Detailed Glycan Structural Characterization by Electronic Excitation Dissociation

Abstract

The structural complexity and diversity of glycans parallel their multilateral functions in living systems. To better understand the vital roles glycans play in biological processes, it is imperative to develop analytical tools that can provide detailed glycan structural information. This was conventionally achieved by multistage tandem mass spectrometry (MSn) analysis using collision-induced dissociation (CID) as the fragmentation method. However, the MSn approach lacks the sensitivity and throughput needed to analyze complex glycan mixtures from biological sources, often available in limited quantities. We define herein the critical parameters for a recently developed fragmentation technique, electronic excitation dissociation (EED), which can yield rich structurally informative fragment ions during liquid chromatographic (LC)-MS/MS analysis of glycans. We further demonstrate that permethylation, reducing end labeling and judicious selection of the metal charge carrier can greatly facilitate spectral interpretation. With its high sensitivity, throughput, and compatibility with on-line chromatographic separation techniques, EED appears to hold great promise for large-scale glycomics studies.

GlycoDeNovo – an Efficient Algorithm for Accurate de novo Glycan Topology Reconstruction from Tandem Mass Spectra

“…Lately, a number of radical-induced dissociation methods have been applied to structural analysis of glycans, many of which were capable of producing more extensive sequence information than CID [33–43]. Among them, the recently developed EED is a particularly powerful method, as it can generate rich structural information for glycan characterization, including linkage differentiation, for a wide variety of glycans, with or without derivatization [40–41,44–45]. Figure 3 shows the CID and EED spectra and cleavage maps of deutero-reduced and permethylated LNFP II, [M+Na] + , with all assigned peaks listed in Supporting Table S1.…”

“…Whereas it is possible to expand the peak list by assigning a fragment ion in n + charge state with either n −1, n , or n +1 (if n is less than the precursor ion charge state) metal cations, this practice not only dramatically increases the computational time by increasing N , but also increases the chance of spurious matches. Since analysis of glycans adducted with a metal cation having a large mass defect can facilitate metal counting [41], the performance of GlycoDeNovo on EED spectra of both sodiated and cesiated glycans was evaluated here. Finally, glycan tandem mass spectra, especially those generated by EED, can be extremely complex.…”

The Challenge and Promise of Glycomics

“…Due to their larger size and extensive branching, the N-glycans are the most complicated non-glycosaminoglycan structures to analyze, since for example, a tetraantennary glycan can have heterogeneous branches that are even isobaric. These individual branches are difficult to resolve using any number of separation and MS-based platforms of analysis, although progress is being made (Yu et al, 2013a). The analysis of N-glycans, whereby they are released from glycoproteins or glycopeptides using PNGase F or chemical means such as hydrazinolysis, is depicted in Figure 6.…”

Mechanistic Study on Electron Capture Dissociation of the Oligosaccharide-Mg2+ Complex

“…While slow-heating fragmentation methods, such as collisionally activated dissociation (CAD) [3–13] and infrared multiphoton dissociation (IRMPD) [14–15], can generate an abundance of glycosidic fragments for deduction of the glycan topology, they do not normally produce sufficient numbers of the cross-ring fragments that are crucial for determining the linkage configuration. Over the past few years, a number of unconventional fragmentation methods have been applied to tandem MS analysis of glycans, including ultraviolet photodissociation (UVPD) [16–19], free radical-activated glycan sequencing (FRAGS) [20], and various electron activated dissociation (ExD) methods, such as electron capture dissociation (ECD) [15, 21–24], electron transfer dissociation (ETD) [25], electronic excitation dissociation (EED) [22, 26], electron-induced dissociation (EID) [27–28], electron detachment dissociation (EDD) [29–30], and negative electron transfer dissociation (NETD) [31]. In particular, ECD appears to be a promising tool for glycomics research as it can provide richer structural information than CAD-based methods, and is fairly straightforward to implement in online liquid chromatography-MS/MS studies.…”