Glycans and their conjugates (proteins, lipids, various anabolites) are found attached to the surface of cells, within cells and in the extracellular matrix. They are integral to an extraordinarily diverse range of biological processes including regulation of cell–cell recognition [1], cell adhesion [2], [3] (including host-pathogen interactions [4], [5], [6]), immune response [7], fertilization [4], [8], [9], [10], trafficking, and intra- and extracellular signalling events. Even subtle changes in carbohydrate structure can result in vastly different interactions, make them susceptible to enzymatic hydrolysis or alter glycoconjugate tertiary structures and thus affect their observed biological response [2], [11], [12], [13], [14], [15], [16], [17], [18]. Interestingly both the identity and levels (or presence) of specific glycan structures within individuals depends on numerous factors including gender [8], [19], blood group [20], age [21], [22], [23], disease state [12], [17], [24], [25], [26], [27], [28] and diet [29], [30]. It remains unclear a priori how changes in glycan structure will affect the resultant biological function [31]. Given their ubiquity in nature, it is unsurprising that aberrant glycan structures have been identified as biomarkers for several disease states including various cancers [11], [14], [26], [32], [33], [34], [35], [36], hereditary disorders [17], [37], acute pancreatitis [24], immune and cardiovascular deficiencies [12], [31], [38], Alzheimer's disease [39], [40] and muscular dystrophy [2].
The incredible amount of three dimensional stereochemical information contained within glycans is biologically very important, as it is required to accommodate for their diverse functionalities even from a relatively small pool of monosaccharide building blocks. This complex ‘chemical information’ arises from: (1) different monosaccharide building blocks; (2) the potential to link these monomers at a number of different positions in two different stereochemistries, namely α- and β-, forming both linear and branched structures; (3) the ability for identical monosaccharides to have been incorporated with different ring connectivities, i.e. pyranose and furanose structures (4) further decoration with chemically diverse functional groups (e.g. phosphate, sulphate, methyl groups) (Fig. 1). To understand and exploit glycan-driven functions, it is necessary to be able to unambiguously elucidate carbohydrate structures, in the absence of a priori information to be able to define structure–function relationships. To develop and exploit differences in glycan structures as potential biomarkers of disease, it will be necessary to define these structures in a high throughput manner to allow characterization on a person-to-person basis, permitting the development of novel therapeutics or diagnostics [41], [42], [43]. The demand for three-dimensional structure determination has resulted in fewer advances in comprehensive structural elucidation of glycans compared with proteins and nucleic acids, especially since glycan structures are not directly encoded by genetic information. Moreover, microheterogeneity, the low amounts available for analysis, and the chemical similarity between monosaccharide building blocks, which are often simple epimers of one another, hinder their full structural elucidation.
The current ‘gold standard’ for glycan analysis typically involves liquid chromatography (LC) separation [11], [12], [31], [44], [45], which can also be coupled with the speed and sensitivity of tandem mass spectrometry (MS2) (Fig. 2) [46], [47], [48]. Glycans are then characterized based on either their retention time against a standard and/or by their MS2 spectrum [45], [49], [50], [51], [52], [53], [54], [55], [56], [57]. However, LC separation can require multiple different columns to gain significant separation of chemically similar glycan species and as a result it is often low throughput. Also, without the benefit of (synthetic) reference standards, LC provides no structural de novo information. MS alone is limited to characterizing the monosaccharide class (i.e. hexose, N-acetylhexosamine, deoxy hexose etc.) and is insufficient to directly identify these monosaccharide units from each other without the use of an orthogonal sequencing approach. Given the intrinsic ability of carbohydrates to be isomeric, these strategies only often work for systems whose biosynthetic pathways are well understood (e.g. N-glycans) to facilitate in their identification. The inability of this analysis strategy to separate and characterize these iso(mer/bar)ic species from complex biological mixtures can hinder the development of specific glycomic approaches for assessing disease state [27].
Recently there has been a surge in the application of Ion Mobility-Mass Spectrometry (IM-MS) to enhance structural characterization and separation of glycans and glycoconjugates. Ion mobility spectrometry (IMS) is an analytical technique that measures the mobility, K, of gas-phase ions under the influence of an electric field in the presence of a buffer gas; the mobility is based on the size, shape and charge of the ions [59], [60], analogous to electrophoresis in the condensed phase, although the timescale is much shorter (high-μs to ms). Ions can be generated by a range of techniques, although for most IMS applications, ions are generated by ESI. This technique has been used for a variety of types of analyses, and has major applications in security and defence for example in airports where it is routinely used to screen against explosives, chemical-warfare agents and drugs. The recent surge in use of ion mobility to analyse glycans is primarily as a result of the commercialization of hybrid IM-MS instrumentation (Synapt HDMS, 2006) and its potential to overcome challenges associated with analysis of glycans; namely separation of often isomeric carbohydrates and structural characterization of carbohydrates in conjugation with molecular dynamics (MD) simulations.
This review assesses the applicability of IM-MS towards biologically relevant glycans and the wealth of additional information that can be obtained from this high-throughput strategy. This will undoubtedly improve our understanding of glycan functions and thus could provide promising tools for further medicinal applications. Initially, the various IMS strategies routinely coupled to MS will be briefly discussed to help the reader appreciate the limitations and advantages of each of the techniques; however for a more thorough background into IMS the reader is encouraged to read excellent previous reviews [60], [61], [62]. Similarly, computational aspects (such as MD simulations) that facilitate elucidation of gas-phase structures are briefly discussed, however, they are more extensively detailed elsewhere [63], [64].