron and Ion-photon Reactions for Structural Characterization of Protein Glycosylation.

Abstract

Glycosylation is one of the most prevalent post-translational modifications (PTMs), playing key roles in various biological activities. Characterization of protein glycosylation faces unique challenges due to the highly diverse structures of glycans and the heterogeneity of glycoforms at a specific glycosylation site. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is an extremely valuable tool for glycosylation analysis, benefiting from high resolution, ultrahigh mass accuracy, and a multitude of tandem mass spectrometric capabilities.

In this thesis, ion-electron and ion-ion based fragmentation techniques such as electron capture dissociation (ECD), electron transfer dissociation (ETD), and electron detachment dissociation (EDD) are explored for structural characterization of glycosylation. Acidic glycans such as sulfated and sialylated glycans have been linked to cancer metastasis. EDD and metal-assisted ECD/ETD are utilized for structural elucidation of acidic and neutral oligosaccharides, and complementary structural information is obtained compared to collision activated dissociation and infrared multiphoton dissociation (IRMPD). Compared to ECD, ETD is less efficient for fragmenting low charge state precursor ions. For sialylated glycans, negative-ion mode analysis is more advantageous due to the abundant signal and the ability of EDD to generate extensive glycosidic and cross-ring cleavages.

We explore EDD and IRMPD of fluorescently labeled oligosaccharides, and compare the influence of different labels. Complementary structural information can be obtained from EDD and IRMPD. Acidic labels promote glycan signal in negative-ion mode, but also introduce competition of deprotonation sites which impedes the formation of cross-ring cleavages. IRMPD and EDD are also utilized for structural characterization of N- and O-linked glycopeptides. For N-linked glycopeptides, EDD fragmentation efficiency decreases with increased peptide length. For O-linked glycopeptides, EDD is able to yield peptide backbone, glycosidic, and cross-ring cleavages in the same spectrum, suggesting EDD as a powerful tool for structural analysis of O-glycopeptides.

Finally, development of LC-FT-ICR MS methods to separate and identify neutral and acidic N-glycans using hydrophilic interaction chromatography and graphitized carbon columns is also described. The methods explored in this thesis can contribute to the process of decoding the glycome, and yield new insights into the highly diverse functions of protein glycosylation.