| Protein glycosylation is the most important post-translational modification which affects both physical and biological properties of proteins. Glycoproteins are widely distributed in animals, plants and microorganisms. To understand the structural basis of a glycoprotein's function, it is essential to characterize its glycosylation state and determine its oligosaccharide structures. The existence of most glycoproteins at very low concentrations in biological sources demands highly sensitive techniques.; The carbohydrate structures can be linked to either asparagine residues (N-glycans), or serine or threonine residues (O-glycans) within the polypeptide backbone. N-glycans can be readily hydrolyzed using certain enzymes. For the analysis of N-glycans, we have developed methodologies which allow us to sequence the oligosaccharides through matrix-assisted laser desorption/ionization mass spectrometry at the level of low micrograms of starting proteins. For example, characterization of N-glycans of a sperm-binding protein from the vitelline envelope has been achieved when just several micrograms of the protein were available. Some glycoproteins from the vomeronasal organ (an organ associated with mammalian pheromone reception) have also been isolated and characterized through different techniques, including mass spectrometry.; Compared with N-glycans, much less progress had been achieved in characterizing the O-linked structures, although they are often more biologically significant as signal recognition determinants. Since there are no enzymes currently available to hydrolyze all types of O-glycans from glycoproteins,{09}β-elimination remains the preferred approach for releasing such structures. However, traditional β-elimination produces oligosaccharide alditols which are missing a reducing end, making it impossible to introduce a fluorophore. Therefore, a modified β-elimination procedure has been developed to cleave the N- and O-glycans with the reducing ends intact, permitting their subsequent labeling with a fluorophore or any other structural entity that may facilitate better separation or detection capabilities. Minimum sample handling and easy reagent removal make it possible to cleave effectively the oligosaccharide chains from only low-microgram quantities of glycoproteins. |
