It is essential to understand the structural information produced in each type of MS experiment7–9
. To begin this process, the reader is referred to for a glossary of glycoscience terms used in this review. Mass measurement produces information on the composition of biomolecules. In a glycomics or glycoproteomics experiment, an accurate mass may be used to calculate the general monosaccharide composition of the detected ions, i.e. Hex, HexNAc, dHex, NeuAc, etc. For this purpose, the more accurate the mass measurement, the greater the certainty of the interpretation of the glycosylation pattern. A single stage of MS is often used to profile glycoconjugate mixtures based on extracted mass and abundance information. If one makes assumptions regarding the glycan compound class (for example N
-glycans), then this information may be elaborated to include the presence of structures, for example the chitobiose core, that are common to all N
-glycans. It is important to recognize that such assumptions derive from information not produced directly in the MS experiment.
A glossary of glycoscience terms used in this article
One may perform tandem MS on some of the ions observed in the profiling experiment. It is very important to understand, however, that such ions will likely be present as isomeric mixtures; the tandem mass spectra will reflect the mixtures present. If desired, one may purify the glycans and perform additional tandem MS experiments. It is possible in some cases to directly interpret the glycan branching and linkage directly from the tandem mass spectra. This task is complicated if more than one glycan positional isomer is present in the sample. As a result, the investigator must judge the purity of the glycan sample. Glycan tandem mass spectra may also be interpreted in reference to glycan standards of known structure. The use of such standards is necessary to validate tandem mass spectral interpretation.
The concept of sequencing is best applied to linear biopolymers. Many glycan classes, however, are branched and as such do not have a linear sequence. The analytical challenge in glycomics is therefore to determine the connectivity of monosaccharides in the glycan and the linkages for each glycosidic bond. Each glycosidic bond has an anomeric position (α or β), but this cannot be determined using tandem MS except under rare circumstances. The monosaccharide linkages present in a purified glycan can be determined using gas chromatography-mass spectrometry linkage analysis10
. In the absence of sufficient quantities of purified glycan material for this method, anomeric positions are often assigned using biosynthetic assumptions. The tandem MS experiment determines Hex, HexNAc, dHex, NeuAc, etc based on mass values. The identity of the Hex and HexNAc monosaccharides may be inferred by making assumptions based on the compound class and biosynthetic rules. These assumptions are often made implicitly in publications rather than stated directly.
There are substantial differences between the two primary mass spectrometric biomolecular ionization methods with respect to analysis of glycans and glycoconjugates. Under typical vacuum source conditions, matrix assisted laser desorption/ionization (MALDI) results in dissociation of labile glycosidic bonds in glycan and glycoconjugate analytes7, 11, 12
. This is of greatest concerns for acidic monosaccharide and substituents (sialic acids, uronic acids, sulfate, phosphate), and for fucose residues. As a result, use of vacuum MALDI for profiling of native glycans or glycoconjugates will typically underestimate the abundances of these labile monosaccharides in the ions detected. Dissociation resulting from the ionization process limits the usefulness of vacuum MALDI tandem MS of native and reductively aminated glycans and glycoconjugates. Permethylated glycans are considerably more stable than their native and reductively aminated counterparts, and may be profiled effectively using vacuum MALDI MS and tandem MS methods.
Because it is considerably gentler than MALDI, electrospray ionization (ESI) MS may be used to profile intact native glycans without dissociation of fragile acidic groups. To be successful in the application of ESI MS, the investigator is advised to purchase glycan standards to verify the performance of the instrument. In addition, it is typically necessary to use source desolvation settings that are gentler than those used for peptides and other analytes.
The peak capacity of MS experiments is increased by the addition of chromatographic separations, and ESI is directly compatible with on-line chromatography8, 13, 14
. On-line liquid chromatography/mass spectrometry (LC/MS) improves the ability to detect low abundance glycans/glycoconjugates relative to analysis of unseparated mixtures. For this purpose, hydrophilic interaction chromatography (HILIC) and porous graphitized carbon chromatography (PGC) may be used for analysis of native and reductively aminated glycans. PGC and reversed phase (RP) chromatography may be used for reductively aminated glycans. RP, PGC and HILIC may be used for glycopeptides15
. Permethylated glycans may be analyzed using RP and PGC16