Glycoprotein Analysis: General Methods

Glycosylation is a common co- and post-translational modification of a protein which may have profound effects on protein structure and function. Many biologically interesting proteins are glycosylated at their asparagine, serine, and threonine residues. Glycosylation has now been recognized as being more ubiquitous and structurally varied than all other types of post-translational modifications combined. The glycosylation pattern of a glycoprotein is not random but is dependent on cell type, physiological conditions and, in some cases, disease states. Carbohydrate chains or glycans attached to the peptide backbone are classified according to the nature of the linkage to the peptide and their structure; both of these parameters allow the definition of common cores. In addition to the extreme structural diversity and complexity of glycan structures, one feature of protein glycosylation is the presence at a single site of any one of a number of glycan structures. This property gives rise to an extremely heterogeneous population of glycoproteins, termed glycoforms. The complexity of the glycan structures, the multiple substitutions (microheterogeneity) at glycosylation sites, and the structural diversity associated with the protein backbone itself represent an enormous task for analytical structural studies. Indirect methods such as immunological approaches using antibodies or lectins provide some structural information concerning the nature of glycans. Prior to their structural study, glycans have to be released from the protein backbone by enzymatic or chemical methods. Different endoenzymes such as N-glycanase® allow a complete deglycosylation of glycoproteins, providing a pool of oligosaccharides which may be further fractionated. Establishment of the site heterogeneity and assessment of the different glycoforms require isolation and analysis of the individual glycopeptides generated after hydrolysis with specific proteases. Fractionation of glycans or glycopeptides can be achieved by different high-performance liquid chromatography (HPLC) or electrophoretic procedures. Lectins, due to their extreme specificities with regard to monosaccharide or oligosaccharide motifs, represent powerful tools for sequential glycan fractionation. Traditionally, a complete structural analysis of the glycan structures, including determination of the carbohydrate sequence and the sugar linkage, has been a tedious, multidisciplinary task. Characterization and determination of the relative amount of individual constituent monosaccharides is generally obtained by gas–liquid chromatography (GLC) after hydrolysis of the oligosaccharide chain. Information concerning the nature of linkages and branching points is furnished by the methylation procedure. Nevertheless, these chemical sequencing methods often necessitate milligrams of material. To progress from this situation, sensitive physicochemical or enzymatic methods have been developed. The nuclear magnetic resonance (NMR) study of oligosaccharides, even of the less sensitive of the physicochemical techniques, is extremely powerful because it allows determination in a mixture of glycans, the complete structure of individual glycans including nature of monosaccharides, sequence, type of linkages, anomericity and branching points. The recent advances in mass spectrometry (MS) techniques, mainly matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI), have provided very sensitive means for the analysis of oligosaccharides and glycopeptides. The gain in sensitivity associated with a minimum number of sample manipulation steps allows work at the submicrogram level. The use of enzyme reagents is rapidly becoming popular in modern carbohydrate analysis because of their inherent selectivities for a sugar substrate and its linkage type. Exoglycosidases can be used in conjunction with other methods such as MALDI/MS or fluorophore-assisted carbohydrate electrophoresis (FACE) separation. Complete structural information is now feasible for low-microgram and submicrogram quantities of a glycoprotein; this quality of performance places the field of carbohydrate analysis closer to the sequencing methods available for other biomolecules, proteins or nucleic acids.