Characterization of protein glycosylation by mass spectrometry

The structural characterization of oligosaccharides on glycoproteins is essential to the understanding of their function. Protein-linked oligosaccharides are generally located at the molecular surface and are therefore optimally situated to modulate biochemical properties such as bioactivity, folding and immunogenicity, and act as determinants in molecular recognition events such as targeting of enzymes or the uptake of glycoproteins by receptors. Conventional strategies involving chemical degradation and nuclear magnetic resonance spectroscopy require nanomole to micromole quantities of isolated glycans, and chemical degradations may alter labile structural features in unforeseen ways. However, newer techniques such as electrospray ionization mass spectrometry can be used to analyze native glycoconjugates intact which have not been subjected to chemical degradations. Enzymatic and mass spectrometric methods have been developed to characterize glycoprotein oligosaccharides while still attached to the protein as glycopeptides, allowing the glycosylation site-specific characterization of picomole to femtomole quantities of glycopeptides. Using these methods, the sites and structures of the O-linked oligosaccharides of recombinant human platelet-derived growth factor and recombinant human p75 nerve growth factor receptor extracellular domain have been characterized. In addition, the sites and structures of the N-linked oligosaccharides of recombinant human platelet-derived growth factor receptor and recombinant human p75 nerve growth factor receptor extracellular domains and human lecithin:cholesterol acyl transferase have been determined. All analyses (with the exception of those for the platelet-derived growth factor which were done using liquid secondary ion mass spectrometry) were performed using electrospray ionization mass spectrometry, with pmole to fmole quantities of glycopeptides. The sensitivity of these techniques is further demonstrated by the additional site-specific characterization and sequencing of the N-linked oligosaccharides from human apolipoprotein D, a protein which co-purified with the human lecithin:cholesterol acyl transferase protein, present at a level approximately one fifth of the lecithin:cholesterol acyl transferase. Knowledge of the site-specific oligosaccharide structures was used to help understand the various known effects of the glycosylation on the specificity and biological activity of these proteins.