Analysis of Glycans by Electrophoretic Approaches: Highly Sensitive Capillary Electrophoretic Separation and Ion Mobility-Mass Spectrometric Structure Assignment

Abstract

Glycosylation is structurally the most complex posttranslational modification of proteins, playing key roles in many biological and disease processes. To understand the biology of glycans and glycoconjugates and to develop biologicals, it is essential to determine exact glycan structures. This thesis describes new electrophoretic analysis approaches for the separation and identification of glycans, based on their mobility in the liquid and gas phase. A new capillary electrophoresis (CE) method for sensitive analysis and ion mobility spectrometry (IMS) methods for exact structure assignment are presented.

An overview of glycan structures, sample preparation and analysis with liquid chromatography, mass spectrometry (MS), IMS and CE is given. Principles of different CE techniques are described and recent developments in instrumentation are discussed. The application of CE with MS and laser induced fluorescence detection for the characterization of biologicals and glycans is shown. Furthermore, a new CE methodology to enrich glycan samples for sensitive analysis is presented. It uses in-line sample trapping with solid-phase extraction sorbents blended with diatomite, which allows for smooth and consistent filling of trap columns and provides higher and sustained flow rates, without affecting injection, elution and other flushing procedures over time. Trap columns where used up to 340 times and applied to glycan analysis using injections of up to 13.6 µL, which is a factor 1000 higher than with conventional CE injection methods. The higher injection volumes enabled the detection of low nM concentrations for N-glycans with CE-IMS-MS.

In addition, a new glycan identification strategy with IMS is presented, which uses the different glycan conformers present in the gas phase. These conformations are measured with IMS to obtain intrinsic arrival time distribution (ATD) fingerprints which are used for the assignment of glycans. We assigned exact structures of protein-derived N-glycans, including all possible Neu5Ac linkage-isomers of a group of biantennary N-glycans, using an ATD database. Such a database will allow the fast identification of N-glycans, including isomers, without the need for MS/MS experiments, which will significantly reduce data processing time.

Another IMS approach, for the exact structure assignment of fucosylated N-glycans, uses ATDs of MS/MS fragments. The fucosylation of glycans leads to diverse structures and is associated with many biological and disease processes. The exact determination of fucoside positions by MS/MS however, is complicated because rearrangements in the gas phase lead to erroneous structural assignments. We demonstrated that the combined use of IMS-MS and synthetic standards can prevent both misinterpretation of MS/MS spectra as well as incorrect structural assignments of fucosylated glycans. We show that fucosyl residues migrate to acetamido moieties of N‐acetylneuraminic acid and N‐acetylglucosamine residues as well as to nucleophilic sites of an anomeric tag, yielding specific isomeric fragment ions. This insight enables the characterization of unique ATDs of the isomers which can be used to accurately determine fucosyl positions in glycans and unambiguous discriminate between MS/MS fragments arising from parent compounds and those that occur due to rearranged fucosyl residues, preventing misinterpretation of MS/MS spectra.